Wednesday, February 11, 2015 489a

Symposium: Membrane Trafficking 2470-Symp Studying Membrane Fusion at the Molecular Level using a Biomimetic 2467-Symp Model System Probing the Structure, Topology, and Oligomerization of -1 Alexander Kros. Kerney Jebrell Glover. leiden institute of chemistry, leiden university, leiden, Netherlands. Chemistry, Lehigh University, Bethlehem, PA, USA. Fusion of lipid bilayers, or membranes, is a ubiquitous process. It occurs in the are highly-curved invaginated micro-domains located in the plasma cells of our body during cargo exchange by membrane vesicles and during viral membrane that play a central role in a variety of cellular processes. infection, but also in industrial processes such as yeast production. While many (1, 2, and 3) are the most important found in caveolae, and are proteins like SNAREs have been identified that play crucial roles in membrane responsible for giving caveolae their unusual ‘‘flask-like’’ shape. Recent fusion, the molecular mechanism of fusion remains unclear. This fascinating evidence has shown that improper regulation and mutant forms of process is unexpectedly complex and our aim is to understand the details of caveolin can result in a variety of diseases including Alzheimer’s, muscular this process using a biomimetic model system which has all the characteristics dystrophy, cancer, and heart disease. Caveolin adopts an unusual intra- of natural membrane fusion; targeted docking, followed by lipid and content membrane ‘‘horseshoe’’ conformation where both its N- and C-termini face mixing in the absence of leakage. the cytoplasm, and this conformation is thought to promote membrane curva- We developed a model system composed of a complementary pair of lipidated ture. In addition, via high-order oligomerization, caveolin is thought to form peptides able to form a heterodimeric coiled coil motif at the membrane inter- a structural backbone which stabilizes the membrane curvature. Using bio- face similar to natural SNARE subunits. The different steps of membrane physical techniques (NMR, fluorescence spectroscopy, and analytical ultra- fusion are currently studied using biophysical and biochemical techniques centrifugation) we have begun to build a structural and topological model with a special focus on peptide-peptide and peptide-lipid interactions. Unravel- of caveolin-1. ling of the molecular mechanism of membrane fusion using a multi-faceted approach of complementary techniques will result in a unique, in depth view 2468-Symp of the process of membrane fusion. Pho85/Cdk5 is a Positive Regulator of Phosphatidylinositol 3,5-Bisphos- To date, it is a major challenge to achieve efficient and targeted liposome de- phate via Direct Phosphorylation of Fab1/PIKfyve livery directly into the cytoplasm of live cells, circumventing endocytotic path- Natsuko Jin, Lois Weisman. ways. In this respect, we recently studied and achieved membrane fusion University of Michigan, Ann Arbor, MI, USA. between liposomes and live cells. Future applications are foreseen in drug de- The signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) plays livery, nanoreactors and membrane engineering to name but a few. critical roles in multiple tissues and multiple organisms. PI(3,5)P2 is regulated in part via a complex that contains the PI3P-5-kinase Fab1, the PI(3,5) P2 lipid phosphatase, Fig4, and the scaffold Vac14. In yeast, as well as in neu- Symposium: Nanopores: Methods and rons, PI(3,5)P2 levels are transiently and rapidly elevated in response to Mechanistic Insights selected signals. However to date, the upstream signaling pathways that regu- late PI(3,5)P2 have not been identified. Here we report that in the yeast 2471-Symp Saccharomyces cerevisiae, the CDK, Pho85 and the cyclin, Pho80 are Polymers through Protein Pores: Single-Molecule Experiments with Nu- required for the acute elevation of PI(3,5)P2 upon hyper-osmotic shock. cleic Acids, Polypeptides and Polysaccharides Pho85-Pho80 directly phosphorylates Fab1 and positively regulates the syn- Hagan Bayley. thesis of PI(3,5)P2. Furthermore, we find that Pho85-Pho80 phosphorylation Univ Oxford, Oxford, United Kingdom. of Fab1 likely generates a conformation change that activates Fab1 lipid ki- When polymers move from one cellular compartment to another, they pass nase activity. Cdk5- p35, the mammalian homologue of Pho85-Pho80, is through protein pores. Nucleic acids, polypeptides and polysaccharides are particularly critical in neuronal physiology and regulate multiple pathways. all transported in this way, stimulating questions about the nature of the trans- Notably, we find that mammalian Cdk5-p35 directly phosphorylates peptides ported polymer (diameter, stiffness, branching, charge, charge distribution), the of mammalian Fab1/PIKfyve, which strongly suggests that PIKfve is a direct driving force (DV, DpH, refolding, binding) and how that driving force is target of Cdk5-p35 in tissue culture cells. These studies reveal a conserved, coupled (direct coupling v diffusion/ ratchet). We have been investigating all new down stream target of Cdk5, and provide insights into how PI(3,5)P2 three classes of biopolymer by current recording through individual transmem- is regulated. brane pores. We have not only made interesting fundamental discoveries about the translocation processes, but also found useful applications of our work, for 2469-Symp example in nucleic acid sequencing and the discovery of antibacterial agents. at the Brink of Fission Jenny E. Hinshaw1, Anna C. Sundborger1, Shunming Fang1, 2472-Symp Jurgen A. Heymann1, Pampa Ray1, Joshua S. Chappie2. Molybdenum Disulfide Nanopores: Why 3 Atoms are Better than One? 1 Laboratory of Cell and Molecular Biology, NIDDK, NIH, Bethesda, MD, Aleksandra Radenovic. 2 USA, Department of Molecular Medicine, College of Veterinary Medicine, EPFL, Lausanne, Switzerland. Cornell University, Ithaca, NY, USA. Atomically thin nanopore membranes are considered to be a promising Dynamin family members are large GTPases that are associated with diverse approach to achieve single base resolution with the ultimate aim of rapid and cellular processes, including -mediated endocytosis, fusion and fission cheap DNA sequencing. Recently, we made advances in using 2 D materials of mitochondria, division of chloroplasts and peroxisomes, cell division, and such as graphene or MoS2. as nanopore platform .Translocation of various types resistance to viral infections. The founding member, dynamin, plays a direct of DNA exhibits a signal amplitude that is five times higher than in the case of role in endocytosis by assembling around the necks of clathrin-coated pits in a solid-state Si3N4 membranes and a SNR of more than 10. These features are helical array. The self-assembly of dynamin into oligomers stimulates its highly desirable for event detection and we take advantage of them by showing GTPase activity, which is necessary for efficient fission during endocytosis. the electric-field induced unfolding of a 48 kbp long DNA molecule within the Recent evidence suggests that a three-bundle helix, near the G domain, un- nanopore which manifests itself in the quantization of the current drop. Unlike dergoes a dramatic hydrolysis-dependent conformational change that may graphene nanopores, no special surface treatment is needed to avoid strong function as a dynamin powerstroke. To understand how the powerstroke prop- interaction between DNA and the surface. Our results imply that MoS2 nano- agates through the helical assembly and contributes to membrane fission, we pore membranes can compete with graphene nanopore membranes in terms solved the helical structure of a transition-state-defective dynamin mutant, of spatial resolution and possibly better performance for transverse detection. K44A, using cryo-electron microscopy and image processing techniques. References The 3-dimensional map of K44A is a 2-start helix with an inner luminal diam- Detecting the translocation of DNA through a nanopore using graphene nanorib- eter of 3.7 nm, reaching the theoretical limit for spontaneous fission. Compu- bons F. Traversi, C.Raillon, S. M. Benameur, K.Liu, S. Khlybov, M. Tosun, D. tational docking of G domains with the three bundle helix into the 3D map, Krasnozhon, A.Kis and A.Radenovic Nature Nanotechnology 8, 939-945 2013 reveals that a GTP ground state of dynamin is sufficient to achieve this Nanopore Integrated Nanogaps for DNA Detection A.Fanget, F. Traversi, ‘super-constricted’ state and shows how the 2-start helical arrangement gener- S.Khlybov, P.Granjon, A.Magrez, L. Forro´, and A. Radenovic Nano Lett., ates the most efficient packing of dynamin around the membrane neck. Addi- 14 (1), pp 244-249 2014 tional structural and biochemical studies are underway to unravel the global Atomically thin molybdenum disulfide nanopores with high sensitivity for DNA conformational changes that are needed to move beyond the final super- translocation constricted pre-fission state. Liu, K., Feng, J., Kis, A. & Radenovic, A. ACS Nano 8, 2504-2511 2014 490a Wednesday, February 11, 2015

2473-Symp with the polypeptide backbone, instead there are interactions with conserved Real-Time Shape Determination and 5-D Fingerprinting of Single Proteins mainchain glutamate and serine residues, as well as backbone carbonyl atoms, Michael Mayer. all of which are likely mediated by a disordered hydration shell. Electrostatic Biomed. Eng. and Chem. Eng., University of Michigan, Ann Arbor, calculations on the structure are compatible with relative cation selectivities of MI, USA. NaþzLiþ>>Kþ,Caþ, which correspond with the ion permeability ratios This talk describes the use of electrolyte-filled nanopores to determine, simul- measured for these channels. Mutation of the conserved glutamate 178 to taneously and in real time, the shape, volume, charge, rotational diffusion aspartate results in reduced sodium ion conductance through the pore. Inter- coefficient, and dipole moment of individual proteins. It introduces the main estingly, our structure of the E178D mutant reveals that it lacks the sodium concepts for a quantitative understanding and analysis of modulations in ionic ion nearest the extracellular vestibule and most closely associated with current that arise from rotational dynamics of single proteins as they move E178 in the native structure, explaining the reduced conductance. These re- through the electric field inside a nanopore. The resulting multi-parametric in- sults provide insight into the biophysical determinants of sodium-selectivity, formation raises the possibility to characterize, identify, and quantify individual which initiates the opening of other ion channels to shape the action potential proteins and protein complexes in a mixture. This approach interrogates single waveform. proteins and determines parameters such as the shape and dipole moment, 2476-Plat which are excellent protein descriptors and cannot be obtained otherwise D from single protein molecules in solution. Therefore, this five-dimensional Conduction and Selectivity in Na Channels Analyzed by Bias-Exchange characterization of proteins at the single particle level has the potential Metadynamics Simulations for instantaneous protein identification, quantification, and possibly sorting Simone Furini1, Paolo Barbini1, Carmen Domene2. 1Department of Medical Biotechnologies, University of Siena, Siena, Italy, with implications for structural biology, proteomics, biomarker detection, 2 and routine protein analysis. Department of Chemistry, King’s College London, London, United Kingdom. 2474-Symp Bacterial Naþ channels have been the subjects of numerous computational Pores with Undulating Diameter for Multipronged Characterization of studies since the first experimental structure of a Naþ selective channel was Single Particles and Cells in Resistive-Pulse Technique solved in 2011. Molecular Dynamics simulations revealed the presence of 2 Zuzanna S. Siwy1, Laura Innes1, Matthew Schiel1, Ivan Vlassiouk2, binding sites for Naþ ions, respectively at the intracellular and extracellular Kenneth J. Shea3, Luke Theogarajan4. entrance of the selectivity filter, separated by low energy barriers. While there 1Dept. of Physics and Astronomy, University of California, Irvine, Irvine, is a general agreement about these features, there are also important differences CA, USA, 2Oak Ridge National Laboratory, Oak Ridge, TN, USA, 3Dept. of among the various computational studies. In particular, ion conduction has Chemistry, University of California, Irvine, Irvine, CA, USA, 4University of been described both as a 2-ions or a 3-ions process, and this difference has California, Santa Barbara, Santa Barbara, CA, USA. been correlated to the direction of conduction, or to the state of the intracellular Single pores in resistive-pulse technique have been successfully used for gate. A current limit of the computational strategies usually adopted to estimate the detection of cells, viruses, particles, and even molecules such as DNA the energy profiles for permeation events, is that the number of permeating ions and proteins. We have investigated application of pores with undulating open- has to be defined in advance. As consequence, it is difficult to compare energet- ing diameter for the detection of particles and characterization of their phys- ically conduction mechanisms characterized by different number of ions, and ical and mechanical properties including size, shape and squishiness. The this could explain the lack of congruence in the literature. In order to overcome resistive pulses generated by polymer spheres passing through these pores this limit, we tested a novel approach for the analysis of ion conduction based had a repeatable pattern of large variations corresponding to these diameter on bias-exchange metadynamics simulations. In bias-exchange, several rep- changes. We showed that this pattern of variations enabled the unambiguous licas of the system are simulated in parallel. A metadynamics simulation is per- resolution of multiple particles simultaneously in the pore, that it could detect formed for each replica, along one or a few collective variable, and at fixed time transient sticking of particles within the pore, and that it could confirm intervals swaps of configurations between replicas are attempted. Using this whether any individual particle completely translocated the pore. These re- approach it was possible to analyze by a single set of simulations the free sults have practical importance for increasing the speed of resistive-pulse energy for permeation events with different number of ions. The analysis re- sensing, optimizing the detection of specific analytes, and identifying particle vealed that several conduction mechanisms are indeed possible for Naþ chan- shapes. We also showed pores with undulating opening diameter developed nels. This computational strategy could find wide applications for the study of local pressure drops, which were sufficiently large to probe mechanical prop- ion channels, in particular to characterize conduction of ion-mixtures, or chan- erties of passing objects. Application to hydrogels as well as biological cells nels that exhibit heterogeneous conduction events. will be discussed. Methods to measure diffusion coefficient and electrokinetic velocity of individ- 2477-Plat ual particles will be discussed in context of performing detection from diluted Inactivation Voltage Sensor S4 in Domain IV of Nav1.2 Controls Immobi- solutions of an analyte. Balancing all forces acting on particles allowed us to lization of S4 in Domain III as Shown by Omega Currents observe random walk of individual particles in a pore and estimate their diffu- Nikolaus G. Greeff, Claudia Lehmann, Hansjakob Heldstab. sion coefficient from the variance of diffusion velocities of a particle. Trapping Biophysics Institute Greeff, Uetikon am See, Switzerland. of particles/cells for a controllable amount of time between few milliseconds The role of S4DIV for inactivation of skeletal muscle Na channel Nav1.4 was and a few minutes will be presented as well. recognized after deciphering the channelopathia Paramyotonia congenita. We showed with point mutations in the rat brain sodium channel Nav1.2 the central role of S4DIV for inactivation (Ku¨hn and Greeff, 1999): The single mutation Platform: Voltage-gated Na and Ca Channels R4H in S4DIV slowed the recovery from inactivation about 20 times in parallel for ionic current and immobilized gating charge. Immobilization concerns 2475-Plat about 50 % of total gating charge returning slowly to the resting state during Sodium Ion Coordination in the Selectivity Filter of a Voltage-Gated recovery while the other half of gating charge returns very quickly. Clearly, Sodium Channel the amount of immobilized charge is more than just the one from S4DIV. So Claire E. Naylor1, Claire Bagneris1, Paul G. DeCaen2, David E. Clapham2, we speculated that S4DIV would control S4s in other domains. Now, we are B.A. Wallace1. able to monitor the return of S4 into the resting position for each domain sepa- 1Institute of Structural and Molecular Biology, Birkbeck College, University rately by recording the leak current of resting-state omega pore mutants (this of London, London, United Kingdom, 2Department of Cardiology, Howard Meeting). We find that S4DIV with the omega mutation RR12QQ shows a Hughes Medical Institute, Children’s Hospital Boston, Boston, MA, USA. fast onset of omega leak current for channels at rest; however, after an inacti- Voltage-gated sodium channels are essential for electrical signalling across vating prepulse, the leak current grows with the time course of recovery as ex- eukaryotic cell membranes. They exhibit strong selectivity for sodium over pected, since this voltage sensor controls the recovery and returns into resting other cations, thus enabling the finely-tuned cascade of events associated position accordingly. Checking the return of S4 in the other domains, we find a with action potentials. A new high resolution crystal structure of the prokary- fast return in DI and DII while in DIII the return follows DIV. Combining these otic sodium channel pore NavMs from Magnetococcus marinus provides the mutations with R4H in DIV, the return in both domains III and IV is about 20 first view of the locations of sodium ions within the selectivity filter of a so- times more slowly. This suggests that immobilization of gating charge across dium channel. The structure reveals three sodium ions are bound within the the domains is most likely achieved by the cytoplasmic loop between DIII selectivity filter. Unlike potassium ions in potassium channels, the sodium and DIV which under control of S4DIV closes the alpha-pore and immobilizes ions in these channels appear to be hydrated and make no direct contact S4DIII. Wednesday, February 11, 2015 491a

2478-Plat 2480-Plat Autosomal Recessive Inheritance of Congenital Myasthenic Syndrome is Live-Cell Imaging of Midchannel Proteolysis of Neuronal Surface L-Type Associated with Skeletal Muscle Sodium Channel Mutations Calcium Channels Karima Habbout1, Serena Guiliano1, Hugo Poulin2, Damien Sternberg3, Ioannis E. Michailidis, Dan Zhang, Jian Yang. Bruno Eymard4,5, Franc¸ois Rivier6, Raul Juntas Morales6, Biological Sciences, Columbia University, New York, NY, USA. Mohamed Chahine2, Sophie Nicole7, Saı¨d Bendahhou1. We recently identified midchannel proteolysis as a novel homeostatic mecha- 1University of Nice Sophia Antipolis, UMR 7370 LP2M CNRS - Labex nism of negative feedback regulation of neuronal voltage-gated calcium chan- 2 ICST, Nice, France, Centre de Recherche, Institut universitaire en sante´ nels (VGCCs): the main body of the pore-forming alpha1 subunit of L-type 3 mentale de Que´bec, Que´bec City, QC, Canada, Inserm, U1127 - CNRS, VGCCs, Cav1.2, is cleaved, resulting in Cav1.2 fragment-channels that separate UMR 7225 -ICM -AP-HP, Hoˆpital de la Pitie´ Salpeˆtrie`re, Fe´de´ration de but remain on the plasma membrane, and can be detected by confocal micro- biochimie, Sorbonne universite´s, UPMC Univ Paris 6, UM 75, Paris, France, scopy in fixed neurons [Michailidis et al, Neuron, 82 (2014), 1045-57]. The pro- 4Inserm, U1127 - CNRS, UMR 7225 -ICM -AP-HP, Hoˆpital de la Pitie´ teolysis is bidirectionally regulated by channel activity, depends on and Salpeˆtrie`re, Sorbonne universite´s, UPMC Univ Paris 6, UM 75, Paris, France, the ubiquitin-proteasome system, and increases with age. To investigate the ki- 5AP-HP, Hoˆpital de la Pitie´ Salpeˆtrie`re, Centre de re´fe´rence des maladies netics of midchannel proteolysis and the fate of the resulting fragment-channels, neuromusculaires Paris Est, Paris, France, 6Service de neurope´diatrie, CHU we performed live-cell confocal microscopy. In live cultured rat hippocampal 7 de Montpellier, Hoˆpital Gui de Chauliac, Montpellier, France, Inserm, neurons, we visualized surface Cav1.2 labeled with extracellular fluorescent U1127 - CNRS, UMR 7225 -ICM, Sorbonne universite´s, UPMC Univ Paris probes placed in different internal repeat domains of the alpha1 subunit. The re- 6, UM 75, Paris, France. sults reveal robust movements of surface Cav1.2 and its domains, suggesting The SCN4A , that encodes the pore-forming a-subunit of the skeletal mus- midchannel proteolysis and continuous dynamic regulation of surface channels. cle voltage-gated sodium channel, has long been associated with dominantly- inherited myotonia, and periodic paralysis mutations. One single severe case 2481-Plat of congenital myasthenic syndrome (CMS) due to a dominantly-inherited Mechanisms of Auxiliary a2d-1 Subunit Regulation of Voltage-Gated SCN4A mutation has been reported. CMS is a clinically and genetically hetero- Calcium Channels Sihui Ma1, Henry M. Colecraft2. geneous group of rare disorders characterized by muscle weakness and fatiga- 1 2 bility. They are due to impairment of neuromuscular transmission and result Pharmacology, Columbia University, New York, NY, USA, Physiology from mutations in encoding for proteins critical for the neuromuscular and Cellular Biophysics, Columbia University, New York, NY, USA. junction. We have identified a novel homozygous SCN4A mutation The voltage-gated calcium (CaV) channel a2d-1 subunit is implicated in a vari- (p.R1454W) in a patient with a rare form of recessively-inherited CMS. The ety of cardiovascular and neurological diseases and is the molecular target of p.R1454W missense mutation is located on the segment S4 of domain IV of gabapentin, a drug used for the treatment of epilepsy and neuropathic pain. the Nav1.4 channel. Expression of p.R1454W mutant Nav1.4 in the human em- The precise mechanisms by which a2d-1 dysfunction causes disease and how bryonic kidney 293 cells induced dramatic changes in the biophysical proper- its targeting by gabapentin is therapeutic are unclear, in part due to incomplete ties of the mutated channel. These include fast and slow inactivation,kinetics of understanding of how a2d-1 binds to and regulates CaV channels. Here, we investigated mechanisms underlying a2d-1 regulation of neuronal CaV2.2 fast inactivation, and recovery from inactivation. A slower current decay com- þ bined with a shift in channel availability at resting potentials can ultimately (a1B b2a) channels reconstituted in HEK293 cells. Using whole-cell electro- physiology, a2d-1 caused a dramatic four to five-fold increase in whole-cell cur- lead to membrane inexcitability and muscle weakness. Our data confirm that þ this CMS phenotype belongs to the group of sodium channel disorders and rent amplitude compared to channels reconstituted with a1B b2a alone. The question the clinical overlapping between periodic paralyses and CMS. We increase in current was wholly accounted for by an increase in channel surface suggest that while Nav1.4 mutations exerting a dominant-negative effect cause density as reported by a quantum dot labeling technique. An optical pulse-chase periodic paralysis, a CMS phenotype may result from recessively-inherited mu- assay showed that the increased surface density was achieved solely through tations that decrease the Nav1.4 availability for muscle action potential genesis reducing the rate of CaV2.2 endocytosis rather than by enhancing the rate of for- at the neuromuscular junction and its propagation along the sarcolemma. ward trafficking. To map the site of interaction between a2d-1 and a1B, we in- serted a small epitope tag into judiciously selected extracellular regions of the a subunit. We determined accessibility of the tag to quantum dot and modu- 2479-Plat 1B lation of whole-cell currents in the presence or absence of a2d-1. From the Optical Tracking of Nav1.5 Conformation Reveals Molecular Mechanisms pattern of this dual readout, we inferred an a2d-1 binding site in the a1B domain b1 Subunit Regulation III extracellular S5-S6 loop. Alanine scanning mutagenesis revealed discrete Wandi Zhu, Eric J. Hsu, Angela R. Schubert, Zoltan Varga, residues in this loop that was necessary for a2d-1 regulation of a1B. These re- Jonathan R. Silva. sults provide novel insights into mechanisms underlying a d-1 regulation of Washington University in St. Louis, saint louis, MO, USA. 2 CaV channels, focus the putative modes of gabapentin action, and offer a new Background: In cardiomyocytes, hNav1.5 co-assembles with b-subunits, which target site for potential development of future therapeutic molecules. are known to modify Nav1.5 gating, cell surface expression, and post-translational modifications. Mutations in b-subunits are linked to many types of arrhythmias, 2482-Plat including Brugada Syndrome, the Long QT Syndrome, and atrial fibrillation. Towards a Common Structure-Function Framework for Our aim is to assess the molecular mechanism of b1-subunit regulation of NaV1.5. Regulation of Calcium and Sodium Channels 1 2 1 1 Methods: The NaV1.5 a-subunit contains four domains (DI-DIV), each with six Manu B. Johny , Paul J. Adams , Billy Kang , David T. Yue . membrane-spanning segments (S1-S6). S1-S4 form voltage sensing domains 1Johns Hopkins University, Baltimore, MD, USA, 2Kwantlen Polytechnic (VSDs) that transduce voltage into channel gating. Previously, we created University, Surrey, BC, Canada. four DNA constructs, each with a cysteine engineered into the extracellular The voltage-gated calcium (CaV) and sodium (NaV) channels represent two ma- S4 of a single channel domain. RNA from these constructs was injected into jor ion-channel superfamilies with distinct biophysical properties that support Xenopus oocytes with or without b1. Heterologously expressed channels diverse biological functions. One similarity between these channels is a homol- were labeled at the introduced cysteine with TAMRA-MTS fluorophores. Ionic ogous region within their carboxy tails, the Ca2þ-inactivating (CI) region. This current and fluorescence emission that reflected changes in VSD conformation segment, composed of two vestigial EF hands and an IQ domain, may be a were simultaneously recorded using the cut-open oocyte configuration. conserved module enabling vital Ca2þ-dependent feedback regulation through 2þ Results: In comparison with a-subunit alone, co-expression with b1 causes a interaction with calmodulin (Cell 157:1657). A single Ca -free calmodulin depolarizing shift in the steady-state inactivation curve, without affecting the (apocalmodulin) preassociates with the CI region, and Ca2þ binding to this channel activation. Observing the b1-subunit consequences for each domain, calmodulin triggers regulation in both channel families. Though this general we noted a significant depolarizing shift in the DIV fluorescence-voltage regulatory scheme appears similar between these channel families, the struc- (F-V) relationship that was induced by b1 (V1/2¼-52.4152.04, k¼13.035 tural likeness of the CI region has remained unclear. Here, we correspond ¼ 5 3.07 S.E.M.), compared with a subunit alone (V1/2 -89.4897 3.29, the first atomic structure of apocalmodulin alone bound to the NaV1.5 CI k¼22.3454.61, p¼0.007 for V1/2). b1 also caused much faster kinetics of domain (Gabelli et al (2014) Nature Communications in press) with function- t¼ 5 DIV voltage sensor activation compared to a alone ( 0.83 0.16 S.E w/ b1 ally relevant apocalmodulin binding residues in CaV1.3. This comparison im- vs t¼2.2350.09 S.E.M. w/o b1,p¼0.03). None of the other three domains plicates an extraordinary structural resemblance between Nav and Cav showed any significant b1 effects. Our results show that b1 alters INa gating channels. But does this resemblance support like functional sequelae? Apocal- by regulating DIV voltage sensor kinetics, consistent with previous studies modulin binding itself enhances peak open probability of Cav channels by indicating b1 interaction with the S5/S6 segment of DIV. Our next aim will severalfold (Adams et al (2014) Cell in press). We now find conservation of be to examine interaction with the b1b, b2, b3 and b4 subunits. this same effect in NaV1.4 channels. NaV1.4 channels bind apocalmodulin 492a Wednesday, February 11, 2015 with high affinity and exhibit a robust open probability ~0.4. Substituting dual quantification of spectrum shape. Here, we show that nanosecond-gated spectral alanines for the ‘IQ’ residues in the IQ element significantly weakens apocal- acquisition - combined with spectrum-shape quantification (such as with spectral modulin binding, and markedly diminishes peak open probability (~0.1). Over- phasor analysis) - can be used to track the physiologic response of a cellular sys- expressing apocalmodulin fully rescues open probability, demonstrating that tem (Saccharomyces cereviseae) to the addition of mitochondrial functional the effects on opening reflect apocalmodulin binding per se. This extensive modifiers (e.g., cyanide) and metabolic substrates (e.g., ethanol and glucose). structural and functional similarity substantiates a striking conservation of We demonstrate that time-gated detection allows for the rejection of short calmodulin regulation across Nav and Cav channels, joint investigation of excited-state-lifetime emission. We observe that the spectrum shape of long which now presents as a genuinely synergistic endeavor. excited-state-lifetime autofluorescence exhibits a different response to chemical additions than that of the time-integrated autofluorescence, even allowing for the discrimination between nominally similar responses. Results are consistent with Platform: Optical Spectroscopy: CD, UV-VIS, the existence of multiple NADH forms, as changes in autofluorescence spectrum shape cannot be accounted for by a two spectra model. Time-gated spectroscopy Vibrational, Fluorescence combined with rapid spectrum-shape analysis may lead to new and useful ap- proaches for the real-time tracking of cellular metabolic state. 2483-Plat Ratiometric Bioluminescence-Based Zinc Biosensor with Nanomolar 2486-Plat Sensitivity Nanoscale Infrared Spectroscopy of Biological Systems Evgenia G. Matveeva1, Graham Franke1, Leslie Bourne1, Eoghan Dillon, Mike Lo, Kevin Kjoller, Qichi Hu, Roshan Shetty. Andrea K. Stoddard2, Carol A. Fierke2, Richard B. Thompson1. Anasys Instruments, Santa Barbara, CA, USA. 1Department of Biochemistry and Molecular Biology, University of Infrared microspectroscopy is a popular technique for investigating biological Maryland School of Medicine, Baltimore, MD, USA, 2Department of structures. It is relatively simple to use, and considered to be a non-destructive Chemistry, University of Michigan, Ann Arbor, MI, USA. technique. By combining atomic force microscopy and infrared spectroscopy In recent years, we and other workers have developed fluorescence-based sen- (AFM-IR), it is possible to resolve chemical differences on the scale of ~100 to sors and biosensors for Zn2þ and other free metal ions in solution, which 200 nm, which often reveals information that could not have been obtained have proven valuable as tools for the study of the biology of metal ions. Using with conventional infrared microspectroscopy. Currently, AFM-IR spectroscopy the sensors in some applications such as in vivo molecular imaging has been has the ability to collect IR spectroscopic information below the diffraction limit challenging, owing to the difficulty of exciting fluorophores deep in tissues with lateral resolution of ~ 100 nm. However, there are still some limitations that which scatter and absorb exciting light effectively. One approach used by inves- prevent its use on many important nanoscale systems. One of the main limitations tigators to minimize this problem is the use of bioluminescent labels for in vivo is the thickness of the sample required for examination (> 100 nm). Overcoming imaging, which perforce require no exciting light. We have employed a biolumi- these limitations has a dramatic impact by enabling widespread use of nanoscale nescence resonance energy transfer approach with our carbonic anhydrase-based IR spectroscopy for spatially resolved chemical characterization. The use of a zinc biosensor to produce a ratiometric bioluminescence-based zinc biosensor. quantum cascade laser (QCL) as the IR source significantly increases the sensi- tivity of AFM-IR. The QCL has repetition rates 1000 times higher than previous 2484-Plat lasers used for AFM-IR. This allows the ability to pulse the laser at the resonant From Charge State to Isosurfaces to Spectra: Unraveling the Mystery of frequency of the AFM cantilever giving rise to a high IR sensitivity mode referred Lys-Trp Dipeptide Fluorescence to as resonance enhanced infrared nanospectroscopy (REINS). Additional Azaria Eisenberg, Laura Juszczak. enhancement of the AFM-IR signal results when a gold-coated AFM tip is Brooklyn College, Brooklyn, NY, USA. used, producing a ‘‘lightning-rod’’ effect which enhances the intensity of the The quest for factors controlling the partition of excitation energy between fluo- exciting electric field at the tip. Furthermore, if the sample is deposited onto a rescence and nonradiative processes in tryptophan is of perennial interest to gold substrate, the local electric field is further enhanced allowing for chemical spectroscopists because its fluorescence has distinct intensity, color and environ- identification of samples as thin as 25 nm. In this presentation we will demonstrate mental sensitivity. Solving the relationship between specifics of molecular envi- the effectiveness of the AFM-IR technique on several biological systems, ronment and characteristics of tryptophan fluorescence would provide the key including; IR spectroscopy of a monolayer film of Halobacterium salinarium for emission spectra interpretation of a wide variety of proteins. Our quantum on a gold substrate and IR chemical imaging of Streptomyces bacteria. mechanical calculations and molecular modeling reveal substantial differences in ground state isosurface charge distribution on the indole ring for Lys-Trp di- 2487-Plat peptides when backbone and residue charge is varied. These isosurfaces repre- Flavonols as Luminescent Probes of Water Activity in Foods and sent the superposition of all ground state molecular orbital contributions to Pharmaceuticals charge distribution and thereby provide a visual representation of the contribu- Siyu (Suzie) Wang, An N. Le, Maria G. Corradini, Richard D. Ludescher. tions of all relevant orbitals. Comparison of Lys-Trp species isosurfaces with Food Science, Rutgers University, New Brunswick, NJ, USA. experimentally derived quantum yields and fluorescence lifetimes reveals a cor- Flavonols are naturally occurring antioxidants with complex photophysical prop- relation: high p-electron density on the indole ring is associated with high quan- erties. Their emission sensitivity to polarity and hydrogen bonding ability of the tum yield and long average lifetime, both of which are found in the zwitterionic local environmentsuggests their potential as luminescentprobes for water activity. and anionic states. Conversely, low quantum yield and short average lifetime is Water activity, aw, is an important parameter for ensuring food safety and quality, associated with low, uneven p-electron density on the indole ring, which is found as it is a determining factor for microbial growth and biochemical reactions. in the cationic and highly anionic state where the indole amine is deprotonated. This study focused on 3-hydroxyflavone (3HF), a synthetic molecule that con- This interpretation explains fluorescence results for tryptophan-containing pro- stitutes the backbone of naturally occuring flavonols. However, the photophys- teins. Where x-ray crystal structures have shown proteinaceous tryptophans to be ical properties and sensitivity to aw of other flavonoid glycosides were also hydrogen bonded at the indole amine, very low quantum yields are observed, and explored. All flavonols were tested in binary solvent mixtures of different aw. isosurfaces resembling those with a deprotonated indole amine are anticipated. 3HF is an excited-state intramolecular proton-transfer probe that exhibits dual Thus isosurfaces reveal losses in p-electron density over the indole ring or a loss fluorescent emission bands corresponding to its normal (lem ¼ 405nm) and of aromaticity for low fluorescing tryptophans. Isosurfaces are the key to solving photo-induced tautomeric (lem ¼ 525nm) form. The normalized intensity of the relationship between molecular environment and tryptophan fluorescence. the photo-induced tautomer was sensitive to aw of binary mixtures, with a marked decrease in intensity at aw > 0.8, likely due to progressive aggregation 2485-Plat of 3HF molecules. Additional sensitivity to aw was observed in terms of the Real-Time Quantification of Time-Gated Autofluorescence Spectrum location of the emission bands. The difference in wavelength between the Shape to Track Mitochondrial Metabolism normal (N) and tautomer (T) forms decreased monotonically at aw > 0.4 Paul Urayama, Jeff Maltas, Zac Long, Arthur Oliva, Jeff Folz, Lana Amer, due to a hypsochromic shift of the tautomer band. The relative position of Dylan Palo. the T* towards N* band as a function of aw was modelled using a log- Physics, Miami University, Oxford, OH, USA. logistic function. A critical aw value above which the sensitivity of 3HF signif- Recent studies by various groups report the use of NADH as an intrinsic optical icantly increased was estimated based on the model. biomarker and metabolic indicator. Using measurements of excited-state life- Quercetin and additional flavonoid glycosides were also responsive to changes time or anisotropy decay, different forms of intracellular NADH were identified in aw; e.g., quercetin’s fluorescence intensity decreased at aw > 0.6. and monitored, with the response of NADH to metabolic conditions being more Although additional validation of these probes in model food systems is complex than a simple concentration change. Recently, we report an approach required, the available data support the potential use of flavonols as probes for the real-time tracking of UV-excited autofluorescence based on the rapid of aw in foods and edible pharmceuticals. Wednesday, February 11, 2015 493a

2488-Plat We present a method of calculating the intensity autocorrelation function from Sensitive Detection of NADD/NADPD via Strong Coupling Fluorescence the arrival times of the photons on the detector that maximizes the information from Silver Nanoclusters content while correcting for the effect of photobleaching to yield an autocorre- Yufeng Yuan1, Yan Chen2, Kehan Huang1, Haifeng Pan1, Sanjun Zhang1, lation function that reflects only the underlying dynamics of the sample. For Jianhua Xu1. this purpose, we determine the overall photobleaching rate by fitting a multi- 1East China Normal University, Shanghai, China, 2Tongji Hospital Affiliated exponential decay to the fluorescence intensity. Then we assign a weight to to Tongji University, Shanghai, China. each photon using the reciprocal of this fit function. The autocorrelation func- The pyridine nucleotides (NADþ, NADPþ) are the major coenzymes partici- tion is then calculated from all photon pairs using the product of the individual pate in multiple redox processes in living cells. Both NADþ and NADPþ are photon weights. This gives late-arriving fluorescence photons that come from a not fluorescent and almost structurally identical, so it is difficult to directly partially photobleached sample a higher weight than those photons that arrived distinguish NADþ or NADPþ via optical methods (such as fluorescence and earlier, compensating for the loss of some of the fluorophores. We demonstrate Raman spectroscopy). We report here a sensitive probe of NADþ/NADPþ the utility of this technique by acquiring artifact-free FCS data from entangled based on fluorescent silver nanoclusters with dual emission band. The silver DNA solutions and from chromosomal DNA in E. coli. nanoclusters with an initial fluorescent emission peak at 410 nm were synthe- sized by etching large size silver nanoparticles. With the addition of NADþ/ NADPþ solution, due to the strong coupling (charge-transfer) between silver Platform: Cell Mechanics, Mechanosensing, and nanoclusters and ligands (NADþ/NADPþ), a new fluorescence emission peak of the silver nanoclusters was found and raised at 550 nm and the fluores- Motility II cence intensity was dependent on the ratio between NADþ and NADPþ. The 2491-Plat time-resolved fluorescence decay (at 550 nm) of silver nanoclusters showed a High Resolution, Large Deformation 3D Traction Force Microscopy single-exponential decay lifetime of 3.9 ns caused by the strong coupling be- Jennet Toyjanova1, Eyal Bar-Kochba1, Cristina Lopez-Fagundo2, tween silver nanoclusters and ligands (NADþ/NADPþ). Meanwhile, the Jonathan Reichner3, Diane Hoffman-Kim4, Christian Franck1. 410 nm emission was also selectively enhanced by the different ratio of 1Engineering, Brown University, Providence, RI, USA, 2Competence Center NADþ /NADPþ molecules. The intensity ratio of fluorescence emission at for Applied Biotechnology and Molecular Medicine, University of Zurich, 410nm and 550nm may be useful to monitor the levels of NADþ /NADPþ Zurich, Switzerland, 3Department of Surgery, Rhode Island Hospital, in aqueous solutions, cellular extracts and living cells. Candidate mechanisms Providence, RI, USA, 4Molecular Pharmacology, Physiology and and the analysis of time resolved emission spectra will be discussed. Biotechnology, Center of Biomedical Engineering, Brown University, Providence, RI, USA. 2489-Plat Traction force microscopy (TFM) is a powerful approach of quantifying Non-Destructive Label-Free Monitoring of Drug Intake in Live Cells using cell-material interactions, which over the last two decades has contributed ATR FT-IR Spectroscopy significantly to our understanding of cellular mechanosensing and mechano- Pedro L. Fale, K.L. Andrew Chan. transduction. In addition, recent advances in three-dimensional (3D) imaging Institute of Pharmaceutical Science, King’s College London, London, United and traction force analysis (3D TFM) have highlighted the significance of Kingdom. the third dimension in influencing various cellular processes. Yet irrespective Drug resistance is one of the major challenges in treatment of cancer. The study of of dimensionality almost all TFM approaches have relied on a linear elastic the- the intake of drugs by cancer cells therefore is vital for elucidating the mechanism ory framework to calculate cell surface tractions. of drug resistance. Currently to quantify the intake of drugs involve destructive This talk presents a new high-resolution 3D TFM algorithm, which utilizes a methods such as lysing the cell at each time point of analysis followed by UV- large deformation formulation to quantify cellular displacement fields with un- Vis or fluorescence measurements. With this approach, different batches of cell precedented resolution. The results feature some of the first experimental evi- are used for each time point, increasing the biological variability, and it is not dence that cells are indeed capable of exerting large material deformations, possible to perform further analysis on the same cell sample. Attenuated total re- which require the formulation of a new theoretical TFM framework to accu- flection Fourier transform infrared (ATR FT-IR) is a promising non-destructive rately calculate traction forces. Based on our previous 3D TFM technique we label-free while chemically specific technique for analysis of biomedical samples. reformulate our approach to accurately account for large material deformation In this work we demonstrated the quantification of drugs (e.g. doxorubicin), in and quantitatively contrast and compare both linear and large deformation situ, using the ATR FT-IR method to obtain the drug diffusion profile in the frameworks as a function of the applied cell deformation. Particular attention live cell. HeLa cells grown on a multi-bounce ATR crystal were treated with is paid in estimating the accuracy penalty associated with utilizing a traditional 20 micromolar of doxorubicin, a concentration level that is relevant to cancer linear elastic approach in the presence of large deformation gradients. studies, and FT-IR spectra were collected in a time course using a MCT detector. Quantification of doxorubicin in the cell was made using the signature peak at 2492-Plat 1284 cm-1 showing the accumulation of the drug in cells as a function of time. Correlative Traction Force Microscopy and Fluorescence Fluctuation The results from the ATR FT-IR measurements have shown that the doxoru- Analysis of Molecular Aggregation and Complex Formation in Cell bicin concentration in the living cell increases from 0 to >50 micromolar after Adhesions in Distinct Microenvironments 2 hours of treatment demonstrating the partitioning effect of the drug in the cell. Alexia I. Bachir1, Jessica Zareno1, Kristopher E. Kubow2, Sangyoon Han3, Furthermore, the cells also present a signature spectrum, which allows to follow Kostadinos Moissoglu4, Gaudenz Danuser3, Enrico Gratton5, Edward Plow6, the cell viability in parallel with the drug intake. This is a fast, cost-effective Alan R. Horwitz1. and chemically selective method which does not require sophisticated equip- 1Cell Biology, University of Virginia, Charlottesville, VA, USA, 2Biology, ment and can be adapted easily. James Madison University, Harrisonburg, VA, USA, 3Cell Biology, UT Southwestern, Dallas, TX, USA, 4Center for Cancer Research, NIH, 2490-Plat Bethesda, MD, USA, 5Biomedical Engineering, UC Irvine, Irvine, CA, USA, Photobleaching Correction in Fluorescence Correlation Spectroscopy 6Molecular Cardiology, Cleveland Clinic, Cleveland, OH, USA. Rudra P. Kafle1, Molly R. Liebeskind1, Jens-Christian Meiners2. The mechanical properties of the cellular microenvironment regulate processes 1LSA Biophysics, University of Michigan, Ann Arbor, MI, USA, 2LSA that include migration, proliferation, and differentiation. These interactions Biophysics and Physics, University of Michigan, Ann Arbor, MI, USA. occur at adhesions, which serve as both traction points and signaling hubs and Fluorescence correlation spectroscopy (FCS) is a fluorescence technique conven- mediate bi-directional sensing and responses to specific features of the surround- tionally used to study the kinetics of fluorescent molecules in a dilute solution. Be- ing extracellular matrix (ECM). Adhesions execute these activities through an ing a non-invasive technique, FCS is now finding wider applications in the study intricate network of putative molecular interactions that largely remain to be of more complex systems like the dynamics of DNA or proteins in living cells and demonstrated and characterized functionally in living cells. The challenge is cell membranes. Unlike an ordinary dye solution, the dynamics of macromole- to capture the highly localized and transient associations that characterize cules like proteins or entangled DNA, in crowded environments is often slow these activities in adhesions and determine how they respond to different micro- and subdiffusive in nature. This in turn leads to longer residence times of the environments. In this study, we use high-resolution fluorescence fluctuation attached fluorophoresin the excitation volume of the microscope. As a result, pho- microscopy to map the formation and stoichiometry of integrin-associated com- tobleaching becomes a problem: the number of photons available to calculate the plexes in the adhesions that populate the leading edge of migrating cells. We intensity autocorrelation function is limited, and the decay of the fluorescence in- focus on putative integrin activating (kindlin and talin) and actin-linking (talin, tensity itself contributes a spurious signal to the autocorrelation function that can vinculin and a-actinin) molecules and show that all molecules are present in ad- easily obscure the signature of the molecular dynamics of interest. hesions as soon as they are visible; however, they form integrin containing 494a Wednesday, February 11, 2015 complexes hierarchically, at different times, with variable stoichiometry within association with the lamina may contribute to the mechanical behavior of meta- the adhesion itself and change as the adhesion matures into larger structures. To zoan nuclei. parse out the effects of the mechanical properties of the ECM on the numbers, aggregation states, and associations of these molecules, we extend these mea- 2495-Plat surements to substrates with variable stiffness and correlate them with high- Three-Dimensional Balance of Cortical Tension and Axial Contractility resolution traction force microscopy (TFM) measurements. We show that Enables Fast Amoeboid Migration 1 2 2 individual and newly formed adhesions at the leading edge of protruding cells Begona Alvarez-Gonzalez , Ruedi Meili , Effie Bastounis , Richard A. Firtel2, Juan C. Lasheras1, Juan C. del Alamo1. transmit forces on soft as well as stiff substrates, with force magnitudes that 1 2 correlate with the integrated intensity of the adhesions and the total number of MAE, UCSD, La Jolla, CA, USA, Cell and Developmental Biology, individual adhesion molecules. These measurements provide novel information UCSD, La Jolla, CA, USA. on complex formation as adhesions evolve and respond to substrate rigidity. Fast amoeboid migration requires cells to apply mechanical forces on their sur- roundings via transient adhesions. However, the mechanistic role of forces in 2493-Plat controlling cell shape changes and cell migration speed remains largely un- Tracking Rotation during Leukocyte Rolling Reveals Asymmetric known. To address these questions, we used three-dimensional force micro- Adhesion Properties scopy to measure the in-plane (tangential) and out-of-plane (normal) forces Isaac T.S. Li1,2, Taekjip Ha1,3, Yann R. Chemla1,2. exerted by cells crawling on flat surfaces. From the measured normal forces, 1Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA, we estimated the cells’ cortical tension using a Young-Laplace’s model. We 2Center for the Physics of Living Cells, Urbana, IL, USA, 3Howard Hughes examined chemotaxing wild-type Dictyostelium cells, as well as mutants Medical Institute, Urbana, IL, USA. with defects in contractility, internal F-actin crosslinking, and cortical integrity, Leukocytes are responsible for fighting infections in the body. When injuries and demonstrated that once the cells initiate their migration and polarize, they occur, selectin molecules are expressed on the surface of nearby blood vessel generate tangential traction forces by myosin II contractility, which requires an walls. These selectin molecules transiently adhere to leukocytes flowing in internal crosslinked F-actin network. Simultaneously, cortical tension provides the bloodstream and capture them, leading to leukocyte rolling towards the an additional mechanism that generates the normal forces and that does not injury site. Rolling adhesion is critical for leukocytes to locate injury sites require myosin II. The 3-D pulling forces generated by both mechanisms are and to activate various signaling pathways for subsequent transmigration and internally balanced by an increase in cytoplasmic pressure that allows cells chemotaxis. to push down on their substrate without adhering to it. These compressive Individual adhesion components involved in rolling adhesion, such as adhe- pressure-induced forces are not associated to adhesion sites, and may allow sion molecules and membrane tethers have been characterized. However, amoeboid cells to push off surrounding structures when migrating in complex models incorporating these properties are still unable to describe fully the roll- three-dimensional environments. Our findings are consistent with a model in ing behavior. Current models assume uniformly distributed adhesion proper- which the two force-generating cellular domains are mechanically connected ties on cell surfaces due to the lack of measurements quantifying this by myosin I crosslinking that enables the communication of forces between distribution. Here, we determined experimentally the spatial distribution of the domains. Furthermore, we found that the balance between axial myosin adhesive properties on leukocyte surfaces. We used dark-field imaging and II contractility and cortical tension is important to produce the cell shape particle tracking techniques to extract not only the translational but also the changes needed for locomotion, as cell migration speed correlates with the ratio rotational motion of a single rolling cell. The additional rotational information of the magnitudes of the tangential traction forces to the normal ones. These allows us to map precisely the whole cell motion and adhesion properties to a results reveal a novel role for 3-D cellular forces in establishing the efficiency particular cell orientation. We find that the adhesion properties of the leuko- of amoeboid cell movement. cyte surface are far from homogenous, with large, localized patches on the 2496-Plat cell surface exhibiting strong or weak adhesive properties. This finding pro- Measuring Mechanical Force during Zebrafish Development using an vides new insight into leukocyte adhesion properties, such as the asymmetric Expressible Tension Sensor distribution of receptor and microvilli on rolling cells, and could lead to better Andrea Hamilton1, Victoria Wu2, Alex Dunn3, Ingmar Riedel-Kruse2. modeling of rolling adhesion. 1Molecular and Cellular Physiology, Stanford University, Palo Alto, CA, USA, 2Department of Bioengineering, Stanford University, Palo Alto, CA, 2494-Plat USA, 3Department of Chemical Engineering, Stanford University, Palo Alto, Chromatin Association with the Nuclear Envelope Supports Stable Nu- CA, USA. clear Mechanics Early vertebrate development is a mechanically dynamic process. Embryos 1 2 3 2 Sarah M. Schreiner , Peter K. Koo , Yao Zhao , Simon G.J. Mochrie , undergo radical morphological changes to mold a ball of cells into the recog- 1 Megan C. King . nizable planes of a frog or fish or mouse. Along with more basic morphogen- 1Cell Biology, Yale University, New Haven, CT, USA, 2Physics, Yale 3 esis, evidence exists that mechanical force can play a determinative role in University, New Haven, CT, USA, Applied Physics, Yale University, New processes such as differentiation and tissue sorting. Yet, while the chemical Haven, CT, USA. and genetic signals underlying early development have long been studied, Although the nuclear lamina is considered to be the primary mechanical de- the mechanical stresses defining the physical landscape of the embryo remain fense of the nucleus, lamins are part of an integrated network of lipids, proteins, poorly understood, particularly at the cell and molecule scale. An expressible and chromatin. Here, we isolate the contribution of chromatin to nuclear fluorescence resonance energy transfer (FRET) based probe has been devel- mechanics by employing fission yeast, which lack a nuclear lamina. We have oped to measure piconewton force levels in vivo (Grashoff et al. 2010). We combined a quantitative imaging platform capable of measuring 3D nuclear adapt this Tension Sensor Module (TSMod) for use in the zebrafish embryo contours in vivo with an in vitro optical tweezers assay to probe the mechanical using the epithelial cell adhesion molecule, EpCAM. In doing so we validate properties of S. pombe nuclei. In live cells, we find that association of chro- FLIM-FRET for use in zebrafish embryos and show that TSMod can be used matin with the inner nuclear membrane (INM) through integral membrane pro- to make meaningful cell and tissue scale force measurements in a developing teins is required for a normal mechanical response to microtubule (MT) forces. vertebrate system. Increasing loss of integral INM proteins results in highly deformable nuclei specifically in response to exogenous MT forces. These nuclei also show a 2497-Plat decreased capacity to recover from mechanical stress. Using optical tweezers, Two Distinct Regimes of Integrin Molecular Forces we find that nuclei lacking integral INM proteins are less stiff than wild type Xuefeng Wang, Taekjip Ha. nuclei and have increased chromatin flow, particularly when force is applied physics, university of illinois at urbana-champaign, Urbana, IL, USA. at rates that recapitulate the kinetics of MT dynamics in vivo. Wild type mitotic Forces transmitted by integrins activate focal adhesion kinase (FAK) and regu- nuclei, in which chromatin is globally released from the INM, are extremely late cell adhesion, spreading and migration. However, the magnitudes and sour- soft and also display increased chromatin flow compared to interphase nuclei. ces of the molecular forces involved with these mechanical regulations remain Interestingly, decreasing the chromatin to nuclear volume ratio without altering largely unknown. Here we discovered two distinct regimes of integrin molec- the association of chromatin with the INM has only a slight effect on stiffness ular forces generated by cell membrane and actomyosin, respectively, and that and does not alter chromatin flow. Together, these findings suggest that associ- they govern different cellular functions. Using integrin ligands linked with ten- ation of chromatin with the nuclear envelope underlies nuclear stiffness. sion gauge tether (TGT), we showed that TGT with 54 pN tension tolerance Further, release of chromatin from the INM allows chromatin to flow into (Ttol) is specifically ruptured at focal adhesions (FA) in CHO-K1 cells or central MT-dependent fluctuations of the nuclear envelope, leading to larger, longer- regions of cell-substratum contact area in neutrophil-like dHL-60 cells, indi- lasting nuclear deformations. Lastly, these results suggest that chromatin cating that remarkably strong forces can be applied to a single integrin. This Wednesday, February 11, 2015 495a force is abolished by blebbistatin treatment which inhibits myosin II, suggest- discussed in the context of proposed NMR models of Ab oligomers and fibrils ing that this >54 pN integrin force is generated by actomyosin. However, bleb- with implications towards further development of therapeutic and diagnostic bistatin treatment does not abolish the rupture of TGT with Ttol of 33 pN and strategies. lower, consistent with the previously reported ~40 pN integrin force generated by cell membrane during cell adhesion. FAK activation and cell spreading 2500-Plat beyond initial adhesion also require ~ 40 pN integrin force. dHL-60 cells Novel Methodologies for the Computational Study of Protein Aggregation 1 2 1 were poorly polarized and migration rate was significantly reduced on 54 pN David Shorthouse , Thomas Gallagher , Mark Sansom . 1Department of Biochemistry, University of Oxford, Oxford, United TGT but not on unrupturable TGT or fibronectin coated surfaces. Collectively, 2 our results demonstrated that cell membrane generates ~40 pN molecular force Kingdom, Lonza Biologics, Cambridge, United Kingdom. on integrins and this force activates FAK and mediates cell initial adhesion and Aggregation is a major challenge in the development of antibody-based thera- spreading, while actomyosin generates a >54 pN force to single integrins and peutics. Therapeutic antibodies are produced and stored in high concentrations this force mediates neutrophil-like cell polarization and migration. The discov- and generated under varying and unfavourable conditions for the stability of ery and decoupling of these two regimes of integrin forces was enabled by TGT monomeric proteins. The aggregation of these proteins in solution can lead which measures molecular forces and also regulates cellular functions by re- to serious consequences for patients in the form of the initiation of immune re- stricting integrin molecular forces under a designed level, providing a global actions, which have the potential to be fatal, and in the loss of clinical potency. molecular force control for cell mechanics study. Further to this, the type of aggregates formed by antibodies, and the processes that lead to their propagation, are relatively poorly understood. By investigating 2498-Plat these molecules as a model system we may find out more about other, more Dynamic Traction Forces of Human Neutrophil Adhesion complex systems known to involve aggregation - including amyloids. Steven J. Henry1, Christopher S. Chen2, John C. Crocker3, Here we present a novel application of coarse-grained molecular dynamics, in a Daniel A. Hammer4. high throughput scheme, coupled with statistical techniques to investigate 1Bioengineering, University of Pennsylvania, Philadelphia, PA, USA, aggregation and self association of proteins. The use of a high throughput meth- 2Biomedical Engineering, Boston University, Boston, MA, USA, 3Chemical odology, developed to be modular, user friendly, and efficient, allows the and Biomolecular Engineering, University of Pennsylvania, Philadelphia, collection of much larger datasets of specific structures than previous work. PA, USA, 4Bioengineering & Chemical and Biomolecular Engineering, These can then be analysed using statistical techniques to gain information University of Pennsylvania, Philadelphia, PA, USA. on specific regions of interactions, and relative rates of interaction. The focus of our presentation will be the mechanics of human neutrophil adhesion and the associated role of the cell cytoskeleton during this process. 2501-Plat The principle tool we employ to measure adhesive forces is microfabricated- Multiscale Simulations Provide Mechanistic Insights into the Effects of Post-Array-Detectors (mPADs). We achieve high spatial and temporal resolu- Sequence Contexts on Early-Stage Polyglutamine-Mediated Aggregation tion of neutrophil adhesion to arrays of sub-micron diameter vertical pillars Kiersten M. Ruff1, Rohit V. Pappu2. 1 functionalized with the adhesive ligand fibronectin (FN). Additionally, we Computational and Systems Biology Program and Center for Biological will present adherent cell profiles (vertical contours) of neutrophils on pillars Systems Engineering, Washington University in St. Louis, St. Louis, MO, 2 via confocal microscopy. The adhesion event is stimulated by haptokinetic USA, Department of Biomedical Engineering and Center for Biological interaction with FN alone, consistent with our previous work on continuous Systems Engineering, Washington University in St. Louis, St. Louis, fields of FN (Henry et al. 2014. Integr Biol. 6:348). Our preliminary data MO, USA. shows the adhesion event is initially a fast (38 5 8 s, mean 5 sd, n ¼ 7 cells) Huntington’s disease is associated with an expanded polyglutamine (polyQ) protrusive front that spreads radially outwards with an average per-pillar force tract in huntingtin. The relevant aggregation prone toxic species encompasses of 32 5 17 pN (mean 5 sd, n ¼ 7 cells). Over the next 2-5 min the cell tran- an N-terminal 17-residue amphipathic stretch (N17), the polyQ tract, and a C-ter- sitions from a protrusive phenotype to a contractile phenotype with the periph- minal 38-residue proline-rich stretch (C38). Experiments have shown that C38 eral pillars being deflected inwards towards the cell center and having an decreases the overall driving force to form aggregates, whereas N17 decreases average per-pillar force of 43 5 26 pN (mean 5 sd, n ¼ 7 cells). Pillar the overall solubility and accelerates fibril formation by destabilizing non- behavior can be dichotomized according to the position of the pillar relative fibrillar species. We present results from multiscale simulations to explain to the cell’s geometric centroid. Peripheral pillars (those residing at the how N17 and C38 accomplish their distinct modulatory behaviors. Our inverse edge of the cell footprint) are highly contractile and persistent in their contrac- Boltzmann procedure yields a systematic coarse-graining method whereby we tility, core pillars are less so. Pretreatment of quiescent neutrophils with a bootstrap against atomistic simulations of monomers and reversible associations variety of small molecule inhibitors of the cell cytoskeleton alter the long- for pairs of molecules. We demonstrate that the species distributions and kinetics time (i.e. minutes) contractile phase of adhesion but have little effect on the of forming early-stage aggregates are governed by the relative favorability of short-time (i.e. seconds) protrusive signature. A notable exception is pretreat- intermolecular interactions between flanking sequence regions and the polyQ ment with Jasplakinolide which rigidifies the quiescent cortical actin shell and tract. Specifically, the two flanking sequences reduce the favorable polyQ inter- abrogates adhesion completely. action surface and hence reduce the frequency of intermolecular interactions be- tween and entanglements of the polyQ tract, thus slowing aggregation kinetics regardless of the molecular architecture at the monomer level. The latter affects Platform: Intrinsically Disordered Proteins (IDP) the morphologies and number of aggregates that form during early aggregation events. Overall, we show that N17 and C38 diminish the formation of large, poly- and Aggregates disperse early-stage aggregates that are intrinsic to polyQ. Our results also show how polyQ expansions can override the effects of flanking sequences. Taken 2499-Plat together with cellular data, we propose that flanking sequences help diminish Sequence Specific Radiolytic Footprinting Study of Monomer, Oligomeric the formation of toxic species and this functionality is overwhelmed by muta- and Fibrillar Amyloid Beta (1-42) tional expansion of polyQ tracts. We use the conceptual framework of Janus col- 1 2 3 Alexandra L. Klinger , Janna Kiselar , Anant Paravastu , loids to propose a connection between our results and other aggregation prone Terrone Rosenberry4. 1 2 systems because they can be partitioned into cores that drive aggregation and University of Pennsylvania, Philadelphia, PA, USA, Case Western Reserve contexts that control the aggregation process. University, Cleveland, OH, USA, 3Florida State University, Tallahassee, FL, USA, 4Mayo Clinic, Jacksonville, FL, USA. 2502-Plat Increasing evidence suggests that soluble aggregates of amyloid-b (Ab) are the New Insight into Amyloid-b Fibril Growth and its Inhibition: Kinetic pathogenic species in Alzheimer’s disease (AD). However, detailed structural Network Analysis of Multi-Scale Molecular Dynamics Simulations information on these species remains scarce due to low levels of endogenous Wei Han1, Klaus Schulten2. Ab oligomers and uncertainties surrounding current in vitro model systems. 1Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, Herein, we describe a hydroxyl radical footprinting (HRF) study of Ab42 fibrils USA, 2Beckman Institute, Center for Biophysics and Computational Biology and a stable and homogeneous preparation of Ab42 oligomers. Specific side and Department of Physics, University of Illinois at Urbana-Champaign, chain solvent accessibilities of individual residues in the aggregated and fibril Urbana, IL, USA. forms of Ab42 are measured with respect to the same residues of Ab42 in a Formation of amyloid fibrils by amyloid-b peptides (Ab) in patient brains is a fully exposed reference state. These data provide residue specific side chain hallmark of Alzheimer’s disease. A major step of Ab fibril formation is elonga- solvent accessibility protection factors and are used in complement with bio- tion of fibrils by unstructured Ab peptides, involving Ab binding and structural physical characterizations and ss-NMR analyses of these systems. Results are transitions. The atomic detail of the structural transitions remains poorly 496a Wednesday, February 11, 2015 understood. Computational characterization of the structural transitions is 2505-Plat limited owing to the long timescale of Ab fibril elongation. We present here HIV-Tat Protein-Amyloid Beta Complex: From Molecular Interaction to our recent effort in overcoming the computational timescale limit by applying Increased Neurotoxicity a novel computational approach combining a multi-scale model, enhanced sam- Alina Popescu Hategan1, Joseph Steiner1, Mario A. Bianchet2, pling techniques and kinetic network analysis to tackle Ab fibril elongation [1]. Elena Karnaukhova3, Emilios K. Dimitriadis4, Avindra Nath1. In the framework of this efficient approach, we are able to perform simulations 1NINDS, NIH, Bethesda, MD, USA, 2Johns Hopkins School of Medicine, reaching the millisecond timescale and determine in a systematic way detailed Baltimore, MD, USA, 3CBER, FDA, Silver Spring, MD, USA, 4NIBIB, NIH, transition pathways and kinetics for Ab fibril elongation. The resulting kinetic Bethesda, MD, USA. model of fibril elongation does not only reveal an atomic picture of pathways Since significant increase in amyloid beta plaque deposition was observed in of Ab fibril elongation not seen before, involving special structures of Ab mono- HIV patients, it is important to examine the direct interaction between HIV- mers as important kinetic intermediates, but does explain also a puzzling exper- Tat protein, largely expressed by HIV virus after entering the cell, and amy- imental observation, namely unidirectional growth of Ab fibrils. Moreover, the loid beta molecules that aggregate and form the plaques. We investigated this kinetic model reveals possible kinetic and thermodynamic effects of ligand bind- interaction under physiological conditions starting with bulk methods and pro- ing on fibril growth, providing new insights into inhibitor design. gressively zoomed in by using methods with fibrilar and molecular resolution. Reference: Circular dichroism of amyloid beta - Tat complexes revealed growth in beta [1] Han, W.; Schulten, K. J. Am. Chem. Soc. 2014, 136, 12450. sheet structure and ThyT bulk fluorescence showed enhanced adhesion of ag- gregates to surfaces. Atomic force microscopy showed that the predominant 2503-Plat typical singular uniform amyloid fibrils turned into a population of double A Role for Unstructured Residues in the Induction of Membrane Poration twisted fibrils followed by populations with predominantly thick unstructured by Pre-Amyloid Assemblies of Islet Amyloid Polypeptide filaments and aggregated large patches in a dose responsive manner, when 1 2 2 Mark A. Brown , Elizabeth Rhoades , Andrew D. Miranker . HIV-Tat was present. The fibers break at regular intervals under air flow 1Department of Chemistry, Yale University, New Haven, CT, USA, 2 and the rupture length increased significantly with HIV-Tat presence, indi- Department of Molecular Biophysics and Biochemistry, Yale University, cating greater mechanical resistance of fibrils. The period of the twists in New Haven, CT, USA. the double fibrils grew with HIV-Tat indicating increased rigidity of the fibers. Amyloid diseases represent a diverse class of pathologies associated with Single fibril fluorescence confirmed the external attachment of HIV-Tat to unique, aggregation-prone proteins. Although association into fibrous amyloid fibrils, which explains the lateral aggregation of fibrils into thick multifibrilar structure represents the end state of such aggregation, it has been observed that structures. Computer simulation indicated that Tat binds to the external side of pre-amyloid states of these proteins may act as membrane-active toxins, the amyloid beta hairpin involving essentially the C terminal region of the providing a potential mechanism for pathology. Islet Amyloid Polypeptide amyloid beta chain. HIV-Tat presence in aggregates increases synergistically (IAPP), associated with Type II diabetes, has been shown to induce cell toxicity the neurotoxicity in primary neuronal cell cultures. Our data suggests that the as well as membrane poration in lipid bilayer models. Although several models major pathway towards cell damage is based on the increased rigidity and of membrane-bound human (hIAPP) and non-amyloidogenic rat (rIAPP) vari- mechanical resistance of the amyloid beta-Tat complexes coupled with their ants have been proposed, a detailed structure of the toxic state of IAPP has re- stronger adhesion capacity due to Tat presence in the fibrils. Together, these mained elusive. In order to obtain a better structural and mechanistic can account for direct mechanical disruption through pore formation in the understanding of IAPP-induced membrane poration, the extent of membrane neuronal cell membranes. association by different sequence regions of rIAPP has been directly compared to the magnitude of membrane poration under matched conditions via modula- 2506-Plat tion of the membrane lipid composition. We find that C-terminal residues pre- The Intrinsically Disordered Membrane Enzymes Selenoprotein S and viously observed to be unstructured in the membrane bound state are Selenoprotein K nevertheless required for efficient poration. Our findings support and extend Liu Jun, zhengqi Zhang, Sharon Rozovsky. a more lipocentric view of peptide-membrane interactions, based on surface Chemistry and Biochemistry, University of Delaware, Newark, DE, USA. and line tension, previously suggested for membrane-active, microbe-derived Selenoproteins constitute a family of enzymes involved in the management and peptide toxins. regulation of reactive oxygen species, signaling molecules that are also affili- ated with molecular damage and disease. The family contains two intrinsically 2504-Plat disordered membrane proteins: selenoprotein S (SelS) and selenoprotein K Keeping it Disordered: A New Mechanism of Protein Quality Control? (SelK). Both of these bitopic membrane proteins reside in the endoplasmic re- Priya R. Banerjee, Ashok Deniz. ticulum and contain the highly reactive amino acid selenocysteine, The precise Integrative Structural and Computational Biology, The Scripps Research function of SelK and SelS is presently still unknown, however it has been Institute, La Jolla, CA, USA. shown that in vivo they participate in anti-oxidant defense. In addition they Molecular chaperones are known for their function in maintaining a healthy pro- are members of the Endoplasmic Reticulum Associated Protein Degradation teome, partly by aiding globular folded proteins to remain in native regions of (ERAD) pathway, which is responsible for dislocation of misfolded proteins their folding landscapes. In contrast, although protein disorder has emerged as from the ER for degradation in the cytoplasm. We have shown that SelK and a significant component of the eukaryotic proteome, little is known about how SelS belong to the very small group of intrinsically disordered proteins that intrinsically disordered protein (IDP) quality control is achieved. The cellular exhibit enzymatic functions. We demonstrate that SelS is an efficient reductase functions of IDPs are often precisely encoded by a complex landscape of coupled capable of reducing disulfide bonds, while SelK has a weak but nevertheless interaction and folding with their binding partners. A key question is therefore relevant lipid peroxidase activity. The latter is particular noteworthy because whether and how cellular protein chaperones can regulate the coupled lipid hydroperoxides are accessible to only a limited set of peroxidases, but interaction-folding events of IDPs, as part of maintaining a healthy protein play a critical role in membrane health. homeostasis. To answer this question, we studied the effects of a stress- inducible, ATP-independent molecular chaperone, Hsp27, on the neuronal, Par- kinson’s diseases (PD)-associated IDP alpha-synuclein (aSyn), which served as Platform: Membrane Active Peptides and Toxins an important and well-characterized model IDP system. Coupled membrane- binding and folding of aSyn has been suggested to play an important role in 2507-Plat the putative function of aSyn in fusion, while aberrant interac- Multiple Driving Forces Contribute to Translocation of Autotransporter tion with lipid-bilayers have toxic effects. Here, using the strengths of single- Virulence Proteins molecule fluorescence to monitor mixtures of multiple conformational protein Igor Drobnak, Esther Braselmann, Patricia L. Clark. states in conjunction with complimentary ensemble experiments, we discovered Department of Chemistry & Biochemistry, University of Notre Dame, Notre that Hsp27 can modulate the membrane interaction and coupled-folding of aSyn. Dame, IN, USA. Intriguingly, we observed that this chaperone favors disorder rather than folding Autotransporter (AT) proteins are a large and diverse class of virulence pro- in this system, and that Hsp27 acts by directly interacting with the binding part- teins from Gram-negative bacterial pathogens that require their own C-termi- ner, rather than the IDP itself. Additional biophysical experiments suggest that nal transmembrane domain to translocate their N-terminal passenger across by modulating the coupled interaction-folding landscape of aSyn, Hsp27 may the bacterial outer membrane (OM). Yet given the unavailability of ATP in directly regulate the role of aSyn in vesicle fusion. Analysis of our biophysical the periplasm or a proton gradient across the OM, the energy source that data point to a potential new model of chaperone regulation of IDP function drives this widespread translocation mechanism is unclear. Here we quantita- and dysfunction via maintenance of the disordered state of the IDP. tively test the feasibility of potential AT OM translocation mechanisms using Wednesday, February 11, 2015 497a computational and experimental techniques. We show directly for the first 2510-Plat time that an AT passenger remains unfolded in the absence of OM secretion. Assessing the Translocation of CPPs using Force Measurement, Single Since OM secretion is also impaired for a folding-deficient passenger mutant, Molecules and Emulsions we conclude that folding and secretion are coupled. We demonstrate that AT Pierre Soule1, Abdou Rachid Thiam2, Fre´de´ric Pincet2, Alain Joliot3, secretion is a kinetically controlled, non-equilibrium process and propose a Sandrine Sagan1, Sophie Cribier1, Nicolas Rodriguez1. model connecting passenger conformation to secretion kinetics. These results 1UPMC, Paris, France, 2ENS, Paris, France, 3Colle`ge de France, Paris, underscore the importance of passenger folding as a driving force for AT France. secretion, but also reveal that other energy sources are required to initiate Gene therapy and inhibition of malign molecules inside abnormal cells rely on secretion. an efficient and specific delivery of drugs into the targeted cells. To do so, disruptive methods such as electroporation or partial destruction of the cell 2508-Plat membrane through chemical reagent are currently used. Deciphering Protein Membrane Interactions Involved in the Translocation An alternative technique is the use of carriers that will help the drugs to cross Process of a Bacterial Toxin, the Adenylate Cyclase (CyaA) Toxin from the membrane, without introducing deleterious effects due to the membrane B. Pertussis disruption. A family of such carriers is known as Cell Penetrating Peptides Orso Subrini1, Johanna Karst1, Ana-Cristina Sotomayor-Pe´rez1, (CPPs). Those peptides are short, about ten amino acids, and often cationic. Audrey Hessel1, Edithe Selwa1, Nicolas Sapay1,Re´mi Veneziano2, They are able to translocate through the membrane with different cargos and Jonathan Pansieri2, Joel Chopineau2, Daniel Ladant1, Alexandre Chenal1. deliver them into the cytosol. However the mechanisms are still unknown. 1Structural Biology and Chemistry, Institut Pasteur, Paris, France, 2Institut We use three different techniques to gain insights into the mechanism leading to Charles Gerhardt, Montpellier, France. the translocation ofa CPP. One method usea force measuring tooladapted toliving The adenylate cyclase (CyaA) toxin, secreted by Bordetella pertussis the path- cells: the Biomembrane Force Probe. It gives us the partners that can be involved in ogenic bacteria responsible for whooping cough, plays a critical role in the such process. A second technique is based on a TIRF microscope where the reflec- early stages of respiratory tract colonization by this bacterium. The CyaA tion is made on a suspended bilayer due to a high optical index differences between toxin is able to invade eukaryotic cells by translocating its N-terminal cata- each side of the membrane. This allows us to follow single peptides close to a sus- lytic domain directly across the plasma membrane of the target cells where, pended bilayer. We managed to observe single CPPs that are in suspension or activated by endogenous calmodulin, it produces supraphysiological levels bound to the membrane. A third method relies on a water-oil emulsion. Two water of cAMP. The molecular process leading to the translocation of the catalytic droplets, one containing fluorescent CPPs and the other only buffer, are generated domain across the plasma membrane remains poorly understood. Here, we in an oil-lipid mixture, so that a lipid bilayer is formed at their interface. We follow have characterized the membrane interacting properties of the CyaA catalytic the translocation of the fluorescent CPPs between the two droplets. domain by a combination of biophysical approaches. We showed that the So far we were able to identify sugars as binding partner of the CPPs and to catalytic domain per se (AC384, residues 1-384) did not interact with mem- image CPP translocation. brane while a longer polypeptide, AC489, was able to insert into and permea- bilize membranes. Moreover, deletion of residues 375 to 485 within CyaA 2511-Plat abrogated the translocation of the catalytic domain into target cells. We Synthetic Molecular Evolution Approach: Discovery and Characterization then further identified within this region a key amphiphilic segment spanning of Novel Antiviral Peptides Jing He1, Gregory Wiedman2, Kalina Hristova2, William C. Wimley1. residues 454 to 485. We showed that a synthetic peptide corresponding to this 1 2 sequence, P454, could bind to membranes containing negatively charged Biochemistry, Tulane University, New Orleans, LA, USA, Materials lipids, adopt an alpha-helical structure oriented in plane with respect to the Science and Engineering, the Johns Hopkins University, Baltimore, lipid bilayer and permeabilize vesicles. All together, these results indicate MD, USA. that this short CyaA region downstream to the catalytic domain is essential Peptide entry inhibitors (PEI) interfere with the key steps by which enveloped for toxin translocation. We propose that after insertion of the hydrophobic re- viruses bind to, fuse with, enter, and eventually infect their target cells. There gion (residues 525 to 715) of CyaA into target cell membrane, the region en- has been increasing interest in engineering new PEIs and improving their bioac- compassing the helix 454-485 may insert into membrane and induces a local tivity to combat rapidly and constantly mutating viruses. Yet the lack of generic destabilization of the lipid bilayer leading to membrane permeabilization, thus molecular design principles has obstructed the development of new PEIs. To favoring the translocation of the catalytic domain across the plasma mem- circumvent this roadblock, we are developing a synthetic molecular evolution- brane of target cells. based approach to select PEIs based on their membrane interactions. Using the Wimley-White interfacial hydrophobicity scale to identify membrane inter- acting sequences, peptide entry inhibitors have been identified from within 2509-Plat several viral fusion proteins. We have hypothesized that some of these peptides Moving along the Free Energy Landscape of Membrane Insertion of the interfere with binding and fusion by interacting with directly viral and cellular Diphtheria Toxin Translocation Domain membranes as well as with hydrophobic portions of viral fusion proteins. This Mauricio Vargas-Uribe, Mykola V. Rodnin, Alexander Kyrychenko, hypothesis leads to the prediction that membrane-active peptides, in general, Alexey S. Ladokhin. will inherently have some activity against enveloped viruses, even when those KUMC, Kansas City, KS, Kansas City, KS, USA. peptides are not derived from viral fusion proteins. To test this idea, we are using The pH-triggered membrane insertion of the translocation (T) domain is critical cell biology and biophysics to assess and compare the antiviral potency of for the entry of the diphtheria toxin into the target cell. Previously we charac- several families of interfacially active peptides that we have discovered. Based terized the kinetic pathway of membrane insertion of the T domain, which on the results of this characterization we will then use synthetic molecular evo- consists of a sequence of conformational changes that convert a water- lution, consisting of iterative library design and high-throughput screening, to soluble state into a transmembrane state. Here we utilize various thermody- identify PEIs that have the needed antiviral potency to be useful therapeutics. namic approaches to determine the changes in the Gibbs free energy associated with these conformational changes. The initial conformational change, which 2512-Plat occurs in solution, was studied by thermal and chemical denaturation using dif- Pore Formation Mechanisms of Melittin-Like Membrane-Active Peptides ferential scanning calorimetry, circular dichroism, and fluorescence spectros- Charles H. Chen1, Gregory Wiedman1, Yukun Wang2, Ayesha Khan3, copy. We found that acidification of solution, which results in the formation Martin B. Ulmschneider1. of the membrane-competent form, reduces the thermodynamic stability of 1Department of Materials Science and Engineering, Johns Hopkins the T-domain by about 3-5 kcal/mol, depending on the experimental condi- University, Baltimore, MD, USA, 2College of Life Sciences and tions. For thermodynamic studies of membrane insertion we applied a novel Biotechnology, Shanghai Jiao Tong University, Shanghai, China, 3Wolfson approach that combines fluorescence correlation spectroscopy with the use of Centre for Age Related Disorders, King’s College London, London, United fluorinated surfactants as chemical chaperones. We estimated that the free Kingdom. energy values for the transition from a membrane-competent state in solution Pore formation by membrane-active peptides is believed to play a key role in the to the interfacial intermediate, and to the final transmembrane state are about mechanism of action of antimicrobial peptides. However, at present the process of 8.2 5 0.2 kcal/mol and 12.0 5 0.2 kcal/mol, respectively. The free energy spontaneous pore formation, the pore structure, and the pore lifetime in the bilayer barrier between the two states is modulated by the presence of anionic lipids. remain unknown. This is largely due to experimental difficulties in resolving tran- We summarize our findings in a proposed free energy landscape for the refold- sient protein structures and dynamics in fluid-phase lipid bilayers. Here we employ ing and bilayer insertion of the T domain. a mixture of unbiased multi-microsecond molecular dynamics simulations and Supported by NIH GM-069783 (A.S.L.), and Fulbright-CONICYT and BRTP experimental techniques to study the process of pore formation and conductance (M.V.U.) of melittin, a pore-forming membrane active peptide from honey-bee venom, as 498a Wednesday, February 11, 2015 well as synthetically evolved gain-of-function variants of melittin, which have target effects may involve a lipid bilayer-mediated mechanism. This under- recently been developed by William Wimley at Tulane University. scores the need to further explore the role of bilayer-mediated mechanism in Using spectroscopic techniques we show that gain-of-function variants are able therapeutic as well as toxic effects of antiarrhythmics agents. to leak larger dyes from unilamellar vesicles than melittin. Like melittin gain- of-function variants result in virtually complete leakage of the dye from vesi- Symposium: Advances in Electron Microscopy cles. However, the concentration of peptide required to achieve leakage is significantly lower for gain-of-function variants than for melittin. This suggests 2515-Symp that these peptides form larger pores than melittin and that these pores are much Cryo-EM of DNA Repair Protein Complexes more stable, remaining functional over the lifetime of the leakage experiment. Hong-Wei Wang. Simulations of both melittin and gain-of-function variants reveal a wealth of School of Life Sciences, Tsinghua University, Beijing, China. atomic detail information about transient processes such as peptide absorption, The most recent technical advancement of cryo-electron microscopy is revolu- folding, and oligomeric assembly, as well as the equilibrium structural tionizing structural biology. Using the new generation of transmission electron ensemble and stability, which were verified using circular dichroism, fluores- microscope and direct electron counting device, we can now solve the structure cence, and electrochemical impedance spectroscopy. of important DNA repair complexes with high efficiency and accuracy so to observe new structural features unseen. 2513-Plat The Curvature Induction of Surface-Bound Antimicrobial Peptides 2516-Symp Piscidin 1 and Piscidin 3 Varies with Lipid Chain Length Structural Maturation of Hepatitis B Core Protein Capsids Bradley S. Perrin1, Alexander J. Sodt1, Myriam L. Cotten2, Bettina Boettcher. Richard W. Pastor1. School of Biological Sciences, University of Edinburgh, Edinburgh, United 1 Laboratory of Computational Biology, NHLBI/NIH, Rockville, MD, USA, Kingdom. 2 Department of Chemistry, Hamilton College, Clinton, NY, USA. Hepatitis B virus consists of a capsid formed by icosahedrally arranged Hepa- The initial steps of membrane disruption by antimicrobial peptides (AMPs) titis B core protein (HBC) and an envelope with three different types of mem- involve binding to bacterial membranes in a surface-bound (S) orientation. To brane integrated surface proteins (HBS). During viral maturation, inside the evaluate the effects of lipid composition on the S state, molecular dynamics sim- capsid the RNA-pregenome is reversely transcribed into a partly double ulations of the AMPs piscidin 1 (p1) and piscidin 3 (p3) were carried out in 4 stranded DNA. The reverse transcription is concomitant with the dephosphor- different bilayers: 3:1 DMPC/DMPG, 3:1 POPC/POPG, 1:1 POPE/POPG, and ylation of HBC. Only after the reverse transcription is completed the viral 4:1 POPC/cholesterol. In all cases, the addition of 1:40 piscidin caused thinning capsid is enveloped. Interestingly a single, naturally occurring point mutation of the bilayer, though thinning was least for DMPC/DMPG. The peptides also I/F97L in HBC causes premature envelopment of the capsid. insert most deeply into DMPC/DMPG, spanning the region from the bilayer mid- We have used electron cryo microscopy and image processing to investigate the plane to the head groups, and thereby only mildly disrupting the acyl chains. In structure of phosphorylated and unphosphorylated HBC cores as well as of the contrast, the peptides insert less deeply in the palmitoyl-oleoyl containing mem- premature envelopment mutant to understand the structural mechanisms of branes, do not reach the midplane, and substantially disrupt the chains; i.e., the capsid maturation. neighboring acyl chains bend under the peptide, forming a basket-like conforma- tion. Curvature free energy derivatives calculated from the simulation pressure 2517-Symp profiles reveal that the peptides generate positive curvature in membranes with Single Particle Cryo-EM of Calcium Release Channels palmitoyl and oleoyl chains but negative curvature in those with myristoyl Irina I. Serysheva1,2. chains. Curvature inductions predicted with a continuum elastic model follow 1Biochemistry and Molecular Biology, Structural Biology Imaging Center, the same trends, though the effect is weaker and a small negative curvature in- The University of Texas Health Science Center at Houston Medical School, duction is obtained in POPC/POPG. These results do not directly speak to the Houston, TX, USA, 2National Center for Macromolecualr Imaging, relative stability of the inserted (I) states or ease of pore formation, which re- Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, quires the free energy pathway between the S and I states. Nevertheless, they TX, USA. þ þ do highlight the importance of lipid composition and acyl chain packing. Ca2 Release Channels (CRCs) are intracellular ligand-gated Ca2 channels that are responsible for an increase of cytosolic Ca2þ levels in response to 2514-Plat diverse stimuli. Two closely related families, ryanodine receptors (RyRs) and A General Mechanism for Off-Target Effects: Studies with Amiodarone inositol 1,4,5-trisphosphate receptors (IP3Rs), constitute this class of the tetra- and other Antiarrhythmics meric ion channels localized in SR/ER membranes of all eukaryotic cells. Radda Rusinova1, Roger E. Koeppe II2, Olaf S. Andersen1. 1 Despite long-established efforts of multiple groups, structural analysis of Physiology and Biophysics, Weill Cornell Medical College, New York, CRCs have proven difficult due to their inherent dynamic nature and their enor- NY, USA, 2Chemistry and Biochemistry, University of Arkansas, New York, mous size (1.3 MDa for IP3Rs and 2.3 MDa for RyRs), making X-ray or NMR NY, USA. techniques poorly suited for structural studies of these membrane proteins. The Atrial fibrillation (AF) is the most prevalent type of arrhythmia and is associ- best current structures of complete CRC, determined by single particle electron ated with significant mortality. Amiodarone, owing to its efficacy and minimal cryo-EM at intermediate resolutions of 10-15 A˚ , reveal some secondary struc- proarrhythmic effects, is the most commonly prescribed antiarrhythmic agent. ture elements in both the cytoplasmic and transmembrane regions of channels. Though amiodarone is considered a class III, repolarization-prolonging, antiar- Recent advances in cryo-EM field, including the use of the direct electron rhythmic that inhibits potassium channels involved in restoring the membrane detection cameras and improved image-processing software, have, in some excitable state, it also alters the function of many other structurally and func- favorable cases, allowed for the determination of near-atomic resolution struc- tionally diverse membrane proteins. This concomitant regulation of multiple tures of integral membrane proteins. Our strategies and recent progress toward membrane proteins by amiodarone results in complex therapeutic and toxicity high-resolution structure determination of the entire CRC will be discussed in profiles. Other antiarrhythmics, such as dronedarone, have similar multiple the context of breakthrough developments in single-particle cryo-EM. targets. Though such a multipronged mechanism for treating This research is supported by grants from the NIH (R01GM072804, AF appears to be desired, it is not clear how amiodarone, and other antiarrhyth- R21AR063255, P41RR002250 and S10OD016279), the AHA mics exert their therapeutic actions and regulate a diverse range of membrane (14RNT1980029) and the MDA (295138) proteins at similar concentrations. Chatelane et al. (1989) found that amiodar- one and propranolol alter lipid bilayer properties, and that amiodarone does so 2518-Symp at therapeutic concentrations. We therefore took advantage of the gramicidin Single Particle CryoEM of Integral Membrane Proteins (gA) channels’ sensitivity to changes in the lipid bilayer properties to deter- Yifan Cheng. mine whether the commonly used antiarrhythmics amiodarone, dronedarone, Biochemistry and Biophysics, University of California, San Francisco, propranolol and pindolol perturb the lipid bilayer properties and at which con- San Francisco, CA, USA. centrations. Using a gA-based fluorescence assay and single-channel electro- The Keck Advanced Microscopy Laboratory, Department of Biochemistry and physiology to explore the antiarrhythmics’ effects on the lipid bilayer, we Biophysics University of California, 600 16th Street, San Francisco, CA 94158 found that amiodarone and dronedarone are potent bilayer modifiers, propran- As a versatile tool in structural biology, single particle electron cryo- olol has intermediate activity, and pindolol is the least potent. Moreover, amio- microscopy (cryo-EM) has achieved milestones of determining near atomic darone and propranolol increase bilayer elasticity. Because amiodarone and resolution three-dimensional (3D) reconstructions of non-enveloped viruses dronedarone alter the lipid bilayer at their therapeutic concentrations, where with icosahedral symmetry. Recent technological breakthroughs in single par- they act on multiple membrane proteins, our results suggest that their multi- ticle cryo-EM, particularly the development of novel dose-fractionated image Wednesday, February 11, 2015 499a acquisition method based on CMOS direct electron detection camera and serine protease expressed in the epithelium of several organs, such as the skin, robust algorithms for correction of motion induced image blurring, are transfor- colon, lung and kidney, and can be released in the extracellular space by the mative. It has enabled near atomic resolution structure determinations of a action of phospholipase C. CAP1/Prss8 was one of the the first of several broad range of proteins complexes without the need of crystals. membrane-bound serine proteases, such as CAP2/Tmptrss4 and CAP3/matrip- One of the major challenges in structural biology is to determine structures of in- tase, found to activate the epithelial sodium channel (ENaC) in-vitro. Since these tegral membrane proteins at different functional states. The bottleneck for X-ray studies strongly suggested a non-enzymatic function of CAP1/Prss8, we over- crystallography is to trap and crystallize the same membrane protein in different expressed the catalytically inactive form of CAP1/Prss8 and asked whether we conformations. Without constrain of crystallization, it is now possible to deter- can negate the obtained phenotype by crossing these mice with mice lacking mine structures of integral membrane proteins at subnanometer to near atomic the protease-activated receptor 2, that we previously identified as downstream resolutions. At this resolution range, it is possible to derive the structural infor- target substrate. Since serine protease activity needs to be tighly regulated, we mation from docking of known or homology model into subnanometer resolu- also asked the question whether the putative serine protease inhibitor nexin-1 tion cryo-EM density maps, or to build de novo atomic structure directly from is able to block CAP1/Prss8 in vitro and in vivo. the 3D density map. We will discuss some of the recent technological advance- Our results demonstrate the ability of a catalytically inactive serine protease to ments specific for structural analysis of integral membrane proteins. induce disease when ectopically expressed, and a novel inhibitory interaction that does not depend on its catalytic site. Symposium: Catalysis in the Membrane 2522-Symp A Molecular Description of Cellulose Biosynthesis 2519-Symp Jochen Zimmer. ZMPSTE24 and Premature Ageing: A Unique Integral Membrane Metal- University of Virginia, Charlottesville, VA, USA. loprotease with a Hole in the Middle Cellulose is the most abundant biological macromolecule and is an extracellular Liz Carpenter. linear polymer of glucose molecules. It is an essential component of plant cell Structural Genomics Consortium, University of OXford, Oxford, United walls but is also found in algae and bacteria. In bacteria, cellulose production Kingdom. frequently correlates with the formation of biofilms, a sessile, multi-cellular The nuclear lamina is an intermediate filament network which underlies the nu- growth form. Cellulose synthesis and transport across the inner bacterial mem- clear membrane, providing rigidity for the nucleus and a binding site for chro- brane is mediated by a complex of the multi-spanning catalytic BcsA subunit and matin and nuclear proteins.The lamina are important in a range of processes the membrane-anchored periplasmic BcsB protein. We determined several crys- including cell division, DNA repair and epigenetic effects. These filaments tal structures of the BcsA-B complex at intermediate states during cellulose syn- are made up of three proteins, lamins A, B and C, two of which, lamins A thesis and membrane translocation. The structures demonstrate how BcsA forms and B, undergo a series of post translational modifications. Failure of lamin a cellulose-conducting channel and delineate conformational changes of the syn- processing leads to a series of laminopathies including the premature ageing thase underlying substrate binding, glycosyl transfer and polymer translocation. syndrome Hutchinson Gilford progeria syndrome and mandibuloacral Furthermore, combining biochemical and structural data, we reveal the mecha- dysplasia. Mature lamin A is formed from its precursor, prelamin A, by farne- nism by which cyclic-di-GMP, a potent stimulator of bacterial biofilms and allo- sylation on a C-terminal CaaX motif, removal of the aaX residues and C-termi- steric activator of bacterial cellulose synthase, regulates BcsA’s activity. nal carboxymethylation. Finally prelamin A also undergoes a proteolytic removal of the C-terminal 15 residues, including the farnesylated Cysteine. ZMPSTE24, a nuclear membrane zinc metalloprotease, can perform both the Platform: Protein-Lipid Interactions III proteolysis steps in this process. Although the first reaction can be performed by RCE1 as well, the second cleavage is only performed by ZMPSTE24. 2523-Plat Our crystal structure of ZMPSTE24 revealed a completely unexpected fold, with a Elucidating the Interaction of 5-Lipoxygenase and FLAP 1,2 1,2 1,2 seven transmembrane a helical barrel, surrounding a huge, intramembrane cham- Ramakrishnan B. Kumar , Hans Hebert , Caroline Jegerscho¨ld . 1Department of Bioscience and Nutrition, Karolinska Institutet, Huddinge, ber (Quigley et al., Science, 339, 1604-7, 2013). One end of the chamber is capped 2 by a zinc metalloprotease. fold, with the active site pointing into the chamber. The Sweden, School of Technology and Health, The Royal Institute of C-terminal farnesylated tail of prelamin A will enter into the chamber from the Technology, Huddinge, Sweden. membrane/ nucleoplasm interface, undergo the first processing, be carboxymethy- Inflammation is one of the innate defense mechanisms exerted by the human lated by ICMT and then reinsert into the ZMPSTE24 chamber to have the final 15 body for protection and to initiate the healing process. Prolonged inflammatory residues, including the farnesylated cysteine, removed. Some laminopathies are reactions can lead to chronic disease conditions like atherosclerosis, asthma and caused by point mutations on ZMPSTE24 and our structure has allowed us to un- myocardial infarction. Leukotrienes (LTs) are one of several pro-inflammatory derstand how these mutations affect the function of ZMPSTE24. lipid mediators involved in such inflammatory diseases and are derived from arachidonic acid (AA). The key enzyme involved in LT biosynthesis is 5 Lip- 2520-Symp oxygenase (5LO), Five lipoxygenase activating protein (FLAP) an integral The Mechanism of Rhomboid Intramembrane Protease membrane protein and Coactosin like protein (CLP) a scaffolding protein. Ya Ha. Upon external stimuli, intracellular calcium concentration increases which Pharmacology, Yale University School of Medicine, New Havent, CT, USA. translocates 5LO from the cytosol to the nuclear membrane and localizes The activities of many membrane proteins, including transcription factors, re- near FLAP. Then 5LO converts the AA to leukotriene A4. The hypothesis is ceptors, growth factors, and transporters, are regulated by a cleavage event that AA is transferred from the nuclear membrane to 5LO by homo-trimeric within their transmembrane domains. The intramembrane proteolytic reaction FLAP and CLP is also involved with 5LO in this stage. Though this hypothesis is catalyzed by highly specialized membrane-embedded proteases belonging has been studied extensively, the association between these proteins in LT to three mechanistic families. The rhomboid serine protease, first discovered biosynthesis is still clouded. To elucidate these assisted interactions, we recon- by Drosophila genetics, represents a large family of functionally diverse mem- stituted the FLAP into ‘‘Nanodisc’’ a membrane mimicking system . We group- brane proteins that share a common core domain of six transmembrane seg- ed the project by first analyzing the interaction of 5LO with ND, to simulate and ments. Extensive crystallographic analysis of the bacterial rhomboid GlpG understand the calcium mediated translocation of 5LO to nuclear membrane in has generated insight into how peptide hydrolysis occurs inside the hydropho- this ND system. We then proceeded to use FLAP-containing nanodiscs (FND) bic environment of membrane bilayers. Here we examine the conformational and repeated aforementioned analyses. We employed biochemical assays and change that takes place during substrate binding and catalysis based on x-ray transmission electron microscopy to characterize the interactions and to create structures of GlpG in complex with inhibitors and peptide analogs. a 3D model of the functional complex of 5LO,CLP and FLAP. Here, we show our results from the above mentioned projects involved in understanding the 2521-Symp interaction of proteins involved in the initiation of leukotriene biosynthesis. Cleavage-Dependent and Independent Role of the Serine Protease CAP1/ Prss8 2524-Plat Edith Hummler. Destabilizing Aquaporin Z Assembly: Effects on Structure, Function and Pharmacology, Lausanne University, Lausanne, Switzerland. Dynamics Serine protease are enzymes involved in the regulation of many biological pro- Victoria Schmidt, Pierre Hubert, Valerie Prima, James Sturgis. cesses, and generally exert their function by cleaving peptide bonds through the LISM - CNRS/AMU, Marseille, France. catalytic triad of histidine, aspartate and serine. The channel-activating protease Aquaporins are membrane proteins, that act as water channels in biological 1 (CAP1/protease serine S1 family member 8 (Prss8) is a membrane-anchored membranes. Members of this family form tetrameric, or rarely pentameric, 500a Wednesday, February 11, 2015 complexes in the membrane. In order to better understand the folding and mul- 2527-Plat timeric assembly of these proteins we have constructed a series of destabilized Role of Mutations in Protein-Protein Interactions proteins by modifying the interface between monomers in Aquaporin Z from Vitaly V. Vostrikov1, Kailey J. Soller2, Kim N. Ha3, Sarah E. Nelson1, E. coli. We have characterized these proteins to test the the effects on the Tata Gopinath1, Gianluigi Veglia1. folding of the monomeric unit, the assembly of monomers into tetramers. 1BMBB, University of Minnesota, Minneapolis, MN, USA, 2Chemistry, We have also examined the consequences modified structures on the function University of Minnesota, Minneapolis, MN, USA, 3Chemistry and of the water channel and the dynamics of the protein. Biochemistry, St. Catherine University, St. Paul, MN, USA. Structure and folding has been examined at the level of the protein topology Calcium regulation in heart muscles is achieved through a fine interplay be- and modifications observable by FTIR spectroscopy. These methods indicate tween a variety of proteins. Of particular importance are sarco(endo)plasmic þ that the surface mutations do not delectably modify the structure of the mono- reticulum Ca2 ATPase (SERCA) and phospholamban (PLN): SERCA trans- meric aquaporin Z. Assembly into tetramers has been investigated by hydrody- fers calcium ions against the concentration gradient and is inhibited by PLN. namic methods, DLS, Fluorescence anisotropy and FCS, FCCS. These methods Inhibition is relieved upon phosphorylation of PLN at S16. Recently mutations suggest that in some mutants tetramers are not properly formed and monomeric in the pln gene have been linked to the progression of cardiomyopathies, raising aquaporins predominate. Some of these mutations can be suppressed by questions about the biophysical basis of the disease. compensatory mutations on the opposite side of the interface. We report the We have investigated the behavior of the phospholamban mutants alone and in the consequences of these changes in assembly. presence of their interaction partners through complimentary techniques. Solution NMR spectroscopy provided insights into ps-ns dynamics of the regions harboring 2525-Plat the mutation. Oriented and magic angle spinning solid-state NMR in lipid bilayers The Role of Lipid Environment on Peptide Structure and Folding were used to probe the topology, conformation and water accessibility of phospho- Renee D. JiJi, Jian Xiong, Anahita Zare, Jason W. Cooley. lamban. SERCA activity assays were performed to assess the inhibitory potency Chemistry, University of Missouri-Columbia, Columbia, MO, USA. of the mutants in their native or phosphorylated forms. Such multiscale approach Membrane proteins are an important class of proteins that are difficult to char- allowed us to build a comprehensive picture of the interactions disrupted through acterize structurally and functionally. In order to gain a better understanding of the mutations (Vostrikov et al. Biochim Biophys Acta 2015). the forces that govern membrane protein folding and structure, a series of sim- Ourdata providesevidence that several essential regulatory functions are disrupted ple leucine-alanine peptides ranging from 12 to 18 residues were designed. The through the naturally occurring mutations. Amino acid substitutions or deletions peptides were anchored by two lysine residues at each end and spontaneously lead to the alteration in the fold, conformation and dynamics of the regulatory do- inserted into negatively charged lipid bilayers. The effects of peptide length, main of PLN. Such changes disrupt the PLN interactions with its binding partners, lipid length, bilayer/micelle composition and the presence of structure breaking shifting the delicate balance of calcium ions transfer. We surmise that the develop- residues glycine and proline on the uniformity of helical structure were evalu- ment of cardiomyopathies elicited by the PLN mutants is linked to a variety of dis- ated. An increase in the intensities of the amide III and S bands in deep-UV rupted protein-protein interactions, rather than affecting one specific target. resonance Raman spectra indicated loss of helical structure. Differences in pep- This work is supported through the American Heart Association fellowships tide hydration were monitored using tryptophan fluorescence. Loss of helical 13POST14670054 to V.V. and 13PRE16950023 to K.S. structure was observed in cases of negative hydrophobic mismatch, increased peptide hydration and upon introduction helix breaking residues. No loss of he- 2528-Plat lical structure was observed in cases of positive hydrophobic mismatch while Single-Molecule FRET Detection of GXXXG-Mediated Transmembrane the shortest peptide adopted beta-sheet structure in instances of negative hydro- Helix-Helix Interactions phobic mismatch. Greater hydration of the peptide, which occurred in surfac- Yoshiaki Yano, Kotaro Kondo, Katsumi Matsuzaki. tant/lipid micelles, magnified the helix breaking- effects of glycine and Kyoto University, Kyoto, Japan. proline. These studies highlight the potential importance of the lipid environ- Helix-helix interactions in lipid bilayers are principal processes that determine ment itself on membrane protein structure. the folding, oligomerization, and conformational change of helical transmem- brane proteins. Not only the amino acid sequence of the protein but also the composition of surrounding lipids significantly affect the stability of the interac- 2526-Plat tion. The GXXXG motif is frequently found at interaction interface of the trans- Investigation on the Interaction between Plexin Intracellular Plus Trans- membrane region, and proposed to mediate helix associations via hydrogen membrane Domains with GTPases and with the Lipid Bilayer using All- bonding between Ca-H donor and the backbone C¼O acceptor. However, ener- Atom Molecular Dynamics Simulations getic/kinetic contributions of the motif have not been well characterized. Liqun Zhang, Buck Matthias. In this study, we investigated the effect of a GXXXG-motif introduced into the cen- Physiology and Biophysics, Case Western Reserve University, Cleveland, ter ofthe host transmembranehelix (AALALAA)3, examinedby a single molecule OH, USA. FRET technique. The host helices are known to weakly self-associate in antipar- Plexins are transmembrane receptors that receive Semaphorin guidance cues by allel orientations in POPC vesicles. In contrast, the GXXXG motif significantly binding them at their extracellular domains and thus are activated by them. stabilized a parallel association of the helices with lifetimes of subseconds. We Plexins function not only in cell migration processes, in neuronal and cardio- also found that cholesterol suppressed the GXXXG-mediated parallel associations, vascular development, but also in cancer metastasis. Plexins are unique, as demonstrating the importance of lipid environment on the helix-helix interaction they are the first example of a receptor that interacts directly with small GTPases, a family of proteins that are essential for cell motility and prolifera- 2529-Plat tion/survival. We previously determined the structure of the Rho GTPase bind- Spontaneous Reconstitution of Bovine Rhodopsin into Artificial ing domain (RBD) of several Plexins and also of the entire intracellular regions Membranes of a Plexin-B1 [1]. Connecting the transmembrane domain to the intracellular Udeep Chawla1, Wan Zheng2, Liangju Kuang2, Yunjiang Jiang2, domain of plexin, we set up a plexin-B1 all atom model bound with small Rho Suchithranga M.D.C. Perera1, Michael F. Brown3, Hongjun Liang2. and Ras GTPases. The C-terminal tails of the GTPases are either farnysylated 1Departments of Chemistry and Biochemistry, University of Arizona, or geranylated, anchoring these proteins to the lipid membrane. Specifically, we Tucson, AZ, USA, 2MT, Colorado School of Mines, Golden, CO, USA, built models of the entire intracellular plus transmembrane regions, starting 3Departments of Chemistry and Biochemistry, Department of Physics, from several crystal structures linked to transmembrane helices whose structure University of Arizona, Tucson, AZ, USA. was predicted using PREDDIMER followed by microsecond-long MD refine- Rhodopsin is a canonical G-protein-coupled receptor (GPCR) that is responsible ment simulations [3]. The simulations probe interactions between the GTPases for vision in dim light. It has the potential to serve as a high-fidelity, light-sensing and with plexin, indicating an allosteric network that changes upon plexin-B1 molecular switch for a broad range of nanotechnologies. Previous studies revealed binding with Rho GTPase. The models and simulations at the lipid bilayer that the photoactivity of rhodopsin depends critically on the native lipid bilayer reveal the origin of Ras and Rho specificity in plexin’s function, as well as environment surrounding this membrane protein [1-3]. It is not yet clear how arti- the importance of the lipid membrane in stabilizing the whole structure of ficial membranes in synthetic systems would affect the activity of rhodopsin [4], plexin. and recent study suggests that membrane moduli may play important roles [4, 5]. References: Partially this uncertainty is due to the fact that it is experimentally challenging to [1] Hota PK, Buck M. (2012) Cell Mol Life Sci. 69(22):3765-805. prepare rhodopsin-supporting artificial proteomembranes with systematicallyvar- [2] Zhang L, Bouguet-Bonnet S, Buck M. (2012) Methods Mol Biol. ied membrane chemistry and physical properties. Here we show that bovine 796:235-59. rhodopsin can be spontaneously reconstituted into a series of well-defined artifi- [3] Zhang L, Polyansky A, Buck M. Submitted to PLoS Computational cial membranes, including both lipid-based (i.e., liposome) and polymer-based Biology. 2014. (i.e., polymersome) membranes, via a charge-interaction-directed reconstitution Wednesday, February 11, 2015 501a

(CIDR) mechanism [5, 6]. Using synchrotron small-angle X-ray scattering The covalently linked domains constituting a multidomain protein typically (SAXS), we show that n-dodecyl-b-D-maltoside solubilized bovine rhodopsin share a similar fold and sequence, are generally stable in isolation, and are is reconstituted spontaneously to form 2-D proteomembrane arrays, which in largely capable of independent folding. However, the elegant interplay of turn are coupled along the trans-membrane direction to form a 3-D multilamellar forces leading an amino acid sequence to its native state is more complex in structure. The lamellar periodicity is ~5.7 nm, which matches closely the trans- proteins with multiple domains because of inter-domain interactions, which membrane dimension of rhodopsin. Using time-resolved UV-visible spectros- complicate the folding energy landscape. copy, we are currently examining the photoactivity of embedded bovine In previous experiments, employing immunoglobulin-like (Ig-like) domains of rhodopsin, and its dependency on the surface charge states and membrane moduli the human multidomain protein titin, we showed that tandem repeats of do- of the artificial membrane. [1] M. F. Brown (1997) Curr. Top. Membr. 44, 285- mains with high sequence identity can form a stable misfolded state upon re- 356. [2] A.V. Botelho et al. (2006) Biophys. J. 91, 4464-4477. [3] A.V. Struts et folding in physiological solution conditions. Conversely, tandem constructs al. (2014) Meth. Mol. Biol. (in press). [4] V. Subramaniam et al. (2005) JACS of natural neighbouring domains, which display a low sequence identity, did 127, 5320-5321. [5] L.J. Kuang et al. (2014) ACS Nano 8, 537-545. [6] D.B. not misfold. This supported the hypothesis that sequence identity between Hua et al. (2011) JACS 133, 2354-2357. neighbouring domains in multidomain proteins is reduced as a result of evolu- tionary pressure to avoid misfolding. 2530-Plat With a combination of single-molecule Fo¨rster resonance energy transfer, Coarse-Grained Modeling of Minute-Timescale Co-Translational Mem- microfluidic mixing, stopped-flow kinetics and molecular dynamics simula- brane Protein Integration via the Sec-Translocon tions, we now demonstrate that Ig-like domains can transiently populate a Michiel J.M. Niesen, Thomas F. Miller III. surprisingly broad range of misfolded conformations on the sub-second time- Chemistry & Chemical Engineering, California Institute of Technology, scale. Using tandem repeats of Ig-like domains, we can resolve both strand- Pasadena, CA, USA. swapped misfolds dominated by native-like interactions and, remarkably, Integration into the cell membrane in the correctly folded structure is essential a non-native-like, largely disordered type of misfolded state which so far for membrane protein function. Membrane protein integration takes place at was never observed experimentally, characterized by promiscuous interac- the Sec-translocon and typically occurs during translation of the protein tions. Even more surprisingly, both types of misfolding are detected also sequence. Although extensive structural data is available, study of the dynamics for the naturally occurring tandem repeat, showing how finely the propen- of Sec-facilitated membrane protein integration is difficult due to roles of long- sities of folding and misfolding have been balanced by co-evolution of timescale dynamics and ribosomal translation. We present a coarse grain simu- adjacent domains to avoid stable misfolded states formation. On longer time- lation approach that is capable of reaching the experimentally relevant (i.e., scales, however, all or most of the protein molecules are able to reach minute) timescales, while retaining the level of detail required for reproducing the native state, demonstrating that the overall free energy surface is still available experimental data. The CG model enables direct simulation of the dy- sufficiently optimized for the protein to efficiently reach its correctly folded namics of co-translational integration at the Sec-translocon and analysis of the state. resulting trajectories provides mechanistic explanations for observed experi- mental data. We will demonstrate use of this CG model to investigate thermody- 2533-Plat namic and kinetic factors that govern membrane protein topology, integration Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy on the efficiency of trans-membrane domains, and hydrophilic loop translocation. Folding Mechanism of B Domain of Protein A Takuhiro Otosu1, Kunihiko Ishii1,2, Hiroyuki Oikawa3, Munehito Arai4, Platform: Protein Folding and Chaperones Satoshi Takahashi3, Tahei Tahara1,2. 1Mol. Spectrosc. Lab., RIKEN, Saitama, Japan, 2RAP, RIKEN, Saitama, 3 4 2531-Plat Japan, IMRAM, Tohoku Univ., Miyagi, Japan, Grad. Sci. Arts. Sci., Univ. Proteomics-Level Identification of Degradation-Resistant Proteins Provide Tokyo, Tokyo, Japan. Insight about their Potential Roles in Organismal Adaptation to Stress Molecular-level description of the folding process of proteins is of fundamental Ke Xia, Jennifer Wilcox, Kayleigh Kobovitch, Brian Ortiz, Areeg Khalil, importance for understanding how proteins acquire their unique conformations Wilfredo Colon. to show various biological functions. Elucidation of the folding mechanism re- Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, quires quantitative analysis with a high time-resolution. To this aim, we NY, USA. recently developed two-dimensional fluorescence lifetime correlation spectros- Some proteins are hyperstable, as demonstrated by their resistance to proteolysis copy (2D FLCS)1,2. This method enables us to examine the microsecond and detergents, and their long half-life. Some of these proteins are characterized conformational dynamics of proteins at the single-molecule level, through by having a high energy barrier toward unfolding that virtually traps them in their the analysis of the fluorescence lifetime correlation. In this study, we applied native state. This property of proteins, known as ‘‘kinetic stability’’ (KS), ap- 2D FLCS to study the folding mechanism of B domain of protein A (BdpA). pears to be a strategy used by ‘‘mother nature’’ to protect certain proteins against This small protein was believed to show a two-state folding process in which aggregation, and premature degradation under stress conditions. In addition to the native state unfolds without exhibiting any intermediate states. However, saving organismal resources by avoiding frequent degradation-biosynthesis of a recent single-molecule study suggested that the folding mechanism of proteins, KSPs could function under extreme stress to maintain vital functions BdpA is not so simple3. To quantitatively analyze the complex folding process needed for survival. On the basis of our observed correlation between KS and of BdpA, we performed 2D FLCS for two FRET mutants of BdpA. The results a protein’s resistance to the detergent SDS, we developed a diagonal two- showed that the conformations of both the native and unfolded states are highly dimensional (D2D) SDS-PAGE method for the proteome-level identification heterogeneous and that the conformational dynamics within each conforma- of KSPs. We applied D2D SDS-PAGE to the lysate of Thermus thermophilus tional ensemble occurs on a time scale shorter than ten microseconds. Fur- and E. coli followed by proteomics analysis, and identified over 100 KSPs. thermore, our results indicated that the conformational distribution in the We also analyzed the cell lysates of various prokaryotic and eukaryotic organ- native and unfolded ensembles gradually change with the change of the dena- isms. Our results suggest that KS was likely a critically important property of turant concentration. proteins for the adaptation and survival of microbial organisms under stress con- [1] Ishii and Tahara, 2013. J. Phys.Chem. B. 117, 11414-11422. ditions. In contrast, the minimal abundance of KSPs in eukaryotic organisms im- [2] Ishii and Tahara, 2013. J. Phys.Chem. B. 117, 11423-11432. plies an evolutionary compromise of KS in favor of more complex cellular [3] Oikawa et al., 2013. Sci. Rep. 3, 2151. defense, function, and regulation. Thus, D2D SDS-PAGE is a simple and power- ful method that may be applied to any complex mixture of proteins to explore the 2534-Plat biological and pathological significance of protein kinetic stability. Conformational Dynamics of Molecular Chaperones Investigated by Single Molecule Multicolor Fo¨rster Resonance Energy Transfer 2532-Plat Lena Voith von Voithenberg1, Anders Barth1, Swati Tyagi2, Surprising Abundance of Misfolding during Refolding of Multidomain Christine Koehler2, Edward A. Lemke2, Don C. Lamb1. Proteins 1Physical Chemistry, Ludwig-Maximilians-Universitaet, Muenchen, Alessandro Borgia1, Katherine R. Kemplen2, Madeleine B. Borgia1, Germany, 2European Molecular Biology Laboratory, Heidelberg, Germany. Robert B. Best3, Andrea Soranno1, Daniel Nettels1, Bengt Wunderlich1, Molecular chaperones of the heat shock protein family 70 (Hsp70) are ubiqui- Jane Clarke1, Benjamin Schuler1. tously expressed and involved in a variety of cellular processes. While partici- 1Biochemistry, University of Zurich, Zurich, Switzerland, 2Chemistry, pating in protein folding and protein complex remodeling, Hsp70 chaperones University of Cambridge, Cambridge, United Kingdom, 3NIKKD, NIH, found in cellular organelles additionally assist in protein import, protection Beteshda, MD, USA. from misfolding, and transfer to the proteosomal machinery. 502a Wednesday, February 11, 2015

An ATP-driven conformational cycle which includes the co-chaperones loops that protrude from every subunit into the lumen of ClpX pore, which J-domain protein and nucleotide exchange factor underlies the chaperone’s contact the polypeptide through a highly-conserved motif: GYVG. We used function in protein folding. During the conformational cycle of Hsp70, both single-molecule assays with optical tweezers to study the effect on the mech- the nucleotide-binding domain (NBD), as well as the substrate-binding domain anochemistry of the motor of Y153A and V154F mutations, which decrease/ (SBD), appear in different conformational states. increase the size of these loops. 1-3 ClpX WT subunits were substituted by Single-pair FRET measurements with multiparameter fluorescence detection either a Y153A or V154F mutant subunit, arranged in different positions rela- (MFD) of conformational changes between the domains and within the SBD tive to each other within the ClpX ring. Since the translocation cycle has two of molecular chaperones from the endoplasmic reticulum and mitochondria phases, a dwell and a burst, we analyzed the effect of these mutations on these of eukaryotic cells suggest nucleotide- and substrate-dependent changes in phases. Y153A mutations decreased the dwell duration compared to WT, Hsp70 conformation leading to allosteric regulation of the SBD by nucleotide increasing translocation velocity, while V154F mutants had longer dwells, binding. reducing translocation velocity. Interestingly, the burst-size remained unaf- To further elucidate the mechanism of chaperone function by conformational fected. ATP-turnover analysis revealed that in Y153A mutants increased changes within and between the domains, we extended the MFD-PIE experi- dramatically, while in V154F it was a smaller increase. We calculated the ments to three colors. Incorporation of nonnatural amino acids into the coupling coefficient (CC), the number of ATP hydrolysis cycles that are proteins enables the attachment of three fluorophores. By placing the fluoro- required for a productive power stroke. For WT the CC is one, Y153A mu- phores on each of the domains as well as on the lid of the SBD, we could tants have a CC of two, while in V154F mutants is between one and two. perform three-color FRET and thereby monitor three FRET efficiencies simul- Finally, we observed that mutant subunits either located across the ClpX taneously. This allows us to detect coordinated motion between the domains ring or intercalated by a WT subunit had more effect in the duration of the and within the SBD of these triple-labeled Hsp70 proteins. By comparing the dwell and in GFP unfolding capability. These results provide missing infor- conformational changes and coordinated motion observed for Hsp70 chaper- mation about the spatial arrangement and inter-subunit coordination of pore ones found in prokaryotes and eukaryotic organelles, we investigate the loops during polypeptide translocation. conserved and divergent behaviors of these different proteins. Thereby, we elucidate details regarding the functional mechanism and specificity of these 2537-Plat chaperones. (DIS)Assembly and Structural Stability of mtHsp60 and its Precursor NaI¨ve Form 2535-Plat Dario Spigolon1,2, Silvia Vilasi1, Maria Rosalia Mangione1, Copper and Zinc Binding Specifically Induce the Aggregation of Human PierLuigi San Biagio1, Donatella Bulone1. g-D Crystallin 1CNR-IBF, Palermo, Italy, 2Department of Biopathology, Medical and Liliana Quintanar1, Evgene Serebryany2, Jose Antonio Domı´nguez-Calva1, Forensic Biotechnologies, University of Palermo, Palermo, Italy. Cameron Haasse-Pettingell2, Jonathan A. King2. Heat shock protein 60kDa is a molecular chaperone (GroEL human homolog) 1Department of Chemistry, Cinvestav (Centro de Investigacion y de Estudios that assists protein folding in mitochondria (mtHsp60). It is synthesized in the Avanzados), Mexico D.F., Mexico, 2Department of Biology, Massachusetts cell cytoplasm as a higher molecular weight precursor form (p-mtHsp60) con- Institute of Technology, Cambridge, MA, USA. taining a N-terminal targeting sequence, that is cleaved after import into the Cataract is the leading cause of blindness in the world, and it is projected to mitochondrial matrix [1, 2]. affect 20-30 million people in 2020. Cataracts are formed upon aggregation It has been established, and demonstrated by various techniques, Hsp60 can of lens proteins into high molecular weight complexes, causing light scattering accumulate in the cytosol, in various pathological conditions (i.e., cancer and and lens opacity (1). A variety of experimental and etiological studies implicate chronic inflammatory diseases). The cytosolical Hsp60 accumulation mecha- metals as a potential etiological agent for cataract. Copper and zinc concentra- nism may occur with or without mitochondrial release concomitantly, so that tions in the cataractous lenses are increased significantly, as compared to in the cytosol the two types of 60 kDa chaperonin proteins, (mtHsp60 and its normal lenses (2). The monomeric all b-sheet Human gD(HgD) crystallin is precursor naı¨ve form, p-mtHsp60) could coexist [3] .It has been recently one of the abundant crystallins in the core of the lens and its non-amyloid ag- observed that in a wide range of concentration, the precursor naı¨ve Hsp60 is gregation is associated with cataracts.1 We find that micromolar concentrations able to assemble in both heptamers and tetradecamers [4]. of Cu(II) and Zn(II) ions exert distinct and specific effects on the conformation Key questions still unanswered pertain to the differences in structure-function of HgD, suggesting site-specific interactions. Circular dichroism studies features that might exist between the well-studied prokaryotic GroEL and the show that Cu(II) induces loss of secondary structure and stability of HgD. largely unexplored eukaryotic Hsp60 proteins. Moreover, studies on human Spectroscopic studies demonstrate that Cu(II) ions can bind at more than one Hsp60 structure and oligomeric state in vitro could help to validate its role in site in the protein. Metal binding to the monomer leads to formation of high physiological or pathological cases. In order to pursue this goal, we investi- molecular weight light scattering aggregates. These metal-induced effects gated the (dis)assembly and thermal stability of mtHsp60, p-mtHsp60 and occur at micromolar concentrations of metal ion and protein, starting at GroEL in vitro, by means of Differential Scanning Calorimetry (DSC) and metal:protein ratios of 1:1. Interestingly, the lens chaperone aB crystallin pro- Isothermal Titration Calorimetry (ITC). Complementar Circular Dicroism tected HgD from metal-induced aggregation. Metal-induced aggregation could (CD) measurements were done to follow the change in the secondary structure be a physiologically relevant phenomenon; understanding its mechanism will due to unfolding. help elucidate the role of metal ions in the aggregation of human crystallins [1] Ellis RJ, 2007, Adv Exp Med Biol., 594: 1-13. and their potential involvement in the development of cataracts. This research [2] Jonathan D, 2000, JHC., 48(1): 45-56. has been supported by: MIT-Seed Funds, Conacyt (grant # 221134 and fellow- [3] D. Chandra, 2007, J. Biol. Chem., 282: 31289-31301. ship to J.A.D.-C.), NIH EY015834, AMC-FUMEC and Fulbright-Garcı´a [4] Vilasi S. et al, 2014, Plos One., 9(5): e97657. Robles fellowships to L.Q. 1. Moureau, L.K.; King A.J. Trends Mol. Med., 2012, 18, 273-282 2538-Plat 2. Curtis, E.D. Exp. Eye Res., 1983, 37, 639-647 Characterizing the Conformational Ensembles of the E. coli Hsp70, DnaK Reveals the Role of the Intermediate State 2536-Plat Alex Liqi Lai1, Mandy Blackburn2, Eugenia M. Clerico3, Peter Borbat1, Role of Pore Loops in the Mechanism of Polypeptide Translocation by a Lila M. Gierasch3, Jack H. Freed1. AAAD Protease Machine 1Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA, Piere Rodriguez-Aliaga1, Luis E. Ramirez2, Frank Kim2, Andreas Martin1,3, 2Chemistry, University of Central Missouri, Warrensburg, MO, USA, Carlos Bustamante1,3. 3Biochemistry and Molecular Biology, University of Massachusetts Amherst, 1Biophysics Graduate Group, University of California at Berkeley, Berkeley, Amherst, MA, USA. CA, USA, 2University of California at Berkeley, Berkeley, CA, USA, DnaK is a molecular chaperone that helps protein folding and protects cells 3Molecular and Cell biology, University of California at Berkeley, Berkeley, from stress. The allosteric mechanism of DnaK enables ATP binding to the CA, USA. N-terminal nucleotide-binding domain (NBD) to alter substrate affinity to Proteases of the AAAþ family play crucial roles maintaining protein the C-terminal substrate-binding domain (SBD) and in turn substrate binding homeostasis in the cell by degrading misfolded/damaged proteins. ClpX is a enhances ATP hydrolysis. Cycling between ATP-bound and ADP/substrate- homohexameric ring-shaped member of this family, which uses ATP to me- bound states requires DnaK to visit an ‘‘ATPþsubstrate’’ state with high chanically unfold native substrates and translocate them into the associated ATPase activity and fast on/off kinetics of substrate binding. The short inter- proteolytic chamber of ClpP. Polypeptide translocation is impelled by pore domain linker transduces the allosteric signal between NBD-SBD and the Wednesday, February 11, 2015 503a b-SBD-a-lid interfaces. We used pulsed dipolar ESR and ion mobility mass dence using drastic interventions, our current work focuses on specific patho/ spectroscopy technologies to characterize the conformational ensembles of physiologically relevant posttranslational modifications of the microtubule DnaK in its allosteric states to better understand how the nucleotide and sub- cytoskeleton. We find that targeting the cleavage of a c-terminal tyrosine on strates modulate the allosteric landscape. We found that DnaK has a narrow alpha-tubulin (detyrosination) alters myocyte mechanical properties without distance distribution in ATP-bound state, but broader distance distributions disrupting overall microtubule structure. Inhibition of detyrosination signifi- in all other states exhibit multiple coexisting components. The ATPþsubstrate cantly alters contractility and blunts mechanical effects on ROS production ensemble reflects the tug-of-war between the forces of NBD-SBD interaction and calcium handling. We also find that detyrosinated microtubules are driven by the binding of nucleotides, and the force of b-SBD-a-lid interaction increased in Duchenne Muscular Dystrophy (DMD), and that inhibition of driven by the binding of substrates. The ATPþsubstrate state contains 24% of detyrosination alleviates dysfunctional and arrhythmias eli- docked and 76% of undocked conformers. The ADPþsubstrate state has a cited by increased mechanical work in a DMD model. Taken together these re- smaller fraction of docked conformers and an additional species, which we sults suggest a critical role for microtubule detyrosination in mechano-chemo may represent as a ‘‘domain rotamer’’ around the unbound linker. Rotation transduction and identify a potential therapeutic target for the treatment of of the NBD and SBD around the interdomain linker may play an important cardiomyopathy. role in the allosteric mechanism. The ATPþsubstrate state releases the SBD’s helical lid from the NBD bound in the ATP-bound state to an SBD 2541-Plat bound position and a ‘‘free’’ position; and the ADPþsubstrate state pushes Serca Located in the Junctional SR Shapes Calcium Release in Cardiac the equilibrium from the free position to the SBD bound position. The Myocytes 1 2 3 3 allosteric states can be modulated by mutations to dissect the energetic Terje R. Kolstad , Mathis K. Stokke , Espen Stang , Sverre H. Brorson , 1 1 contributions. Louch E. William , Ole M. Sejersted . 1Institute for Experimental Medical Research, Oslo University Hospital Ulleva˚l, University of Oslo, Oslo, Norway, 2Clinic for Internal Medicine, Lovisenberg Diakonale Hospital, Oslo, Norway, Oslo, Norway, 3Department Platform: Excitation-Contraction Coupling of Pathology, Oslo University Hospital, Rikshospitalet, Oslo, Norway. Calcium-induced calcium release is in principle an all-or-nothing mechanism 2539-Plat and self-propagation of Ca2þ waves is a pathological manifestation of this pos- Identification of a -1 Mutation in a Human Vacuolar itive feedback loop. However, since local release of Ca2þ only elicits elemen- Myopathy tary Ca2þ release events (Ca2þ sparks) that do not develop into Ca2þ waves, 1 1 1 1 Daniela Rossi , Bianca Vezzani , Valeria Del Re , Virginia Barone , Ca2þ release can be graded. This local control is possible because of the spatial 1 1 1 Simone Spinozzi , Alessandra Gamberucci , Stefania Lorenzini , arrangement of L-type Ca2þ channels and clusters of ryanodine receptors at the 2 2 3 1 Cecilia Paolini , Feliciano Protasi , Carlo Reggiani , Vincenzo Sorrentino , sites of Ca2þ release (dyads) (Stern 1992). We propose that the sarcoplasmic Lucia Galli4. 2þ 1 reticulum (SR) Ca -ATPase (SERCA) also contributes to local control by Department of Molecular and Developmental Medicine, University, Siena, 2þ 2 limiting diffusion of Ca . Six days following conditional SERCA knockout, Italy, Department of Neuroscience, Imaging, and Clinical Sciences, Ca2þ sparks exhibited broadened geometry and slowed kinetics (increase in University G.D’Annunzio, Chieti, Italy, 3Department of Biomedical 4 spark width, time to peak, and duration by 33%, 33%, and 51%, respectively). Sciences, University of Padova, Padova, Italy, U.O.C. Molecular Medicine, At this time point, SERCA protein levels are reduced by 53% and SR Ca2þ con- Azienda Ospedaliera Universitaria Senese, Siena, Italy. tent is decreased by 25% (Stokke et al. 2010). To determine the precise local- Calsequestrin-1 (CASQ1) is the major calcium binding protein of the sarco- ization of SERCA we employed cryo immuno-gold electron microscopy on plasmic reticulum (SR) of skeletal muscle cells. It is mainly localized in sections from the mouse papillary muscle. Normal cardiac myocytes showed the junctional domain of the SR where it is part of a quaternary complex, preferential SERCA expression near the Z-lines (nearly twofold higher labeling which includes the calcium release channel, junctin and 2þ density at the Z-line than the A-band) with SERCA molecules clearly located in triadin. Calsequestrin-1 can modulate Ca release by either directly bind the junctional SR in close proximity to the dyads. The conditional SERCA the ryanodine receptor and/or by binding to junctin and triadin. We recently knockout resulted in preferential loss of labeling at these locations. From identified a D244G mutation in CASQ1 in patients with a myopathy charac- resin-embedded cross-sections of papillary muscles, we observed more abun- terized by the presence of vacuoles containing aggregates of SR proteins. The dant SR in the I-band compared to the A-band (by factor of 1.7 and 1.6 for con- mutation affects a conserved aspartic acid located in one of the high-affinity 2þ trol and KO respectively). These results suggest that SERCA contributes to Ca binding sites of CASQ1. We found that muscle fibers from patients car- 2þ 2þ 2þ local control of Ca release by limiting diffusion of Ca from the dyad. rying the CASQ1 mutation show alterations in the Ca release kinetics, thus 2þ 2þ Thus, the processes of Ca release and re-uptake are closely linked by a pop- suggesting that the D244G mutation may alter the intracellular Ca signaling ulation of SERCA molecules in the junctional SR. in the affected fibers. Interestingly, mutations in the CASQ2 protein identified in patients affected by catecholaminergic polymorphic ventricular tachycardia 2542-Plat þ þ (CPVT) were shown to alter either Ca2 buffering or Ca2 release properties Large Amplitude Rate-Dependent Mechanical Alternans may Precede of cardiac muscle cells and to reduce the ability of calsequestrin to bind junc- Arrhythmogenesis in Human Heart Failure and are Linked to Electrical tin and triadin. In order to understand the cellular mechanisms responsible for Alternans via Abnormal Calcium Handling þ alterations of Ca2 release kinetics in skeletal muscle cells carrying the Melanie Zile, Natalia Trayanova. D244G mutation, interactions between mutated and wild type CASQ1 and Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. the ryanodine receptor type 1, junctin and triadin are being investigated. Microvolt T-wave alternans (MTWA) testing identifies patients at risk for lethal ventricular arrhythmias. However, stratification of low and high risk pa- 2540-Plat tients with MTWA is challenging due in part to poor signal-to-noise ratio Microtubule Detyrosination Modulates Stretch-Dependent X-ROS (SNR) of MTWA measurements. Since microscopic systolic pressure alternans Signaling in Heart (MSPA) has a higher SNR than MTWA, and is also associated with abnormal Patrick G. Robison1, Jaclyn P. Kerr2, Alexey I. Bogush1, Daniel A. Harki3, calcium handling, we hypothesized that rate-dependent MSPA also precedes Christopher W. Ward4, Benjamin L. Prosser1. arrhythmia and may be an alternative approach for arrhythmia risk stratification 1Physiology, University of Pennsylvania Perelman School of Medicine, in heart failure patients. To test this hypothesis, we investigated mechanical al- Philadelphia, PA, USA, 2Physiology, University of Maryland School of ternans, a surrogate for MSPA, and its proposed link to arrhythmogenesis via Medicine, Baltimore, MD, USA, 3Medicinal Chemistry, University of abnormal calcium handling. Electromechanical models of single human myo- Minnesota, Minneapolis, MN, USA, 4BioMET, University of Maryland cytes were constructed. Key features of remodeling were incorporated to simu- School of Nursing, Baltimore, MD, USA. late abnormal calcium handling in human heart failure. A dynamical pacing Mechano-chemo transduction, the conversion of mechanical signals into chem- protocol was used to investigate intracellular calcium concentration ([Ca]i), ical ones, is a topic of accelerating interest in striated muscle. Axial stretch of a voltage, and force for different pacing rates. In normal myocytes, [Ca]i, cardiac myocyte triggers an array of effects, including generation of reactive voltage, and force alternans were not found for pacing rates <200 bpm. In oxygen species and sensitization of the ryanodine receptor calcium release the presence of deranged calcium handling common in heart failure (sarco- complex through a process termed X-ROS signaling. These phenomena are plasmic calcium reuptake reduced below 74%), [Ca]i, AP phase II voltage, paralleled in skeletal muscle, and in both tissues are known to depend on the and force alternans developed at moderate pacing rates <120 bpm and microtubule cytoskeleton. While past work has shown a microtubule depen- increased in magnitude with increased pacing rate. For all pacing rates, the ratio 504a Wednesday, February 11, 2015 of force alternans to peak force was at least tenfold larger than the ratio of AP ryanodine receptors. Support: OTKA-NN107765, FWF P23479, W1101, phase II voltage alternans to maximum AP phase II voltage. In conclusion, TA´ MOP-4.2.4.A./2-11-1-2012-0001. electrical and mechanical alternans are both rate-dependent and linked via abnormal calcium handling, but mechanical alternans has the greatest ampli- 2545-Plat tude across all pacing rates. Thus, mechanical alternans, due to their greater Calcium Channel Dysfunction in a Mutant Mouse Model of Malignant SNR, may be better predictors for arrhythmogenic propensity in heart failure Hyperthermia(CaV1.1 R174W) 1 1 2 2 patients than electrical alternans. Donald Beqollari , Christin F. Romberg , Wei Feng , Jose R. Lopez , Manuela Lavorato3, Stefano Perni4, Philip M. Hopkins5, 2543-Plat Clara Franzini-Armstrong3, Isaac N. Pessah2, Paul D. Allen2, Kurt G. Beam4, D D Na /H Exchange Blockers Reveal the Existence of a Skeletal Muscle Roger A. Bannister1. Ca2D/HD Exchanger, which is Altered in Malignant Hyperthermia 1Medicine-Cardiology, University of Colorado Denver-AMC, Aurora, CO, Muscle Cells USA, 2Molecular Biosciences, University of California-Davis, Davis, CA, Gaelle Robin1, Francisco Altamirano1, Eric Esteve2, Isaac N. Pessah1, USA, 3Cell and Developmental Biology, University of Pennsylvania, Paul D. Allen1, Jose R. Lopez1. Philadelphia, PA, USA, 4Physiology and Biophysics, University of Colorado 1Molecular Biosciences, University of California at Davis, Davis, CA, USA, Denver-AMC, Aurora, CO, USA, 5Leeds Institute of Biomedical and Clinical 2Laboratoire HP2, INSERM U1042, Institut Jean Roget, BP170, Grenoble, Sciences, University of Leeds, Leeds, United Kingdom. France. Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder In resting skeletal muscle fibers intracellular pH (pHi) is kept constant at a rela- of skeletal muscle that is triggered by exposure to volatile anesthetics. MH tively alkaline level. The transporters involved in maintaining muscle pHi at has been studied extensively in mice and pigs carrying causative mutations rest are the Naþ/Hþ exchange system (NHE), and to a lesser extent the in the type 1 ryanodine receptor (RyR1). However, no in vivo information ex- Naþ- and Cl–dependent bicarbonate dependent transport systems. Many ists regarding how mutations in the skeletal muscle L-type Ca2þ channel studies conducted in nerve and smooth cells have suggested a link between (CaV1.1) precipitate MH crises. For this reason, we generated a mouse line car- changes in intracellular free Ca2þ ([Ca2þ]i) and changes in intracellular pH rying the R174W mutation. Homozygous R174W mice ambulated efficiently, (pHi) suggesting a Ca2þ/Hþ exchange (Daugirdas et al, 1995). Malignant hy- reproduced and had normal lifespans. When exposed to isoflurane, homozy- perthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered by gous R174W mice entered a hypermetabolic state ending ultimately in death. inhalational anesthetics and depolarizing neuromuscular blocking agents. We On the ultrastructural level, R174W muscle displayed limited and variable have measured [Ca2þ]i and pHi simultaneously in Wt and heterozygous changes: some variability of the SR calsequestrin content, displacement of R163C myotubes using double barreled Ca2þ-selective and single barreled mitochondria in some soleus fibers of aged mice and occasional accumulation pHþ-selective microelectrodes. MH cells have a higher [Ca2þ]i and a lower of SR stacks. On the cellular level, homozygous R174W muscle had elevated pHi than Wt cells. Treatment of both MH and Wt cells with the NHE inhibitors resting myoplasmic Ca2þ levels that were greatly increased upon exposure to dimethylamiloride (DMA) or cariporide, caused an increase in [Ca2þ]i and a isoflurane. Flexor digitorum brevis (FDB) fibers dissociated from homozygous decrease in pHi in a dose dependent manner. These effects were more promi- R174W mice lacked L-type Ca2þ current even though intramembrane charge nent in MH than Wt myotubes. YM-244769, a high affinity reverse mode movements of were of similar magnitude and voltage-dependence to those re- NCX3 blocker, did not modify [Ca2þ]i overload or the drop in pHi elicited corded from wild-type fibers. Ca2þ released from the SR in response to depo- by DMA or cariporide in either cell type, suggesting that the change in larization was substantially reduced in homozygous R174W fibers suggesting a [Ca2þ]i was not the consequence to an activation of the reverse form of the depleted SR Ca2þ store. Lipid bilayer recordings showed that the Po of RyR1s exchanger. Gd3þ and dantrolene respectively were able to partially inhibit isolated from homozygous R174W mice was significantly increased at all cis or fully reverse the DMA or cariporide-mediated elevation of [Ca2þ]i and Ca2þ concentrations (Feng et al., this meeting). Taken together, our results sup- acidification in both types of cells. These results suggest the existence of a port a mechanism for MH susceptibility in which CaV1.1 R174W promotes SR Ca2þ/Hþ exchange in skeletal myotubes, which appears to be altered in MH Ca2þ leak without affecting EC coupling per se. This work was supported by muscle cells. grants from the NIH (AR055104 to KGB, AR052534 to PDA, KGB, PMH, CFA and INP) and MDA (MDA277475 to KGB). DB received a stipend 2544-Plat from 2T32AG000279-11. Calcium Sparklets in Intact Mammalian Skeletal Muscle Fibers Express- ing the Embryonic CaV1.1 Splice Variant 2546-Plat Beatrix Dienes1, Nasreen Sultana2, Janos Vincze1, Monika Sztretye1, Spatially Localized Disruptions of Voltage Activated Calcium Release in Peter Szentesi1, Bernhard E. Flucher2, Laszlo Csernoch1. Mtm1-Deficient Muscle Fibers 1University of Debrecen, Debrecen, Hungary, 2Medical University of Candice Kutchukian1, Karine Poulard2, Anna Buj-Bello2, Innsbruck, Innsbruck, Austria. Vincent Jacquemond1. The embryonic splice variant of the voltage-gated L-type calcium channel 1Centre for Molecular and Cellular Genetics and Physiology, UCB Lyon 1, (CaV1.1e) displays an altered voltage-dependence and gating kinetics as UMR CNRS 5534, Villeurbanne, France, 2Department of Research and compared to that expressed in adult skeletal muscle. Because the adult Development, Ge´ne´thon, INSERM, Evry, France. CaV1.1a only opens slowly at strong depolarizations, its contribution as a Mutations in the gene encoding the phosphoinositide phosphatase myotubu- source of calcium influx during action potentials is negligible. In contrast, cal- larin (Mtm1) are responsible for myotubular myopathy. We previously cium influx through the embryonic CaV1.1e substantially contributes to showed that muscle fibers from Mtm1-deficient mouse suffer from defective depolarization-induced calcium transients in fetal muscles and in cultured excitation-contraction coupling. Here we measured voltage-clamp activated myotubes. In a genetically modified mouse (CaV1.1aE29), which exclusively Ca2þ signals in fibers from the flexor digitorum brevis muscles of 4 week- expresses the embryonic CaV1.1e variant also in adult muscle the calcium old wild type (WT) and Mtm1-ko mice under line-scan confocal microscopy influx component is maintained throughout life. Utilizing this mouse model, using the dye rhod-2. Ca2þ release in the diseased fibers was deficient over the calcium release events - calcium sparklets - were recorded in enzymatically iso- full range of activation: fitting the voltage dependence of the peak rate of rise lated, intact adult skeletal muscle fibers from the m. flexor digitorum longus of the line-averaged rhod-2 F/F0 signals with a Boltzmann function gave mean using the fluorescent calcium probe fluo-8 and the fast confocal scanner (Zeis- values for maximum rate, midpoint voltage and slope of 0.29 5 0.02 and 1 sLive) in the x-y mode. While control animals did not display such events, 0.14 5 0.02 F/F0.ms , 8.8 5 1.5 and 0.95 5 2.3 mV, 7.0 5 0.4 and CaV1.1aE29 mice spontaneously generated sparklets with a frequency of 9.2 5 0.7 mV in WT (n¼22) and Mtm1-ko fibers (n¼15), respectively. 9.2*10-456*10-4 Hz/mm2 (19 fibers; mean5SEM). The role of external cal- Furthermore, the mean time to peak rate was significantly delayed by 5-10 cium as the trigger was tested by either removing calcium from the external ms in Mtm1-ko as compared to WT fibers. These global alterations were asso- solution or by the application of 5mM nizoldipine to block the calcium current ciated with severe spatial inhomogeneity of Ca2þ release in the diseased fibers through CaV1.1e. Both interventions resulted in a complete loss of the events. with rhod-2 transients yielding localized disruptions along the scanned line Identified sparklets (n¼311) were characterized by an average amplitude including reduced peak amplitude but also delayed or slower rate of onset, (aF/F0) of 0.28750.005, a full-width at half-maximum of 3.0550.05 mm, suggestive of specific alteration of the early peak component of the rate of and duration of 23554 ms, clearly different from the properties of calcium Ca2þ release. In fibers treated with wortmannin and LY294002, the properties sparks on saponin-permeabilized adult mammalian skeletal muscle fibers. of the line-averaged rhod-2 F/F0 signals were unchanged in WT fibers but the These findings indicate that the sustained expression of the CaV1.1e splice mean maximum rate of rise of the rhod-2 signal was enhanced by 60% in variant gives rise to spontaneous calcium entry events (sparklets) in adult mus- Mtm1-deficient fibers. Results show that Mtm1-deficiency provokes EC cle fibers and that their properties are distinct from calcium sparks arising from coupling failure through accumulation of spatially localized disruptions of Wednesday, February 11, 2015 505a

Ca2þ release. They also suggest that pharmacological inhibition of PtdIns-3- notion of a cryptic protein binding site uncovered by force does not only pro- kinase activity has the potency to alleviate defective EC coupling in the vide the molecular basis for the long-standing observation of shear-dependent diseased fibers. platelet binding during blood clotting, but it might also help to explain shear- induced VWF self-assembly. Platform: Mechanosensation 2549-Plat Structural Study of a Novel Partial Calcium-Free Linker and a Positively 2547-Plat Selected Variation in Protocadherin-15: Implications for Hearing and Cell The Primary Cilium is a Self-Adaptable, Integrating Nexus for Mechanical Adhesion Stimuli and Cell Signaling Robert E. Powers1, Rachelle Gaudet2, Marcos Sotomayor3. An M. Nguyen1, Yuan N. Young2, Christopher R. Jacobs1. 1Biophysics Graduate Program, Harvard University, Boston, MA, USA, 1Biomedical Engineering, Columbia University, New York, NY, USA, 2Department of Molecular and Cellular Biology, Harvard University, 2Mathematical Sciences, New Jersey Institute of Technology, Newark, Cambridge, MA, USA, 3Department of Chemistry and Biochemistry, The NJ, USA. Ohio State University, Columbus, OH, USA. Mechanosensation is critical for cells to maintain homeostasis and in devas- Protocadherin-15 (PCDH15) is a non-classical cadherin that interacts with tating diseases, including atherosclerosis, osteoporosis and cancer. Although Cadherin-23 (CDH23) to form a heterophilic filament called the ‘‘tip link’’. several cellular mechanosensing structures have been described, none have The tip link is essential for hearing as it conveys sound-induced forces to been shown to adapt to mechanical stimuli or be regulated in non-excitable the mechanosensitive ion channels responsible for the generation of electrical cells. Primary cilia are ubiquitous chemo-mechanical sensors and function signals involved in sound perception. CDH23 and PCDH15 are part of the as mechanosensors in several tissues, including kidney, liver, cartilage, larger family of cadherin proteins that typically adhere cells together in a bone, and the embryonic node, deflecting in response to mechanical stimuli. calcium-dependent manner and contain extracellular cadherin (EC) repeats. Several groups have shown cilia length adapts in response to mechanical stim- Recent research has identified an aspartate to alanine variation (D414A) uli while others have shown relatively small changes in length can affect cilia within the linker region between EC3 and EC4 of PCDH15 that is under pos- deflection and downstream load-induced changes in gene and protein expres- itive selection in East Asian populations. The EC3-4 linker region of PCDH15 sion. Collectively, this suggests cilia mechanosensitivity may be modulated. also lacks several conserved calcium-binding residues, placing it in a recently þ Here, we show that both mechanical and chemical mechanisms can alter identified class of cadherin linker regions that may be unable to bind Ca2 ciliary rigidity. We exposed mouse inner medullary collecting duct cells trans- ions at one or more of the canonical binding sites. To study the implications fected with a live-cell marker for primary cilia to flow. Cilium bending of the D414A variation and the noncanonical calcium-binding motifs, atomic behavior was captured with high-speed confocal microscopy and modeled resolution structures of the EC3-5D414 and the EC3-5A414 fragments of as a beam anchored by a torsional spring. We found exposure to flow stiffened PCDH15 were determined by x-ray crystallography. The structures revealed the cilium up to 4-fold (n¼12), deflecting less in response to subsequent ex- a novel partial calcium-free linker region between EC3 and EC4 that only þ posures to flow. We hypothesized that modifying tubulin may mimic this stiff- binds 2 Ca2 ions. Preliminary molecular dynamics simulations revealed ening. Post-translational modifications of tubulin, such as acetylation, have an increased flexibility for the EC3-4D414 linker region as compared to þ been shown to stiffen microtubules. Interestingly, acetylation can increase canonical cadherin linker regions that bind 3 Ca2 ions. Furthermore, the sim- with mechanical stimuli. Using a potent pharmaceutical agent and a siRNA ulations predict that the D414A variation may suppress this flexibility and knockdown to alter acetylation, we showed that through acetylation the cell rigidify the EC3-4 linker region, which could be advantageous given the can biochemically regulate ciliary stiffness up to 4-fold (n¼5/group). We role of PCDH15 in force transduction. The new structures represent a first further showed that this altered stiffness directly affects the sensitivity of step in determining the structural and dynamical properties of non- the cell to mechanical signals, resulting in a 2-fold change in gene expression canonical EC repeats of PCDH15 with positively selected variations that (n¼5/group). We demonstrated, for the first time, a potential mechanism are relevant for sensory perception. through which the cell can regulate its mechanosensing apparatus. 2550-Plat 2548-Plat Calcium Influx through TRPV1 Inhibits Piezo Channels via Phosphoinosi- Mechanosensitive Von Willebrand Factor Protein-Protein Interactions tide Depletion Regulate Hemostasis Istvan Borbiro, Doreen Badheka, Tibor Rohacs. Camilo A. Aponte Santamarı´a1, Volker Huck2, Sandra Posch3, Dept. of Pharm. and Phys., Rutgers NJMS, Newark, NJ, USA. Agnieszka K. Bronowska1, Sandra Gra¨ssle2, Maria A. Brehm4, Capsaicin, the activator of the noxious heat sensor TPRV1 is clinically used to Tobias Obser4, Reinhard Schneppenheim4, Peter Hinterdorfer3, relieve chronic pain. Desensitization of TRPV1 involves downstream PLCd Stefan W. Schneider2, Carsten Baldauf5, Frauke Gra¨ter1. activation, but the mechanism how capsaicin treatment can also alleviate me- 1Molecular Biomechanics, Heidelberg Institute for Theoretical Studies, chanical allodynia is unknown. Piezo2 encodes rapidly adapting mechanically Heidelberg, Germany, 2Experimental Dermatology, Medical Faculty activated currents in sensory neurons. Expression of these novel mechanosen- Mannheim, Heidelberg University, Mannheim, Mannheim, Germany, sitive ion channels in a subset of TPRV1 positive neurons suggests their role in 3Institute of Biophysics, Johannes Kepler University, Linz, Austria, pain related sensory mechanotransduction. 4Pediatric Hematology and Oncology, University Medical Center Our study shows that capsaicin application inhibited rapidly adapting mechan- Hamburg-Eppendorf, Hamburg, Germany, 5Theory, Fritz-Haber-Institut der ically activated currents in TRPV1 expressing sensory neurons. We also show Max-Planck-Gesellschaft, Berlin, Germany. that TRPV1 activation inhibited heterologously expressed Piezo1 and Piezo2 A recurring theme of mechanosensitive proteins is a cryptic binding site that ion channels in whole-cell patch clamp experiments. Inclusion of either phos- gets uncovered by an external force. Here, we test the role of such mechanism phatidylinositol 4,5-bisphosphate [PI(4,5)P2] or its precursor PI(4)P in the in the adhesion of platelets at sites of vascular injury, a key process mediated patch pipette alleviated this inhibitory effect. Activation of PLCb by stimu- by the von Willebrand factor (VWF). Our data from atomistic simulations, lating muscarinic receptors only marginally inhibited mechanically activated atomic force microscopy (AFM), and microfluidic experiments demonstrate Piezo1 currents. Experiments using phosphoinositide sensors revealed that acti- that the VWF A2 domain binds to the VWF A1 domain, such that it buries vation of PLCd by a robust calcium influx through TRPV1 severely depleted the platelet binding site located at A1. This implies inactivation of VWF PI(4,5)P2 and PI(4)P. On the other hand, muscarinic stimulation of PLCb for the binding of platelets by a direct protein-protein interaction between significantly decreased PI(4,5)P2 levels, but only induced a small decrease of the VWF A1 and A2 domains. During force-probe simulations and AFM ex- PI(4)P. This differential activation of PLC isoforms may explain the difference periments, a stretching force uncovered the platelet binding site, by dissoci- between the inhibitory effects of these PLC pathways. Targeted depletion of ating the A1-A2 complex. This process was accompanied with only a PI(4,5)P2 and PI(4)P using a chemically inducible lipid phosphatase replicated partial unfolding of the A2 domain causing minor exposure of its cleavage the inhibition of Piezo1 currents. Additionally, PI(4,5)P2 and PI(4)P applied to site. Our data thus suggest that activation for platelet binding and degradation excised inside-out patches inhibited the rundown of Piezo1 activity further by cleavage are coupled through the interaction of A1 with A2, and force emphasizing the significance of these phosphoinositides in Piezo channel guarantees that VWF gets ready for activation before cleavage. Microfluidic regulation. experiments with an A2-deletion VWF mutant corroborate the critical inacti- Here we demonstrate that the activity of Piezo channels require the presence of vation role of the A2 domain in vitro. Overall, inactivation of VWF by force- either PI(4,5)P2 or PI(4)P and severe depletion of both phosphoinositides by dependent inter-domain A1-A2 interactions answers the question of how TRPV1 activation limits channel activity. In conclusion our data may explain platelets are prevented to bind to VWF under equilibrium conditions. The how capsaicin alleviates mechanically induced pain. 506a Wednesday, February 11, 2015

2551-Plat phys. Biophys. Chem. 16: 455-478). This sensitivity is at least partly attributed Piezo1 Transduces Extracellular Matrix Mechanical Cues to Direct to the mechanisms associated with gating of hair cell mechanotransduction Human Neural Stem Cell Fate (MET) channels, where at rest approximately 50% of the channels are open, Medha M. Pathak1, Jamison L. Nourse2, Truc Tran1, Jennifer Hwe1, positioning the channels at the steepest part of their activation curve (Johnson Janahan Arulmoli3, Dai Trang T. Le1, Elena Bernardis4, Lisa A. Flanagan2, et al. 2011. Neuron 70, 1143-1154). Previously, a calcium-regulated adaptation Francesco Tombola1. process was thought to control the resting open probability of MET channels 1Department of Physiology & Biophysics, UC Irvine, Irvine, CA, USA, (Farris, Wells, and Ricci. 2006. J. Neurosci. 26: 12526-12536). However, 2Department of Neurology, UC Irvine, Irvine, CA, USA, 3Department of mammalian cochlear hair cell adaptation is not driven by calcium and low Biomedical Engineering, UC Irvine, Irvine, CA, USA, 4Department of external calcium effects on resting open probability occur via an uncharacter- Pediatrics, Section of Dermatology, Children’s Hospital of Philadelphia, ized extracellular mechanism (Peng, Effertz, and Ricci. 2013. Neuron 80: 960- Philadelphia, PA, USA. 972). Upon further characterization of the extracellular mechanism, we find Mechanical cues are powerful determinants of cell behavior. For instance, that large radii divalent ions act similarly to calcium intimating a nonspecific mechanical properties of the extracellular matrix direct neural stem cell fate, divalent ion mechanism. We show that hair cell depolarization also increases determining whether they differentiate along a neuronal or glial lineage. The mo- the resting open probability, and acts through a similar mechanism as the lecular mechanisms linking matrix mechanics to intracellular signaling pathways external calcium. Finally, we find that GsMTx4, a purported lipid-mediated that influence lineage choice remain unclear. Here we use a multi-disciplinary stretch-activated channel modifier, blocks both the voltage and the low external approach to uncover a new player in mechanosensitive lineage commitment. calcium effects on resting open probability with only a minor decrease in adap- We find that the stretch-activated ion channel, Piezo1, generates a tation. These results suggest a new, probably lipid-mediated, mechanism of cal- mechanically-induced ionic current in human neural stem/progenitor cells. In cium modulation of the hair cell MET channel independent of adaptation. the absence of externally applied mechanical force, Piezo1 activity is elicited Additionally, these results suggest that auditory mechanotransduction channel by cellular traction forces and manifests as spontaneous calcium transients may have similar mechanisms to other mechanically-sensitive ion channels. that vary with substrate stiffness. Piezo1 knockdown evoked nuclear exclusion This work was supported by RO1 DC0003896 to AJR, F32 DC010975 and K99 of the mechanoreactive transcriptional co-activator Yap, suggesting a down- DC013299 to AWP, and NIDCD Core Grant P30-44992. stream effector of channel activity. Suppression of Piezo1 activity by the phar- macological inhibitor GsMTx-4 or by siRNA-mediated knockdown reduced 2554-Plat neurogenesis and enhanced astrogenesis, demonstrating a role for Piezo1 in Probing the Structure and Function of TMC1 in Sensory Hair Cells using neural stem cell fate. Mutagenesis and Cysteine Modification We propose that the mechanically-gated ion channel Piezo1 is an important Xiao-Ping Liu1, Bifeng Pan1, Yukako Asai1, Kyoto Kurima2, Andrew J. Griffith2, Jeffrey R. Holt1. determinant of mechanosensitive lineage choice in neural stem cells and that 1 2 it may play similar roles in other multipotent stem cells. Boston Children’s Hospital, Boston, MA, USA, NIDCD/NIH, Bethesda, MD, USA. 2552-Plat Transmembrane channel-like genes 1 and 2 (Tmc1 and Tmc2) have recently Mechanosignalling of Focal Adhesion Kinase been implicated as components of the sensory transduction channel in mamma- Jing Zhou1, Camilo Aponte-Santamaria1, Agnieszka Bronowska1, lian hair cells. Mice deficient in both Tmc1 and Tmc2 have complete loss of Frauke Gra¨ter1,2. hearing and balance function and lack sensory transduction, despite intact 1HITS gGmbH, Heidelberg, Germany, 2Interdisciplinary Center for Scientific hair bundles and tip-links (Kawashima et al., 2011). Mice that express only Computing, Heidelberg, Germany. TMC1 or TMC2 have distinct single-channel conductances and calcium selec- Mechanosensing plays a prominent role at focal adhesions (FAs), sites at which tivity. A methionine-to-lysine substitution at position 412 in TMC1 reduces the external forces are integrated into cellular signaling pathways, thereby deciding single-channel current amplitude and calcium permeability of transduction upon vital processes such as cell proliferation and differentiation. While an (Pan et al., 2013). These results suggest that TMCs participate as essential com- increasing number of FAs molecules has been recognized as force sensors, a ponents of the sensory transduction channel in auditory and vestibular hair force-sensitive enzyme that can ultimately translate force into gene expression cells, but their exact role is not yet clear. They may form a vestibule at the levels, analogously to titin/twitchin kinases in muscle [1], has yet to be uncov- mouth of the pore, the pore of the ion channel itself or both (Holt et al., ered for FAs. 2014). Alternatively, TMCs may function as non-essential accessory subunits We present results from molecular dynamics (MD) and biochemical network sim- (Beurg et al., 2014). Progress toward understanding the structure and function ulations that suggest focal adhesion kinase (FAK) as a mechano-sensing enzyme of TMC proteins has been limited by the lack of homology to known genes or in FAs. An increased FAK activation in tensile-stress subjected cells has been domains (Kurima et al., 2003); the low quantity of native protein (50-100 func- observed previously, however, evidence of direct force-sensing by FAK, instead tional channels/cell); and poor membrane localization in heterologous cells. To of upstream molecules, is lacking. We here show that tensile forces, propagating circumvent these limitations, we combined electrophysiological recording with from the membrane through the PIP2 binding site of the FERM domain [2] and mutagenesis and cysteine modification in native hair cells. To probe TMC1 from the cytoskeleton-anchored FAT domain, activate FAK by relieving the structure and function, mutant Tmc1 sequences were introduced, via viral trans- occlusion of the central phosphorylation site of FAK (Tyr576/577) by the autoin- fection, into organotypic cultures harvested from Tmc1/Tmc2 doubly-deficient hibitory FERM domain [3]. Extensive control simulations with varying loading mice. We find that the mutant constructs restore sensitivity to hair bundle de- rates, pulling directions and membrane PIP2 concentrations further corroborated flections in virally-transfected hair cells and that acute application of cysteine the specific opening of the domain interface, due to its lower mechanical stability modification reagents alters the biophysical properties of sensory transduction than both the mostly helical domains themselves and the membrane-PIP2-FERM within seconds. The data further support a direct role for TMC1 in sensory link. We established a mechano-biochemical network model to connect force- transduction in mammalian hair cells. dependent FAK kinetics based on extrapolated MD results to the expected downstream RasGTP concentrations. Our computational study provides direct evidence for a mechanosignalling role of FAK, by broadcasting force signals Platform: DNA Replication and Transcription through Ras to the nucleus, with predictions directly testable by cell stretching ex- 2555-Plat periments and single molecule force spectroscopy. [1] Puchner, EM. et al, (2008) PNAS. 105(36):13385-90. Initiation of Asymmetric Rolling-Circle Plasmid Replication by RepD Studied using Magnetic Tweezers [2] Goni,} MG. et al. (2014). PNAS. 111, 3177-3186. Algirdas Toleikis, Simone Kunzelmann, Gregory I. Mashanov, [3] Lietha, D. et al, (2007) Cell. 129(6):1177-87. Martin R. Webb, Justin E. Molloy. 2553-Plat MRC NIMR, London, United Kingdom. Modulation of Rat Auditory Hair Cell Mechanotransduction Channel Bacterial plasmids, like the ones of T181 family, are replicated by an asym- Resting Open Probability Implicates a Role for the Lipid Bilayer metric rolling-circle mechanism. The replication is initiated by the protein, Anthony Peng1, Radhakrishnan Gnanasamdandam2, Frederick Sachs2, RepD, which binds to the origin of replication (oriD) on the plasmid and Anthony Ricci1,3. makes a single strand nick forming a covalent complex with the DNA 1Otolaryngology, Head & Neck Surgery, Stanford University, Stanford, CA, ‘‘plus-strand’’. This enables the helicase, PcrA and the DNA polymerase, USA, 2Physiology and Biophysics, SUNY Buffalo, Buffalo, NY, USA, Pol-III to bind the short length of exposed single-stranded DNA allowing 3Molecular and Cell Physiology, Stanford University, Stanford, CA, USA. replication of the ‘‘minus-strand’’ to commence. In the final stage of DNA The auditory system is the most sensitive mechanosensory system known, able replication, RepD terminates the process by religating the two ends of the to detect movement down to atomic dimensions (Bialek. 1987. Annu. Rev. Bio- plus-strand leaving behind the, newly formed, double-stranded daughter Wednesday, February 11, 2015 507a plasmid and the circular plus-strand, which is later replicated by a different Remodelling of the stalled RNAP transcription bubble occurs in a sequence mechanism. We used a custom-built magnetic tweezers device to observe of two mechanical steps with formation of a long-lived intermediate. In the in- initiation (nicking) by RepD. The initiation site, oriD, consists of three in- termediate state, mechanical signatures of protein-DNA interactions are insuf- verted complementary repeats that are predicted to form secondary DNA ficient to clearly identify which protein partners are interacting with the DNA, structures (hairpins). When the circular plasmid is damage-free DNA gyrase as they could involve RNAP, Mfd, or a combination of the two. We combine converts the relaxed circular DNA into a compact, negatively supercoiled, single-molecule nanomanipulation and single-molecule fluorescence in a form which favours extrusion of secondary structure motifs. Using magnetic TIRF field to monitor, in real-time, the arrival and departure of the different tweezers to artificially supercoil a length of DNA containing the oriD components of the reaction, via fluorescence, while we simultaneously record sequence, we found RepD nicking activity is highly sensitive to the degree the chemo-mechanical state of the protein-DNA complex, via nanomanipula- and sign of supercoiling. Positive supercoiling (over-winding) strongly in- tion. This allows us to correlate the catalytic state to the molecular composition hibits nicking whereas negative supercoiling stimulates nicking. We propose of this dynamic, multicomponent DNA repair complex. that DNA supercoiling acts as a gate-keeper to ensure plasmids are damage-free before DNA replication is initiated and the time required for 2559-Plat daughter plasmid supercoiling may regulate plasmid copy number. To corrob- Functional Implications of the RecQ Helicase - Topoisomerase III - SSB orate this idea we used magnetic tweezers to directly observe formation of Complex: Insights from Single Molecule Measurements DNA secondary structure in the absence of RepD and we have preliminary Maria Mills, Yeonee Seol, Keir Neuman. results showing how the structures change in the presence of RepD. Laboratory of Molecular Biophysics, NHLBI/NIH, Bethesda, MD, USA. RecQ Helicases are a highly conserved class of ATP-dependent DNA helicases 2556-Plat that perform multifunctional roles in genome maintenance. In E. coli, RecQ is Mechanistic Studies of DNA-Protein Interactions in Bacteriophage T4 known to physically and functionally interact with Topoisomerase III, a type IA DNA Replication Complexes at Single-Base Resolution topoisomerase. The coupling of helicase activity and topoisomerase activity Davis Jose, Lee Wonbae, Gillies P. John, Marcus H. Andrew, that results from this interaction is responsible for resolving complex DNA Peter H. von Hippel. structures such as double Holliday junctions. Similar interactions have been University of Oregon, Eugene, OR, USA. demonstrated in homologous proteins in other organisms, including the human Combining biophysical measurements on the function and control of T4 bacte- RecQ helicase BLM and TopoIIIa, and the yeast helicase SgsI and TopoIII. riophage replication complexes with detailed structural information can throw There is also evidence that single-stranded DNA binding protein (SSB) inter- light onto the mechanism of action of these ‘macromolecular machines’. In this acts with RecQ and is a necessary component of this complex in vivo. We study we use the low energy circular dichroism and fluorescent properties of sought to explore the mechanism by which RecQ stimulates TopoIII activity, site-specifically introduced fluorescent base analogues and single molecule and by which SSB stimulates RecQ activity, as well as the individual contribu- (sm) FRET experiments with cyanine dyes to monitor the binding and interac- tions of these proteins to topological changes in DNA using single molecule tions of gene 59 protein (gp59) with gene 41 protein (gp41). Gp59 is the experiments. To investigate the roles of RecQ, TopoIII, and SSB, individually helicase loader protein of the T4 DNA replication system, and gp41 is the hex- and in a ternary complex, we measured their effect on DNA hairpin unwinding americ helicase that drives duplex DNA unwinding. Using smFRET experi- and refolding using magnetic tweezers. We also conducted experiments on ments we are able to monitor the stoichiometry and dynamics of gp59 over- and under-wound double-stranded DNA to probe the roles of RecQ binding to different DNA constructs and our results show that gp59 binds as and SSB in activation of TopoIII relaxation of supercoiled DNA. This tech- a single subunit to the fork junction of a model DNA replication fork. CD nique allows us to probe changes in DNA topology as a consequence of enzyme and fluorescence experiments with base analogue probes show that the binding activity in real time. Our findings demonstrate a complex set of interactions and of gp59 to a forked DNA construct perturbs the bases at the junction and sug- provide a framework for understanding the mechanism of the resolvase activity gests that once the gp41 helicase hexamer has been loaded this conformational of the RecQ-TopoIII- SSB complex. perturbation extends deep into the double helix. In addition these experiments suggest that the stoichiometry of gp41 to gp59 subunits in the functional 2560-Plat helicase-loader complex is 6:1. Possible molecular mechanisms by which these Transcription Kinetics Heterogeneity of Highly Mobile Identical Genes proteins might work together in setting up various functional interactions with Revealed by Simultaneous Measurement at the Single Cell Level the other components of the DNA replication complex will be discussed. Enrico Gratton, Paolo Annibale. Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. 2557-Plat Fluorescence microscopy was used in combination with molecular constructs DNA Translocations in Real-Time: Insights into Non-Homologous End to measure in real time and in living cells the transcriptional activity of individ- Joining Pathway ual genes and their displacement within the nucleus. The question whether local Andrea Candelli1, Ineke Brouwer1, Gerrit Sitters1, Stephanie Heerema2, mobility of individual chromatin regions is correlated to their transcriptional Mauro Modesti3, Erwin J.G. Peterman1, Gijs J.L. Wuite1. output is still the subject of active research. There is a consensus that the local 1VU University Amsterdam, Amsterdam, Netherlands, 2Delft University of chromatin environment and physical accessibility may play a role in modu- Technology, Delft, Netherlands, 3Inserm, Marseille, France. lating transcription, possibly determining the large heterogeneity observed in DNA translocations are key factors in deregulating cell growth. Currently RNA Polymerase II (PolII) elongation rates measured from fluorescence as- known translocations are estimated to drive at least 20% of cancer cases. In says. Employing high-speed fluorescence nanoimaging we measure here at vitro single-molecule assays of DNA translocations are technically challenging ms-temporal resolution PolII elongation and we are able to resolve the minute since translocations occurs often at random sites of distinct DNA elements. displacements of identical active genes surrounding a labeled transgene array. Here we present a dual-DNA manipulation and fluorescence visualization assay We observe a correlation between the transcriptional activity and sub- using Correlative optical Tweezers-Fluorescence Microscopy (CTFM) where micrometer movements of the active genes that is evidence of an active molec- we reconstitute an in vitro model of a translocation between two gene-sized ular mechanism determining displacements of the active genes following DNA molecules by first inducing DNA break at controlled locations and sub- transcriptional bursts. Furthermore, we detect a significant heterogeneity in sequently observing, in real time, the repair of DNA catalyzed by two core the kinetics of identical genes measured simultaneously and at sub-second tem- elements of the human Non-Homologous End Joining (NHEJ) pathway: XLF poral resolution within the same cell. Together these observations rule out cell and XRCC4. We find that XLF and XRCC4 efficiently catalyze the formation to cell variability as the underlying cause for the observed kinetic heterogeneity of a bridge between broken DNA molecule having extraordinary strength and and point to the local chromatin environment and physical accessibility as the stability. source of PolII elongation variability. Work supported in part by NIH P41- GM103540 and NIH P50-GM076516 2558-Plat Correlative Nanomanipulation and Colocalization of Single-Molecules to 2561-Plat Study Transcription-Coupled DNA Repair Dynamics of GreB Interactions with RNA Polymerase: How a Regulatory Evan T. Graves, Camille Duboc, Jun Fan, Terence Strick. Protein may Patrol the Genome for Transcription Complexes to Rescue Institut Jacques Monod, Paris, France. Larry E. Tetone1, Larry J. Friedman1, Melisa L. Osborne1, Harini Ravi1, In transcription-coupled repair (TCR), E. coli RNA polymerase (RNAP) stalled Scotty Kyzer2, Rachel A. Mooney2, Robert Landick2, Jeff Gelles1. on a DNA lesion is removed by the Mfd tanslocase, which then recruits 1Biochemistry, Brandeis University, Waltham, MA, USA, 2Biochemistry, downstream repair components UvrA/UvrB. Mfd-RNAP interactions can be University of Wisconsin-Madison, Madison, WI, USA. observed in real-time using the magnetic trapping of single DNA molecules RNA synthesis in all organisms is regulated throughout transcript initiation and to monitor states of the transcription bubble created by RNA polymerase. elongation. The secondary channel (SC) of multi-subunit RNA polymerases 508a Wednesday, February 11, 2015

(RNAPs) allows access to the enzyme active site and is a nexus for regulation. -mediated actin filament nucleation mechanism where protein-protein In E. coli, multiple regulatory proteins bind in the SC and reprogram the cata- interactions trigger ‘nuclei’ instability necessary for filament elongation. lytic activity of RNAP, but how these factors function without interference and the dynamics of their interactions with RNAP are unclear. GreB is an SC pro- 2564-Plat tein that promotes endonucleolytic transcript cleavage in elongation complexes Actin Filament Nucleation is Influenced by Electrostatic Interactions with that are backtracked by nucleotide misincorporation. Here we used multi- the Bni1p Formin FH2 Domain 1 2 3 wavelength single-molecule fluorescence microscopy to characterize in vitro Joseph L. Baker , Naomi Courtemanche , Daniel L. Parton , Martin McCullagh4, Thomas D. Pollard5, Gregory A. Voth6. GreB effects on transcript elongation and the dynamics of GreB interactions 1 with elongation complexes. Contrary to previous dogma but consistent with Department of Chemistry, The College of New Jersey, Ewing, NJ, USA, 2Department of Molecular, Cellular and Developmental Biology, Yale structural data, we found that high GreB concentrations substantially slow tran- 3 script elongation. During steady-state elongation, GreB binds RNAP at near- University, New Haven, CT, USA, Memorial Sloan-Kettering Cancer < Center, New York, NY, USA, 4Department of Chemistry, Colorado State diffusion-limited rates, but remains bound for 1s, approaching the duration 5 of the nucleotide addition cycle and much shorter than needed to produce a University, Fort Collins, CO, USA, Department of Molecular, Cellular and Developmental Biol.; Mol. Biophys. and Biochem.; Cell Biol., Yale complete mRNA. To examine whether GreB specifically selects backtracked 6 complexes, we reconstituted static complexes stabilized in backtracked and University, New Haven, CT, USA, Department of Chemistry; Inst. for non-backtracked configurations. By verifying the functional state of each mo- Biophysical Dynamics; James Franck Inst.; Computation Inst., The lecular complex measured, we showed that both configurations bind GreB at University of Chicago, Chicago, IL, USA. rates similar to actively elongating complexes, and that backtracking does Formins catalyze the nucleation and growth of actin filaments. Here we study not kinetically stabilize bound GreB. Our results exclude models in which both the structure and interactions of actin with the FH2 domain of budding GreB is selectively recruited to backtracked complexes or is ejected from yeast formin Bni1p. We built an all-atom model of the formin dimer on an RNAP by catalytic turnover. Instead, they suggest that GreB binds rapidly Oda actin filament 7-mer and studied structural relaxation and interactions be- and randomly to all elongation complexes to patrol for those requiring nucleo- tween formin and actin by molecular dynamics simulations. These simulations lytic rescue, and that its short residence time serves to minimize inhibition of produced a refined model for the FH2 dimer associated with the barbed end of non-backtracked elongation complexes and to prevent interference with the the actin filament and revealed the presence of electrostatic interactions be- function of other SC factors. tween the formin knob and actin target-binding cleft. Mutations of two formin residues which contribute to these interactions (R1423N, K1467L or both) 2562-Plat reduced the interaction energies between the proteins, and in coarse-grained A Single Molecule Perspective of Elongation by RNA Polymerase I simulations the formin lost more inter-protein contacts with an actin dimer Suleyman Ucuncuoglu1, David A. Schneider2, David D. Dunlap3, than with an actin 7-mer. Biochemical experiments confirmed that there is a Laura Finzi1. strong influence of these mutations on Bni1p-mediated actin filament nucle- 1Physics, Emory University, Atlanta, GA, USA, 2Biochemistry and ation, but not elongation. This suggests that there are different interactions Molecular Genetics, University of Alabama at Birmingham, Birmingham, that contribute to these two functions of formins. AL, USA, 3Biology, Emory University, Atlanta, GA, USA. RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for 2565-Plat more than 60% of total transcription in a growing cell. Despite its central Three Dimensional Reconstruction of the Native Cardiac Thin Filament role in transcription and fundamental role in cell growth and proliferation, a Decorated with Myosin-Binding Protein C Fragment: Implications for detailed understanding of kinetics of transcription by Pol I is lacking. We pre- Cardiac Regulation sent the first direct characterization of Pol I transcription elongation at the sin- Samantha Harris1, Betty Virok2, Howard White2, Vitold E. Galkin2. 1 gle molecule level. Tethered particle microscopy shows that the pause-free rate Department of Cellular & Molecular Medicine, University of Arizona, 2 measured for Pol I, approximately 50 nt/s, is comparable to the in vivo rate Tucson, AZ, USA, Department of Physiological Sciences, Eastern Virginia estimated from the number of active genes, the cell division rate and the num- Medical School, Norfolk, VA, USA. ber of engaged polymerases determined from EM images. Furthermore, com- Mutations in the genes encoding myosin, the molecular motor that powers car- parison of elongation traces in the presence or absence of RNAse A & T1, diac muscle contraction, and its accessory protein, cardiac Myosin Binding reveals strong interactions between the nascent RNA and the DNA tether Protein-C (cMyBP-C), are the two most common causes of hypertrophic car- that might modulate transcription elongation in vivo. In the presence of RNAse, diomyopathy (HCM). While much is known regarding the structure and func- Pol I processivity is higher than in the absence of RNAse. Furthermore, when tion of myosin, relatively little is known regarding how cMyBP-C interacts the nascent transcript is not digested by RNAse, large tether length fluctuations with the myosin or the thin filament (TF) to affect cardiac contraction. are noticed which yield an apparently slower average elongation rate of approx- cMyBP-C is comprised of 11 Ig- or Fn- like folded domains numbered C0- imately 20 nt/s. C10 starting from the N-terminus. The N-terminal portion of cMyBP-C is composed of three actin binding Ig-domains (C0, C1 and C2), the PA linker be- tween domains C0 and C1 and the M linker between domains C1 and C2. We Platform: Actin Filaments and Microtubules have used cryo electron microscopy and image processing to classify the fila- ments as to whether they contain tropomyosin and either domains C0, C1 or all 2563-Plat three proteins in addition to actin. This approach reveals the position of the C0- Direct Monomer-By-Monomer Observation of Gelsolin-Mediated Actin C1 tandem Ig-domains on the surface of the native cardiac thin filaments (TF). Filament Nucleation Both the C0 and C1 domains bind to multiple sites on F-actin, but each domain Alvaro H. Crevenna, Maria Hoyer, Don C. Lamb. has its preferred site of binding on the TF when both Ig-domains are present. Chemistry and Biochemistry, LMU Munich, Munich, Germany. Despite a low amino acid identity (27%) between C0 and C1 both Ig- Actin nucleation is a fundamental biological process involved in several domains bind to F-actin in the TF through a conserved region. Our data also cellular events. Actin filament nucleation requires several hundred nano- demonstrate that only the C1 domain directly interacts with the tropomyosin, molars to micro-molar amounts of polymerizable actin monomers. This and that this interaction influences the mode of binding of the tandem Ig- requirement makes the impossible use of standard single molecule techniques. domain to F-actin. We propose a model of how C0 and C1 domains of Here, we use zero-mode waveguides to reduce the observation volume and cMyBP-C activate the TF and the role of the PA linker in the activation monitor, with single molecule resolution, the initial steps in actin filament for- mechanism. mation. We immobilized gelsolin, a capping and nucleating protein at the bot- tom of the waveguides and added up to several micro-molar amounts of fully 2566-Plat labeled actin monomers in polymerization conditions. We observe the first New Models for Regulation of Vinculin by Actin and Phospholipids steps during filament formation as step-wise increases in detected fluorescence Peter M. Thompson1, Hyunna T. Lee1, Laura Kim2, intensity. Mean reaction rates are then estimated for each oligomer size. Arrival Srinivas Ramachandran1,3, Arpit Tandon1, Raul Mendez-Giraldez1, time distribution of individual steps reveals the presence of kinetic intermedi- Gregory M. Alushin2, Nikolay V. Dokholyan1, Sharon L. Campbell1. ates. Furthermore, analysis of oligomer size distribution as a function of actin 1Biochemistry and Biophysics, University of North Carolina at Chapel Hill, concentration reveals transitions at around 600 and 900 nM where the system Chapel Hill, NC, USA, 2Laboratory of Macromolecular Interactions, dynamics change concomitant with the appearance of filament elongation. The National Heart Lung and Blood Institute, Bethesda, MD, USA, 3Fred change in dynamics at the transition is associated with an increase in dissocia- Hutchinson Cancer Research Center, Seattle, WA, USA. tion rates. Such transition is blocked by Latrunculin A, suggesting a functional Vinculin is an essential cytoskeletal protein that is a prominent component role for ATP hydrolysis and/or monomer flattening. Our results point towards a of focal adhesions and adherens junctions. It exists in an autoinhibited Wednesday, February 11, 2015 509a conformation that masks interactions of ligands with the head (Vh) and tail (Vt) events promote fluctuations in tip composition and structure, most notably domain. Upon activation, vinculin functions as a scaffold to regulate cellular in the number of protofilament curls and lateral tubulin-tubulin contacts at events resulting in cell migration, cell survival and embryogenesis. Vinculin the tip. We propose that microtubule aging is a property of the complex sto- null cells display tumorigenic properties and mutation or loss of vinculin is chastic system, representing fluctuating microtubule tip, to evolve slowly and associated with cardiac disease. The interaction between vinculin and actin asymptotically towards the steady-state levels of occupancy of these rare plays a pivotal role in linking transmembrane receptors to the cytoskeleton, configurations. which, in turn, is important for controlling cellular cell morphology, force transmission and motility. Binding of F-actin to Vt causes a conformational 2569-Plat change that induces formation of a cryptic dimer necessary for actin filament Intrinsically Disordered Map Tau Mediates both Short-Range Attraction bundling, however, the conformational change that occurs and dimer that is and Long-Range Repulsion between Microtubules 1 2 3 4 4 formed is unknown. It is also unclear how vinculin recognizes PIP2, inserts Peter J. Chung , M.C. Choi , Uri Raviv , Herb P. Miller , Les Wilson , 4 1 into membranes and is regulated by this interaction. We have now obtained a Stuart C. Feinstein , Cyrus R. Safinya . 1Physics and Materials, University of California, Santa Barbara, Santa sub-nanometer resolution reconstruction of the Vt/actin complex which sheds 2 light on actin-induced conformational changes necessary for vinculin dimeriza- Barbara, CA, USA, Bio and Brain Engineering, Korean Advanced Institute of Science and Technology, Daejeon, Korea, Republic of, 3Biochemistry and tion and actin filament bundling, and have integrated computational and exper- 4 imental approaches to generate and test models for the actin-induced vinculin Molecular Biology, The Hebrew University, Jerusalem, Israel, Molecular, dimer and vinculin/PIP2 membrane interaction and assess their significance in Cellular, and Developmental Biology, University of California, Santa vinculin function both in vitro and in cells. Barbara, Santa Barbara, CA, USA. Neurofibrilliary tangles, the hallmark intracellular symptom of Alzheimer’s 2567-Plat disease, are the aggregated form of hyperphosphorylated tau. To give insight Towards a Model of the Tau-Tubulin Complex on the diseased state, the complete understanding of the healthy, physiological Xiaohan Li1, Jacob Culver2, Elizabeth Rhoades3,4. function of tau is necessary but difficult due to the intrinsically disordered na- 1Department of Chemistry, Yale University, New Haven, CT, USA, 2Ball ture of the protein. All isoforms of tau are often found bound to microtubule State University, Muncie, IN, USA, 3Department of Molecular Biophysics surfaces, but the N-terminal tail (with isoform-dependent lengths) is thought and Biochemistry, Yale University, New Haven, CT, USA, 4Department of to project off the microtubule surface and interact with the N-terminal tail of Physics, Yale University, New Haven, CT, USA. tau proteins on other microtubule surfaces. The forces effected by tau-coated Tau is a microtubule-associated protein which functions to maintain microtu- microtubules in the physiological composition range were investigated using bule stability as well as promote microtubule polymerization in the axons of small-angle X-ray scattering (SAXS). Samples were osmotically stressed using neurons. Its self-association and deposition as neurofibrillary tangles is also depletants to replicate the packed axoplasmic environment in which tau and mi- one of the primary pathological features of Alzheimer’s disease and a broad crotubules are found. array of other neurodegenerative disorders. We previously showed that altered In going from no coverage of tau to high coverage (1:10 tau-to-tubulin molar interactions between tau and tubulin heterodimers are associated with impaired ratio, near the physiological limit) isoforms with longer N-terminal tails steri- microtubule polymerization and that the interaction between tau and soluble cally stabilized microtubules, preventing bundling up to z10,000 Pa (in com- tubulin may play an important role in both tau function and dysfunction. How- parison to microtubule bundling at z1,000 Pa in absence of tau). In striking ever, a detailed description of the tau-tubulin complex is lacking. This is in part contrast, coverage by tau isoforms with the shortest N-terminal tails did not due to the challenges associated with characterizing this complex, in particular change the bundling pressure (z1,000 Pa), even at high coverages (1:10 tau- the highly dynamic character of the intrinsically disordered tau as well its to-tubulin molar ratio). Surprisingly, in the high-pressure limit, the polyampho- ability to accelerate tubulin polymerization. Here, we studied the interaction lytic nature of tau brought about a coverage-dependent and irreversible between tau and tubulin heterodimers using fluorescence correlation spectros- electrostatic attraction between microtubules. The unique manifestation of copy (FCS) and acrylodan fluorescence which allow us to propose a detailed both short-range electrostatic attraction and long-range steric repulsion by model of the complex. Our results provide insight into differential roles of tau on microtubules gives insight to both the physiological function of tau the individual microtubule binding repeats in mediating the tau-tubulin interac- and the design of biologically-inspired materials with multiple interaction tion and further illuminate the mechanism by which tau promotes tubulin motifs. polymerization. 2570-Plat 2568-Plat Eb1: A Highly Dynamic and Diffusive Microtubule DTip-Tracking Multiple Reversible Molecular Events at the Microtubule Tip Drive the Protein Age-Dependent Microtubule Catastrophes Benjamin J. Lopez, Megan T. Valentine. Pavel Zakharov1, Nikita Gudimchuk2, Vladimir Voevodin3, Mechanical Engineering, University of California, Santa Barbara, Santa Alexander Tikhonravov3, Fazly I. Ataullakhanov2, Barbara, CA, USA. Ekaterina L. Grishchuk1. We use the nonhydrolyzable GTP analogs GMPCPP and GTPgS to polymerize 1University of Pennsylvania, Philadelphia, PA, USA, 2Center for theoretical microtubules that recapitulate the plus-end binding behavior of EB1 along the problems of physicochemical pharmacology, Moscow, Russian Federation, entire length of the microtubule. Through the use of single-molecule TIRF im- 3Moscow State University, Moscow, Russian Federation. aging we find that EB1 is highly dynamic (with a sub-second characteristic A growing microtubule can undergo a catastrophe, a sudden stochastic switch binding lifetime) and continuously diffusive while bound to the microtubule. to shortening. Catastrophe rate increases slowly during the first few minutes of We measure the diffusion coefficient through linear fitting to mean-squared microtubule elongation in vitro but then reaches a plateau, a phenomenon displacement of individually labeled proteins, and the binding lifetime by called microtubule ‘‘aging’’. Two different models have been proposed fitting a single exponential decay to the probability distribution of trajectory previously to explain aging: one based on gradual accumulation of permanent lifetimes. In agreement with measurements of other diffusive microtubule asso- wall defects and the other based on a progressive increase in tapering of ciating proteins, we find that the diffusion coefficient increases and the charac- the growing microtubule end. To examine these hypotheses we have teristic binding lifetime decreases with increasing ionic strength. We also find used molecular dynamics approaches to simulate tubulin-tubulin interactions that the diffusion coefficient is sensitive to the choice of GTP analog: EB1 pro- and the resulting evolution of the microtubule tip structure. Although the in teins bound to GTPgS polymerized microtubules have a diffusion coefficient silico microtubule does not accumulate any microtubule wall defects or half of that found with GMPCPP polymerized microtubules. To compare these show a monotonic increase of tip tapering, the model still exhibits realistic single-molecule measurements to the bulk binding behavior of EB1, we use microtubule aging and the plateau of catastrophe rate. It also provides good TIRF imaging to measure the intensity of microtubules coated with EB1- quantitative description of other key experimental findings, including the GFP as a function of EB1 concentration. We find that EB1 binding is cooper- effects of soluble tubulin concentration and sudden tubulin dilution. Soon ative and both the quantity of EB1 bound and the dissociation constant are after the initiation of tubulin assembly in this model, the average tip charac- sensitive to GTP analog and ionic concentration. The correlation between bind- teristics, such as the number of GTP tubulins and the extent of tip tapering, ing affinity and diffusion coefficient and the cooperative nature of EB1- reach steady states. The tip, however, continues to fluctuate quickly and microtubule binding leads to a decrease in diffusion coefficient with increasing reversibly among a large and varied range of configurations. Microtubule EB1 concentration. Interestingly, we also find an increase in binding lifetime at catastrophes result from rare coincidences of multiple short-lived molecular high EB1 concentrations, consistent with attractive EB1-microtubule interac- events, each of which does not precipitate a catastrophe on its own. These tions driving the cooperativity. 510a Wednesday, February 11, 2015

Posters L-PGDS was inhibited by PGJ2, a metabolite of PGD2 metabolite. Therefore, the structural information regarding the of L-PGDS/PGJ2 complex should pro- vide potential information for the drug development of drugs for treating sleep Protein Structure and Conformation IV disorders. In this study, we investigated the three-dimensional structure of re- combinant mouse L-PGDS complexed with PGJ2 by means of NMR and eval- 2571-Pos Board B1 uated the binding mechanism from the deduced molecular structure. Biochemical State of the Aryl Carrier Protein Directs Sequential Domain- The Chemical shift assignments for the of backbone and side-chain protons of Domain Interactions in the Yersiniabactin Synthetase System the L-PGDS/PGJ2 complex was made carried out using two-dimensional 1 15 Andrew C. Goodrich, Dominique P. Frueh. H- N HSQC, three-dimensional HNCACB, CBCA(CO)NH, HBHA(CO) Biophysics and Biophysical Chemistry, Johns Hopkins University School of NH, CCH-TOCSY, and HCCH-TOCSY. NOEs were collected from three- 13 Medicine, Baltimore, MD, USA. dimensional C-edited NOESY data. In addition, to investigate the binding Nonribosomal peptide synthetases (NRPSs) are modular enzymatic systems site of PGJ2, we compared the HSQC of L-PGDS/PGJ2 complex with that of responsible for the production of complex secondary metabolites in bacteria apo-L-PGDS. Significant changes in the chemical shifts of the catalytic and fungi. Each module is comprised of (at least) three core domains whose Cys65 and other residues which are locatedexist in the bottom area of the bind- combined action leads to the selection, activation, and incorporation of a single ing pocket of L-PGDS were observed upon PGJ2 binding, indicating that PGJ2 small molecule into a growing peptide. Central to each module is the carrier competitively binds to the substrate binding site. protein (CP), which is first primed via attachment of a 40-phosphopantetheine 2574-Pos Board B4 moiety (ppant arm) to a conserved serine to generate the active holo form. The Origin of CDR H3 Structural Diversity The ppant arm then covalently harbors activated monomers and growing pep- Brian D. Weitzner1, Roland L. Dunbrack2, Jeffrey J. Gray1. tides and shuttles them between the active sites of catalytic domains in both the 1Chemical & Biomolecular Engineering, Johns Hopkins University, same and adjacent modules. During CP priming and peptide elongation, a CP Baltimore, MD, USA, 2Institute for Cancer Research, Fox Chase Cancer thus exists in multiple different post-translational states and must interact with Center, Philadelphia, PA, USA. multiple catalytic domains. Understanding how NRPSs are able to efficiently Antibody CDR H3 loops are critical for adaptive immunological functions. orchestrate this series of sequential protein-protein interactions between a CP Although the other five CDR loops adopt predictable canonical structures, and its partner catalytic domains is key to understanding the molecular mech- H3 conformations have proven unclassifiable, other than an unusual C-terminal anism of NRP synthesis. In functionally analogous fatty acid synthases (FAS) ‘‘kink’’ present in most antibodies. To determine why the majority of H3 loops and polyketide synthases (PKS), the post-translational state of a CP (holo vs. are kinked and to learn whether non-antibody proteins have loop structures substrate loaded) has been implicated in directing the sequence of interactions similar to H3, we searched a set of 15,679 high-quality non-antibody structures in these systems. However, the role these modifications play in modulating for regions geometrically similar to the residues immediately surrounding the protein-protein interactions in a NRPS has not previously been explored. loop. By incorporating the kink into our search, we identified 1,030 H3-like Here, we provide evidence that the biochemical state of a CP (apo vs. holo loops from 632 protein families. Some protein families, including PDZ do- vs. monomer loaded) alters the affinity of the CP for its partner catalytic do- mains, appear to use the identified region for recognition and binding. Our re- mains in a NRPS system. Our results demonstrate that each partner catalytic sults suggest the kink is conserved in the immunoglobulin heavy chain fold domain preferentially binds to a different biochemical state of the CP and sug- because it disrupts the b-strand pairing at the base of the loop. Thus, the gests a means by which directionality in protein-protein interactions is achieved kink is a critical driver of the observed structural diversity in CDR H3. in NRPSs. 2575-Pos Board B5 2572-Pos Board B2 Biophysical, Biochemical and Functional Studies of a Novel Fungal Tec1 Systematic Perturbation of Protein:Protein Interfaces may Aid in Paralog Functional Classification Lenka Slachtova1, Matthew Lohse2, Sandy Johnson2, Cameron J. Jones, Ambreen Qureshi, Sanjana Sudarshan, Brian Beck. Sudha Veeraraghavan1. Biology, Texas Woman’s University, Denton, TX, USA. 1Dept. of Pharmaceutical Sciences, University of Maryland, School of Protein:protein interactions play vital roles in many biological reactions. In a Pharmacy, Baltimore, MD, USA, 2Departments of Microbiology, previous study we constructed a database of protein:protein interfaces (FLIPdb) Immunology, Biochemistry and Biophysics, University of California, San and have shown that calculations of the computational alanine scanning (CAS) Francisco, CA, USA. energy of residues along the interface can distinguish functional categories of Tec1 is a member of TEA/ATTS transcription factors family and a key regu- proteins. lator of cell development and adhesion in fungi. In Candida albicans, Tec1 Here, to further understand the underlying principles of protein interactions, we is required for yeast to filamentation switch and virulence. It also plays a examine the effects of systematically translating one interfacial subunit over a role in biofilm formation. Here, we report the identification of a novel Tec1 pa- grid in relation to the other stationary subunit for the structures in FLIPdb. A ralog, Tec2, which has been implicated in white/opaque switching. First, using three-dimensional potential energy surface was generated from the change in the NMR and X-ray crystallographic structures of the DNA-binding TEA spatial coordinates and energy of alanine substitution that accompanies each domain, solved in the Veeraraghavan laboratory, we constructed three- shift in conformation. Specific characteristics of the potential energy surfaces, dimensional structural models of the Tec1 and Tec2 proteins to gain insights including the volume within the surface, maximum depth, width at half depth, into their activities. We then investigated the DNA binding activity of Tec2 and energy per residue were found to discriminate between different functional and compare it with that of Tec1. Finally, we report on the potential biological classes of protein interfaces with an accuracy of approximately seventy-six role for Tec2, determined using knockout and overexpression studies in C. al- percent. These results may suggest that Functionally-Linked Interfaces of Pro- bicans. We are currently investigating the three-dimensional structure of Tec2. teins (FLIPs) are more sensitive to perturbation and are thus more specific than Functionally uncorrelated Contacts (FunCs). 2576-Pos Board B6 Structure and Molecular Dynamics of the Ig58/58 Domains of Obscurin 2573-Pos Board B3 Tracy A. Caldwell, Nathan T. Wright, Rachel A. Policke, Isaiah Sumner, Structural Analysis of Lipocalin-Type Prostaglandin D Synthase Christopher E. Berndsen. Complexed with Prostaglandin J2 Chemistry and Biochemistry, James Madison University, Harrisonburg, Yuta Nakahata1, Shigeru Shimamoto1, Tadayasu Ohkubo2, VA, USA. Kosuke Aritake3, Yoshihiro Urade3, Yuji Hidaka1. Obscurin (800-900 kDa) is a giant muscle protein vital to muscle cell mainte- 1Science and Engineering, Kinki University, Higashi-osaka, Japan, nance and organization. It is the only known connection between the contractile 2Pharmaceutical Science, Osaka University, Suita, Japan, 3International apparatus and the sarcoplasmic reticulum and also binds to specific cytoskel- Institute For Integrative Sleep Medicine, University of Tsukuba, Tsukuba, etal, signaling, or membrane-associated proteins. Obscurin domains Ig58/59 Japan. bind to titin ZIg9/10, which is hypothesized to stabilize the sarcomeric cyto- Lipocalin-type prostaglandin (PG) D synthase (L-PGDS), is a member of the skeleton. Mutations in this obscurin region lead to malformed muscle architec- lipocalin superfamily, is found and exists in the brain. L-PGDS catalyzes the ture and, eventually, to hypertrophic cardiomyopathy (HCM). For obscurin/ isomerization of PGH2, a common precursor molecule of various prostanoids, titin binding to occur, all four of these domains must be present. In order to to produce PGD2, a potent endogenous somnogen. L-PGDS-produced PGD2 is fully characterize this physiologically important region of obscurin, and by sleep-inducing and to accumulates in the brain during prolonged periods of extension determine the molecular factors that drive HCM, here we present wakefulness. In addition, We have recently found that the enzyme activity of the Ig58 and Ig59 structure using both X-ray crystallography and heteronuclear Wednesday, February 11, 2015 511a multidimensional NMR spectra. Low-resolution structure and dynamics of 2580-Pos Board B10 these tandem domains, along with how these domains react to physical stress, Single Molecule Fo¨rster Resonance Energy Transfer Studies of the Effect are also discussed. of Deglycosylation on the Structure of Immunoglobulin G Cathrine A. Southern, Mark S. Piraino, Jihad Aburas, Alan J. Mlotkowski. 2577-Pos Board B7 Chemistry, DePaul University, Chicago, IL, USA. Structural Studies of Obscurin Ig2 The deglycosylation of immunoglobulin G (IgG) antibodies with the bacterial Matthew C. Oehler, Nathan T. Wright, Christopher E. Berndsen. enzyme EndoS has been suggested as a potential treatment for some autoim- Chemistry and Biochemistry, James Madison University, Harrisonburg, mune disorders as this process leads to a diminished immune response. The VA, USA. reduction in immune response is thought to arise from weakened binding of Obscurin (720-900 kD) is a giant sarcomeric signaling protein that plays a effector molecules to the fragment crystallizable (Fc) region of IgG antibodies crucial role in the arrangement of the basic contractile unit of muscle. Muta- as a result of a conformational change in the Fc region. The nature of this struc- tions to Obscurin and to Obscurin binding partners have been linked to human tural alteration is uncertain due to conflicting results obtained from x-ray crys- muscle diseases such as hypertrophic cardiomyopathies and muscular dystro- tallography and small-angle x-ray scattering studies. To further examine the phy. These diseases likely occur due to the rescindment of specific molecular impact of deglycosylation on the structure of the Fc region, we have examined interactions necessary for suitable function. The modular arrangement of the both glycosylated and EndoS deglycosylated IgG antibodies using single mole- independently folding domains of Obscurin allows for select analysis of each cule Fo¨rster Resonance Energy Transfer (smFRET). The FRET efficiency his- of these independent binding events. Here, we present the high-resolution crys- tograms obtained from studying freely-diffusing, dye-labeled antibodies tal structure of the Obscurin Ig2 domain. This region binds to the extreme C- suggest that the flexibility of the Fc region increases upon deglycosylation. terminus of MBPC-slow variant, although how it does this is unknown. This structure is a canonical Ig-like fold, consisting of two beta sheets coming 2581-Pos Board B11 together to form a beta sandwich. Probing Structural Implications of Unnatural Amino Acid Incorporation into Green Fluorescent Protein 2578-Pos Board B8 Nicole Maurici, Andrew Dippel, Melanie Liskov, Scott Brewer, The Unusual Heme Coordination of THB1, a Hemoglobin from Chlamydo- Christine Phillips-Piro. monas Reinhardtii Chemistry, Franklin & Marshall College, Lancaster, PA, USA. Selena L. Rice1, Matt R. Preimesberger1, Jamie L. Schlessman2, The ability to study local protein structure and dynamics has been greatly Lauren E. Boucher3, Jurgen Bosch3, Juliette T.J. Lecomte1. enhanced by the genetic incorporation of unnatural amino acids (UAAs) that 1Johns Hopkins University, Baltimore, MD, USA, 2U.S. Naval Academy, 3 contain spectroscopic reporters. An important characteristic of an effective Annapolis, MD, USA, Johns Hopkins University Bloomberg School of spectroscopic reporter UAA is the ability to probe local protein environments Public Health, Baltimore, MD, USA. in a relatively non-invasive manner. Here we have investigated the structural THB1 is a ~15 kDa ‘‘truncated’’ hemoglobin (Hb) found in the cytoplasm of the consequences of the genetic, site-specific incorporation of the spectroscopic re- unicellular green alga Chlamydomonas reinhardtii. As for other Hbs within porter UAA 4-cyano-L-phenylalanine (pCNPhe) into distinct sites in super- photosynthetic microbes, THB1 is thought to be involved in the management folder green fluorescent protein (sfGFP) by X-ray crystallography. This UAA of reactive oxygen/nitrogen species. To complement physiological studies was selected since it can serve as both a vibrational and fluorescent reporter aimed at determining the function of THB1, we are pursuing the characteriza- of local protein structure and dynamics. X-ray crystal structures of sfGFP con- tion of the recombinant protein (rTHB1) [1] with special attention to the heme structs containing pCNPhe will be presented and the structural impacts of the environment. At pH ~ 5, ferric rTHB1 has an optical spectrum characteristic of incorporation of this UAA into sfGFP will be discussed. a six-coordinate, high-spin complex with a histidine and a water molecule as axial ligands to the heme iron. When the pH is raised near neutrality, the protein 2582-Pos Board B12 undergoes a transition (apparent pKa of 6.5) to a six-coordinate, low-spin com- Engineering the Cysteine Motif ‘CXXC’ into a Protein Imparts It Novel plex. Mutagenesis and NMR data collected in the diamagnetic ferrous state pro- Properties vide direct evidence for the ligation of a lysine (K53) on the distal side of the Likhesh Sharma. heme [1]. Here, we continue the study of this unusual iron coordination scheme. Molecular Biophysics Unit, Indian Institute of Science, Banaglore, India. The distal lysine, like the distal histidine of cyanobacterial globins (GlbNs), fa- We introduced the widely occurring cysteine motif CXXC into the maltose cilitates electron transfer. However, unlike the histidine of GlbN, the distal binding protein (one-at-a-time in five alpha-helices, at the N-termini) to test lysine of THB1 does not protect ferric heme from oxidative damage caused three hypotheses: 1) Does a disulphide bond form at the N-terminus? 2) by H2O2. Analysis of ferrous THB1 NMR data reveals a neutral lysine amino Does the protein acquire any oxido-reductase activity? 3) Does it acquire group with highly upfield-shifted 15N signal. Fast-exchange averaging of the new metal-binding properties? two amino protons is a further indication of a dynamic distal coordination. We confirmed: 1) Each cysteine pair forms a stable intrahelical disulphide bond Additional structural information was obtained from diffraction data collected under non-reducing conditions. 2) The five mutant proteins acquire consider- at beamline X25 (National Synchrotron Light Source, Brookhaven National able oxidoreductase activity, tested by the insulin aggregation assay. 3) The þ þ þ laboratory). Crystals of ferric rTHB1 grown at pH 9.5 diffracted to 1.9 A˚ res- mutants acquire novel metal-binding properties for Ni2 ,Cd2 , and Zn2 olution (P6122 space group) and the data were used to inspect the structural fac- upon reduction. We observed that introducing the CXXC motif neither desta- tors allowing lysine coordination. bilizes the protein nor affects its global structure. [1] Johnson et al. (2014) Biochemistry 53:4573 Our results demonstrate that introduction of CXXC motifs can be used to probe Supported by NSF grant MCB-1330488 alpha-helix start sites and to introduce oxidoreductase and metal binding func- tionality into proteins. 2579-Pos Board B9 Characterizing Steric Limitations of the Heme Pocket in the Gas-Binding 2583-Pos Board B13 Tt H-Nox Protein using Site-Specific Incorporation of Unnatural Amino Deciphering the Glycosylation Code Acids Christopher Ellis1,2, Will Noid2. Lukasz T. Olenginski1, Christine M. Phillips-Piro2. 1University of Maryland, Baltimore, MD, USA, 2The Pennsylvania State 1Biochemistry and Molecular Biology, Franklin and Marshall College, University, University Park, PA, USA. Lancaster, PA, USA, 2Chemistry, Franklin and Marshall College, Lancaster, Asparagine-linked glycosylation, the cotranslational attachment of a carbohy- PA, USA. drate to an asparagine sidechain, dramatically impacts protein folding, stability, Heme Nitric Oxide and/or Oxygen (H-NOX) binding proteins are bacterial O2 and structure. However, the ‘‘glycosylation code’’ that relates these effects to and/or NO gas-sensing proteins involved in signaling a variety of functions to protein sequence remains unsolved. This work investigates the underlying the cell. Much work has been done to characterize the heme-binding pocket in mechanism of an experimentally observed asx- to beta-turn conformational Thermoanaerobacter tencongensis H-NOX (Tt H-NOX) using site-directed switch that is induced by the glycosylation of a short peptide using atomistic mutagenesis with the 20 naturally occurring amino acids. We aim to further molecular dynamics simulations. In order to distinguish between the effects characterize the heme-binding pocket of Tt H-NOX by incorporating unnatural of specific and nonspecific interactions with the carbohydrate, these simulations amino acids (UAAs) into the H-NOX scaffold, shedding light on both ligand considered model peptides that were N-linked to a disaccharide and also to a discrimination and the tuning of ligand affinity. Currently, we are working to steric crowder of the same shape. The simulations suggest that nonspecific ste- understand the steric limitations in this pocket by incorporating halogenated ric crowding by the N-linked disaccharide does not significantly alter the pep- phenylalanine residues and characterizing the spectroscopic, gas-binding, and tide free energy surface. However, the combination of steric crowding with structural properties of these proteins. specific interactions, e.g. hydrogen bonding and aromatic-glycan stacking, 512a Wednesday, February 11, 2015 dramatically impacts the peptide ensemble and stabilizes the conformational (SAXS) allowed us to unambiguously attribute apparent changes in radius of switch. Motivated by these results, sequence specific effects of N-linked glyco- gyration to changes in the structure of SOD. We find that SOD is highly sylation were investigated by additional simulations of peptides with the central compressible for changes in volume up to 9%. Resistance to deformation sequon Pro-Asn-Gly/Ala-Thr-Trp/Ala. The simulations suggest that sequences beyond 9% increased dramatically. with glycine adjacent to the glycosylation site readily form compact beta-turns upon glycosylation, while sequences with alanine in this location appear to be 2587-Pos Board B17 much less influenced by the glycan. Somewhat surprisingly, the simulations Conformational Dynamics and Functional Asymmetry of ABCE1, a Ribo- suggest that aromatic-glycan interactions are less significant for this conforma- some Recycling Protein tional switch. In order to corroborate these simulations, structural analysis of a Hadas Leonov, Bert L. de Groot, Helmut Grubmueller. dataset of glycoprotein structures was performed. This analysis indicates that Theoretical and Computational Biophysics, Max Planck Institute for the simulations of short glycopeptides are quite predictive of the conformations Biophysical Chemistry, Go¨ttingen, Germany. adopted by glycosylated sequences in the context of full length glycoproteins. Ribosome recycling is enabled by ABCE1, a twin-ATPase protein attached to an iron sulfur (FeS) cluster. ABC proteins typically adopt an ATP-bound closed 2584-Pos Board B14 state and an open state, however, for ABCE1, only the structure of an open or a Interactions between Thrombomodulin and Complement Component C3 semi open state are experimentally resolved. Furthermore, despite the structural Studied by Fluorescence Resonance Energy Transfer similarity of the two nucleotide binding domains (NBDs), they were reported to Gavin Palowitch, Caroline Gambone, Julia R. Koeppe. show functional asymmetry in mutations of the functional glutamates in both Chemistry, Ursinus College, Collegeville, PA, USA. NBDs. In our study we have modeled the ATP bound conformation relying Interactions between the lectin-like domain of thrombomodulin (TMD1) and on the similarity to other ABC proteins combined with essential dynamics sim- the complement system may provide a link between coagulation and inflamma- ulations. We then used thermodynamic cycles to explore interface mutations tion. Thrombomodulin is an integral membrane protein that is most studied for that shift the equilibrium towards a closed conformation in order to guide ex- its role as a down-regulator of blood clotting, but the lectin-like domain has periments and stabilize the protein in its closed state so that a better basis more recently been shown to interfere with complement proteins within the can be provided for its crystallization. In addition, we examine the question innate immune system. Of these complement proteins, specifically component of whether the asymmetric function of ABCE1 observed in mutations at paral- 3 (C3) has been shown to interact with TMD1. C3 is at the center of three lel positions in the NBDs might stem from the change in its open-closed different modes of activation of the complement system and may provide a equilibrium. good target for regulation of the system. By understanding the interactions be- tween TMD1 and C3, we would be able to target the causes of many inflamma- 2588-Pos Board B18 tory diseases. C3 has been isolated and purified from bovine blood plasma, and Moving Macromolecular Surfaces under Hydrophobic/Hydrophilic Stress TMD1 was expressed in Pichia pastoris and then purified. The stability of both David V. Svintradze. TMD1 and C3 was studied using urea-induced unfolding, revealing intrinsic Department of Physics, Tbilisi State University, Tbilisi, Georgia. tryptophan fluorescence. After labeling TMD1 with a fluorophore and C3 Hydrophobic and hydrophilic interactions can be described as dispersive inter- with a quencher, interactions were studied using Fluorescence Resonance En- actions throughout the molecules, interaction between permanent or induced ergy Transfer (FRET). The use of FRET will not only verify that the two pro- dipoles and ionic interactions. Hydrophobic effect is synonymous with disper- teins are interacting, but will also eventually allow for the calculation of sive interactivity and hydrophilic one is synonymous with polar interactivity. binding constants to measure the binding affinity of TMD1 and C3. Future Unification of all these interactions in one interaction is electromagnetic inter- studies will include using protein pull-down assays and hydrogen/deuterium action’s dependence on interacting body’s geometries. Hydrogen bonding is exchange coupled with mass spectrometry to further study the interaction. direct implication of such geometric dependence. Given the uniqueness of the problem, which is obvious for two surfaces, we only focus on two- 2585-Pos Board B15 dimensional surfaces embedded in the higher four-dimensional Minkowskian Structural Analysis of the Ectodomain of the Anti-Viral Protein BST-2 ambient space. Though, the analysis can be easily extended to hypersurfaces Kelly E. Du Pont, Aidan M. McKenzie, Samantha B. Chinn, of any dimension. Limitation by two surfaces, embedded in four space-time, Christopher E. Berndsen. which is necessary to describe electromagnetism, is consequence of specificity James Madison University, Harrisonburg, VA, USA. of processes that takes place on macromolecular surfaces. In the following pa- Human BST-2/tetherin is a host factor that inhibits release of HIV-1, HIV-2, per we discuss equations for the dynamic of macromolecular surfaces under the and SIV from the cell surface. Viruses can evade this inhibition through antag- influence of potential energy consisting from four-potential time four-current onistic viral protein interactions with BST-2. Structurally, full-length BST-2 and contraction of electromagnetic tensor. The macromolecular surfaces are consists of an N-terminal cytoplasmic domain, a transmembrane domain, an ec- modeled as a two-dimensional surface with a variable surface mass density. Ki- todomain, and a C-terminal membrane anchor. The N-terminal side of the ec- netic energy is calculated according to calculus of moving surfaces. Definition todomain contains three cysteine residues; each can contribute to the formation of Lagrangian by subtracting potential energy from kinetic energy and setting of cysteine-linked dimers. We explored the ectodomain of BST-2 to further un- minimum action principal are yielding nonlinear equations for moving surfaces derstand the flexibility of the protein as it relates to function. Recent cellular under hydrophobic-hydrophilic interactions. The equations can describe studies suggest BST-2 is flexible with regards to the dimerization and ability uniqueness and specific functionality of proteins and nucleic acids. Shape mini- to function properly. However, X-ray crystallography suggests the ectodomain mization problems as well as minimum surface problems are also discussed. is rigid. Through limited proteolysis, molecular dynamics and small-angle x- ray scattering, we showed that the ectodomain of BST-2 is flexible. However, 2589-Pos Board B19 the flexibility of the membrane bound BST-2 and the interaction between Dynamics of Gal80p in the Gal80p-Gal3p Complex Differ Significantly the HIV-1 viral antagonist protein, Vpu, is still unknown. To investigate the from the Dynamics in the Gal80p-Gal1p Complex: Implications for the flexibility and the interaction between BST-2 and Vpu, we are optimizing Higher Specificity of Gal3p Towards Transcriptional Induction of Gal conditions for purifying and crystallizing the full-length BST-2, Vpu and the Genes BST-2/Vpu complex. These studies will show how the innate immune system Sanjay K. Upadhyay, Jakob P. Ulmschneider. protein, BST2, interferes with viral budding. Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai, China. 2586-Pos Board B16 Expression of the GAL gene in Sacharomyces cerevisiae is regulated by three Macromolecular Crowding of a Protein Complex by Small Angle Neutron proteins; Gal3p/Gal1p, Gal80p and Gal4p. Both Gal3p and Gal1p act as tran- Scattering and Small Angle X-Ray Scattering scriptional inducers, though Gal3p has a higher activity than Gal1p. The differ- Ajith Rajapaksha1, Christopher B. Stanley2, Brian A. Todd1. ence in activity may depend on the strength of the interaction and dynamical 1Physics and Astronomy, Purdue University, West Lafayette, IN, USA, behavior of these proteins during complex formation with the repressor protein 2Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Gal80p. To address these queries we have modeled the binding interface of the Ridge, TN, USA. Gal1p-Gal80p and Gal3p-Gal80p complexes. The comparison of the dynamics Macromolecular crowding can alter the structure and function of biological of these proteins in the complex and in the Apo protein was carried out. It was molecules. We used small angle scattering to measure the change in size of observed that the binding of Gal3p with Gal80p induces significant flexibility in a protein complex induced by macromolecular crowding. Crowding of the Gal80p on a surface different from the one involved in binding with Gal3p. homodimer, superoxide dismutase (SOD) was induced using polyethylene gly- Several other differences at the interface between the Gal3p-Gal80p and the col - 400, triethylene glycol, methyl-a-glucoside and trimethylamine N-oxide. Gal1p-Gal80p complex were observed, which might permit Gal3p to act as a Parallel small angle neutron scattering (SANS) and small angle x-ray scattering transcriptional inducer with higher activity. Further, on the basis of our finding Wednesday, February 11, 2015 513a we have proposed the dynamical event and plausible mechanism of complex solved the structure of PfAtg8, we performed two screens against this protein: formation of Gal3p and Gal1p with Gal80p at the molecular level. a screen of the MMV Malaria box of compounds and a virtual library screen. We pursued hits from both screens and tested them via SPR against five pairs 2590-Pos Board B20 of Atg3 and Atg8 homologues: Plasmodium falciparum, Homo sapiens, Cryp- Death Effector Domain Flexibility in Mediating Protein-Protein Inter- tosporidium parvum, Eimeria tenella, and Neospora caninum. We have now actions also obtained protein crystals for the Atg8 homologues in E. tenella, C. parvum, Yufeng Wei. and N. caninum, which will facilitate future virtual library screens against these Institute of NeuroImmune Pharmacology, Seton Hall University, South specific proteins. Orange, NJ, USA. PED/PEA-15 (phosphoprotein enriched in diabetes/astrocytes, 15 kD) is a 2593-Pos Board B23 small, non-catalytic, death-effector domain (DED) containing protein, that is Probing Conformational Change in the First Actin-Binding Domain of widely expressed in different tissues and highly conserved among mammals. Dystrophin PED/PEA-15 has been found to interact with several protein targets in various Michael E. Fealey, David D. Thomas. pathways, including FADD and procaspase-8 (apoptosis), ERK1/2 (cell cycle Biochemistry, Molecular Biology & Biophysics, University of Minnesota, entry), and PLD1/2 (diabetes). We have previously reported a surprising Minneapolis, MN, USA. conformational change of PED/PEA-15 DED upon interaction with ERK2 us- We have used time-resolved EPR and fluorescence to resolve structural transi- ing NMR dynamics and residual dipolar coupling (RDC) data. In the complex, tions of dystrophin upon actin binding. Dystrophin (Dys) is a muscle cytoskel- PED/PEA-15 utilizes helices a1, a5, and a6 of the DED, in addition to the C- etal protein that binds to filamentous actin (F-actin) and the dystroglycan terminal tail, to binding to ERK2, while helices a2, a3, and a4 are highly flex- complex in the sarcolemmal membrane. Dys acts to dissipate mechanical forces ible in the complex, and adopt a distinct relative orientation comparing to the generated during the contraction and relaxation of muscle thereby maintaining free-form conformation. We have additionally modeled the PED/PEA-15 con- sarcolemmal membrane integrity and protecting from tears. The protein-protein formations in the ERK2 complex using CS/RDC-Rosetta protocol. Based on interactions and allostery underlying this function of Dys have not been well our NMR model of PED/PEA-15 complex with ERK2, we propose that the studied in the context of conformational change and thermodynamics, partly conserved charge triad motif on DED surface, comprising of D19-R72- because acquisition of structural and thermodynamic detail on large and flex- D74L, which is located at a hinge position between these two dynamically ible proteins is difficult. Two techniques capable of measuring large-scale distinct segments, mediates the necessary conformational changes to accom- conformational changes are dipolar electron-electron resonance (DEER) and modate ERK2 binding. We believe that various conformations of the DED, time-resolved fluorescence resonance energy transfer (TR-FRET). Using a facilitated by large number of surface polar interactions, may attribute to its combination of DEER and TR-FRET, we placed a single label (nitroxide ability to interact with structurally and functionally diverse proteins. or fluorescent) in each CH domain Dys ABD1 and subsequently measured the interprobe distance to assess conformational change upon association 2591-Pos Board B21 with F-actin. To probe the allosteric network of Dys ABD1, we also subjected Inhibition of GPR18 through Docking of known Antagonists using a the protein to differential scanning calorimetry. Homology Model Jenn L. Reynolds. 2594-Pos Board B24 biochemistry, uncg, greensboro, NC, USA. The Interaction between L-PGDS and its Substrates or Products, as Deter- The Class A G-protein coupled receptor, GPR18, is a newly de-orphanized mined by Isothermal Titration Calorimetry and NMR cannabinoid receptor that binds the endogenous ligand, N-arachidonoyl glycine Shigeru Shimamoto1, Yutaro Fukuda1, Takahiro Maruno2, Yuji Kobayashi2, (NAGly). As part of a larger project to model the interaction of ligands with Tadayasu Ohkubo2, Kosuke Aritake3, Yoshihiro Urade3, Yuji Hidaka1. GPR18, we undertook the conformational analysis of NAGly. Because 1Kinki University, Higashiosaka, Japan, 2Osaka University, Osaka, Japan, NAGLY is an arachidonic acid derivative and arachidonic acid has been shown 3University of Tsukuba, Tsukuba, Japan. to be highly flexible, we began by investigating the conformational flexibility Lipocalin-type Prostaglandin D synthase (L-PGDS) catalyzes the isomeriza- of this endogenous ligand. To this end, the Conformational Memories (CM) tion of prostaglandin H2 (PGH2) to produce prostaglandin D2 (PGD2), which method was employed. This method combines Monte Carlo exploration of acts as a somnogen in the brain. This enzyme belongs to the lipocalin super- the dihedral angle and bond angle space with simulated annealing (MC/SA) family which consists of transporter proteins for lipophilic substances in the to determine the range of values that each dihedral angle and bond angle is extracellular space. Our previous studies suggested that L-PGDS is comprised capable of exploring in a broad temperature range (Whitnell J Comp Chem of a b-barrel structure with a hydrophobic pocket and the active thiol group of 2007). Similar to previously reported CM results for arachidonic acid (Bar- Cys65 is located in this pocket and faces the inside of the pocket. A number nett-Norris J Med Chem 1998), NAGly’s conformers could be divided into of studies of L-PGDS, as a drug target for treating sleep disorders, have been four groups: linear, U-shape, J-shape, and helical. These results will be used reported, in attempts to understand its catalytic mechanism, and several sub- to explore the binding site interactions of NAGly at GPR18. strate recognition models of L-PGDS have been proposed. However, details of the mechanism by which L-PDGS recognizes its substrates and products 2592-Pos Board B22 are obscure, since essential information, such as its binding affinity and stoi- Targeting Apicomplexan Atg8 for Rational Drug Design chiometry of the interactions between L-PGDS and its substrate and product 1 2 2 Alexia S. Miller , Adelaide U.P. Hain ,Ju¨rgen Bosch . remains unclear. To address this, the binding properties of the molecule were 1Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, 2 examined by isothermal titration calorimetry (ITC) and NMR experiments. Baltimore, MD, USA, Biochemistry and Molecular Biology, Johns Hopkins The results of the ITC measurements revealed that, not only the substrate School of Public Health, Baltimore, MD, USA. analog, but also the product bind to L-PGDS in a stoichiometry of 2 to 1 Apicomplexan pathogens present significant health and economic burdens and that L-PGDS possesses two binding sites (high and low affinity sites). globally. Species of Plasmodium, the causative agent of malaria, result in In addition, NMR titration and ITC experiments of L-PGDS mutants indi- more than half a million deaths annually, with the primary victims being chil- cated that the active Cys65 residue is located at the high affinity-binding dren under the age of five. Other organisms in this phylum present a threat to site and plays a critical role in the binding of the substrate and product to immunocompromised individuals (e.g., Toxoplasma gondii, Cryptosporidium L-PGDS. spp.) or to the productivity of the livestock industry (e.g., Eimeria spp., Neo- spora caninum). Autophagy is the process by which cells recycle intracellular 2595-Pos Board B25 material by encapsulating them in a double membrane bound vesicle, the auto- Investigating Interactions between the Lectin-Like Domain of Thrombo- phagosome, which then fuses with the lysosome. The autophagy pathway has modulin and Complement Component 3 been shown to be essential to parasite survival in both Plasmodium and Toxo- Daniel DeHelian, Thomas Holt, Nathan Fritzinger, Julia R. Koeppe. plasma. Autophagy-related protein 8 (Atg8), which is required for the forma- Chemistry, Ursinus College, Collegeville, PA, USA. tion of the autophagosomal membrane, is a ubiquitin-like protein whose Protein-protein interactions are vital to the proper functioning of numerous bio- conjugation pathway is conserved in Apicomplexa. Within Apicomplexans, logical systems. Thrombomodulin (TM) is a protein that is involved in the Atg8 has a conserved loop region not present in human homologues. In Plas- down-regulation of coagulation induced by the clotting protein thrombin. Com- modium falciparum, this loop was shown to be essential to the interaction be- plement component 3 (C3) is a vital component of the complement system, tween Atg8 and its E2 conjugating enzyme, Atg3, and may represent a viable which is involved in innate immunity against bacteria and viruses. However, pan-Apicomplexan drug target. We are pursuing this possibility using x-ray dysregulation of C3 can lead to the degradation of host cells. Evidence suggests crystallography, SPR, and virtual library screening to identify small compound that the lectin-like domain of TM (TMD1) may interact with active C3 (C3b) to drug leads which can then be chemically diversified and optimized. Having inactivate it, thus preventing host cell degradation. The research conducted 514a Wednesday, February 11, 2015 herein required the expression and purification of TMD1 in yeast cells and the surface of the PH domain. Simultaneous stimulation of PLC-b2byGbg and isolation and purification of C3 from bovine blood plasma. A protein pull-down Rac1 indicates that they bind non-competitively, thus excluding a large swathe assay was used to verify that the two proteins interact. To aid in specific immo- of PH domain surface as a potential Gbg binding site. Although several PH do- bilization of the TMD1, two lysine residues were converted to methionine by mains bind Gbg, the PH domain of GRK2 is the only one whose Gbg-binding site-directed mutagenesis via polymerase chain reaction leaving only one surface is known. In PLC-bs, this surface does not overlap the Rac-binding sur- free amine available for future reactions. The wild type and mutant TMD1 pro- face, but it is buried against the adjacent EF hand domain, suggesting that Gbg teins were characterized by mass spectrometry and urea-induced unfolding. binding to PLC-b requires significant domain motion. We show that crosslink- The proteins were also both used in pull-down assays with C3. Once interac- ing the PH domain to the EF hand domain inhibits PLC-b activation by Gbg but tions are confirmed, hydrogen/deuterium exchange followed by matrix assisted not Gaq, supporting the hypothesis that Gbg binds to an occluded site on the PH laser desorption and ionization time of flight mass spectrometry (MALDI-TOF domain to directly regulate enzyme activity. PLC-bs accelerate hydrolysis of MS) will be performed to determine which regions of each protein are involved GTP bound to Gaq, and this GAP activity is inhibited by Gbg. PH-EF hand in binding. Additionally, interactions between the two proteins will be charac- crosslinking does not alter GAP inhibition by Gbg. This is consistent with terized using fluorescence resonance energy transfer (FRET) experiments. our suggestion that Gbg inhibits the GAP activity of PLC-b and other GAPs by binding Gaq-GTP, competitively, inhibiting GAP interaction with its G pro- 2596-Pos Board B26 tein substrate. Thus, Gbg employs distinct mechanisms to regulate different Probing Buffer-Specific Effects on Nucleotide Binding to RecA using PLC-b activities. Difference Ftir Joshua E. Temple, Gina MacDonald. 2599-Pos Board B29 Chemistry and Biochemistry, James Madison University, Harrisonburg, Localization of Small Ligands in a Metabolic Protein Complex using Cryo- VA, USA. Electron Microscopy The Escherichia coli protein RecA catalyzes the strand exchange reaction used Mario J. Borgnia1, Soojay Banerjee1, Prashant Rao1, Alberto Bartesaghi1, in DNA repair and genetic recombination. Previous studies in our lab have Allan Merk1, Jason Pierson2, Sriram Subramaniam1, Jacqueline Milne1. shown buffer-specific changes in RecA stability and unfolding transitions. 1Lab of Cell Biology, NCI/NIH, Bethesda, MD, USA, 2FEI Company, However, these studies suggest only minimal buffer dependent changes in Hillsboro, OR, USA. nucleotide binding and secondary structure that did not explain the large buffer Revolutionary advances in cryo-electron microscopy are rapidly driving the dependent differences in RecA stability and unfolding profiles. These observa- determination of structures of small protein complexes to near-atomic resolu- tions led to further investigations of how the four common biological buffers tion. Automation of image acquisition together with streamlined image pro- Tris, HEPES, MES, and PO4 alter RecA structure and nucleotide binding. cessing pipelines now offer the opportunity to localize the binding of ligands Here we have employed circular dichroism (CD) and infrared (IR) spectros- with low molecular weight to their target proteins using largely automated pro- copy to further discern if buffers influence nucleotide binding to RecA. CD cedures that could be useful for pharmaceutical drug development. To test the spectra of RecA were obtained in the presence and absence of ADP in each practicality of this approach, we used automated procedures and advances with buffer condition. Laser-induced photolysis of caged nucleotides was used in direct electron detectors to localize ADP (427 Da) and other small ligands on conjunction with difference IR to generate RecA-ADP minus RecA difference glutamate dehydrogenase, a clinically significant 365 kDa enzyme that is a rele- infrared spectra in each of the four buffers. These studies detected buffer- vant pharmaceutical target for cancer and diabetes. specific changes in nucleotide binding to RecA including possible perturbations Data from 385 images from a single specimen in a single session recorded on a in Gln, Glu, Asp, Asn, Tyr, and Lys residues and unique secondary structural Falcon II detector using EPU data collection software provided enough infor- transitions. These differences between RecA-ADP minus RecA difference mation to localize ADP in the ligand-protein complex at ~ 4.5 A˚ resolution. spectra will be discussed. The validity of the results was confirmed by comparison with the crystallo- 2597-Pos Board B27 graphic coordinates of the GDH-ADP complex. Using these methods we Revisiting Allostery in the Lac Repressor Protein were able to localize the binding site of other physiologically relevant ligands Matthew A. Stetz, Marie V. Carter, Kathleen G. Valentine, A. Joshua Wand. in different conformations of the enzyme. These methods thus provide a Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, streamlined path to localize nucleotides and other low molecular weight. PA, USA. The lac repressor protein (lac) is an allosterically regulated transcription factor which controls expression of the lac operon in bacteria. Binding of a small Protein Folds molecule inducer to a site 40A˚ away from the DNA-binding domain relieves repression through what is thought to be local unfolding of the hinge helix. 2600-Pos Board B30 Despite decades of characterization, our understanding of this allosteric transi- Urea Interactions with Native and Unfolded Proteins: A Volumetric Study tion remains incomplete_mostly inferred from partial crystal structures. In prin- Ikbae Son. ciple, high-resolution solution NMR could provide detailed structural and University of Toronto, Toronto, ON, Canada. dynamical information unobtainable by crystallography. However, due to We describe a statistical thermodynamics-based approach to analyzing urea- lac’s high molecular weight (70 kDa free, 85 kDa operator-bound), low solubi- dependent volumetric properties of proteins. The analysis produces the ther- lity, and transient stability, such studies have been limited to the non-allosteric, modynamic properties of elementary reactions in which a urea molecule isolated DNA-binding domain. We present, for the first time, a high-resolution interacts with a protein while also yielding an estimate of the effective solution NMR study of intact wild-type lac in all of its relevant functional solvent-accessible surface areas of the native and unfolded protein states. states, including the poorly characterized lac-inducer-operator ternary com- We carried out high precision measurements of the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease plex. Non-uniform sampling was employed extensively in order to obtain back- bone resonance assignments. Our initial results suggest that induction may not A, and a-chymotrypsinogen A at 25 C as a function of urea. The resulting proceed simply through unfolding of the hinge helix but rather involves a more volumetric data were analyzed within the framework of a statistical thermo- complex interplay between changes in structure and dynamics. Research sup- dynamic formalism. Lysozyme remains folded, while apocytochrome c is ported by NIH GM102447. unfolded between 0 and 8 M urea. In contrast, ribonuclease A and a-chymo- trypsinogen A exhibit urea-induced unfolding transitions. Thus, our data 2598-Pos Board B28 permit us to characterize urea-protein interactions in both the native and Activation of Phospholipase C-b by Gbg Involves Interactions with the unfolded states. We interpreted the urea-dependent volumetric properties of PH-EF Hand Domain Interface urea in terms of equilibrium constant, k, and changes in volume, DV0, and Ganesh Kadamur, Elliott M. Ross. compressibility, DKT0, for an elementary reaction of urea binding to protein UT Southwestern Medical Center, Dallas, TX, USA. with a concomitant release of two water molecules from its hydration PLC-bs signal downstream of Gq- and Gi-coupled GPCRs to synthesize two shell. Comparison of the values of k, DV0, and DKT0 with the similar data second messengers, IP3 and DAG, from PIP2.Gi regulates PLC-bs through obtained for small molecules mimicking protein groups reveals the lack of its Gbg subunit. The N-terminal PH domain of PLC-b appears to be the cooperative effects involved in urea-protein interactions. Urea-dependent primary Gbg sensor because its deletion abolishes stimulation by Gbg and volumetric data enable one to evaluate the extent of solvent exposure of because replacement of the PLC-d PH domain by the PLC-b PH domain con- protein groups in both the native and unfolded states. We emphasize that fers Gbg sensitivity. We find that the isolated PLC-b PH domain inhibits the volumetric approach offers a practical way for evaluating the effective PLC-b activation by Gbg, suggesting that the PH domain binds Gbg directly. solvent accessible surface area of biologically significant fully or partially PLC-b2 is also stimulated by Rac1, and the Rac1 binding site is on an exposed unfolded polypeptides. Wednesday, February 11, 2015 515a

2601-Pos Board B31 (delta4 and delta6) of an immunoglobulin fold - ddFLN, the F-actin cross- Prediction of Co-Translational Protein Folding in Living Cells linking gelation factor from Dictyostelium discoideum - are allowing us to Edward P. O’Brien, Daniel A. Nissley. present, the energy landscape that emerges from these co-translational folding Department of Chemistry, Penn State University, University Park, PA, USA. mimetics In my work I provide atomistic details to this energy landscape by Protein folding, the self-assembly of a protein molecule or domain into a ter- carrying out bias exchange metadynamics simulations with chemical shifts tiary structure, can occur as a protein molecule is being synthesized by the ribo- used as structural restrains. Using this approach I overcome, in large part, some in a process referred to as co-translational folding. The most convincing the two main limitations of molecular dynamics simulations in structural demonstration that co-translational folding occurs inside cells comes from studies of proteins, firstly the inaccuracies in the use of force field alone and pulse-chase experiments in which the synthesis of the cytosolic Semliki Forest secondly the limitations inherent in sampling of conformational space. virus protein (SFVP) was monitored in Chinese hamster ovarian cells [1]. My study presents a structural and dynamical characterization of the free en- SFVP is composed of four distinct protein segments, including an N-terminal ergy landscape of this C-terminal truncation as well as changes in the land- protease segment (referred to as ‘‘C protein’’) that auto-catalytically cleaves scape, while a protein is folding in the vectorial manner. Hence, it is itself from the SFVP molecule once folded. The pulse-chase experiment re- providing insights into a better understanding of co-translational folding, which vealed that the cleaved C protein appeared well before synthesis of the full- still represents a major open problem in molecular biology. length SFVP was completed, demonstrating that C protein does indeed fold co-translationally in vivo. Here, we show that the time course of such co- 2604-Pos Board B34 translational folding can be accurately predicted by a chemical kinetic model Rare Example of a Protein where an Isolated Domain is More Stable than using a domain’s bulk folding and unfolding rates and the average rate at which the Full-Length codons are translated in vivo; such quantities have been reported in the litera- Swati Bandi, Surinder Singh, Krishna Mallela. ture for a number of different proteins and cell types, suggesting this theoretical Department of Pharmaceutical Sciences, University of Colorado Anschutz approach can be applied to a wide variety of proteins. The model explains the Medical Campus, Aurora, CO, USA. essential features of co-translational folding time courses, and provides a means Studying the contribution of individual domains to protein structure and func- for predicting how varying the translation rate at different codon positions tion is of considerable interest over the years. Domains are in general less stable along a transcript’s open reading frame affects this self-assembly process in than the corresponding full-length proteins. Here, we report an exceptional case vivo. of utrophin tandem calponin-homology (CH) domain. Isolated C-terminal CH [1] Nicola, A. V., Chen, W. & Helenius, A. Co-translational folding of an al- domain (CH2) is both thermodynamically and kinetically more stable than the phavirus capsid protein in the cytosol of living cells. Nat. Cell Biol. 1, 341-5 full-length tandem CH domain. Reversible, equilibrium denaturant melts using (1999). both circular dichroism and protein fluorescence signals show that the CH2 is thermodynamically more stable by 4.0 kcal/mol when compared with the full- length tandem CH domain. Thermal melts indicate that CH2 unfolds at a higher 2602-Pos Board B32 Elucidating the Folding Pathways of Calcium-Binding Proteins temperature (15 C) than the full-length protein. Stopped-flow kinetics indicate Apurva Shah1, Daniel Goldman2, Lisa Alexander2, Carlos Bustamante2. that the CH2 unfolds slower (by 3 times) and folds faster (by 7 times) than the 1University of Maryland, Baltimore County, Baltimore, MD, USA, full-length protein, suggesting the higher kinetic stability of CH2. Analytical 2University of California, Berkeley, Berkeley, CA, USA. ultracentrifugation, size-exclusion chromatography, and dynamic light scat- During translation, ribosomes synthesize proteins according to the messenger tering show that both CH2 and the full-length protein are monomers in solution, RNA template. The polypeptide chain, specified by the template, acquires its confirming that the higher stability of CH2 is not due to formation of oligomers. three dimensional structure either co- or post-translationally in a process Thus, the utrophin tandem CH domain is a rare example in which an isolated termed ‘‘folding’’. Understanding folding mechanisms is important since pro- domain is more stable than the corresponding full-length protein. tein structure is critical for biological function, and misfolded proteins are 2605-Pos Board B35 correlated with cell stress and disease. Here, we map the folding pathways of Global Contacts Direct Hydophobic Collapse in Protein Folding two distantly related proteins: Calmodulin, a eukaryotic, calcium-dependent Loan K. Huynh1, Chris Neale2,Re´gis Pome`s1,3, Hue Sun Chan1. signaling protein, and Calerythrin, a prokaryotic, calcium-buffering protein. 1Biochemistry, University of Toronto, Toronto, ON, Canada, 2Department of These proteins have a highly conserved sequence and structure dictated by their Physics, Applied Physics, and Astronomy, Rensselaer Polytechnic Institute, similar calcium-binding function. Both proteins have two domains, and each Troy, NY, USA, 3Molecular Structure and Function, The Hospital for Sick domain is composed of two ‘‘EF-hand’’ motifs. To probe the folding of these Children, Toronto, ON, Canada. proteins, we utilize a focused laser beam to form an optical trap and exert me- Protein folding research aims to decipher how the amino acid sequence of a chanical force on the molecule, while measuring the molecule’s response to protein encodes its ordered or intrinsically disordered three-dimensional struc- force via its change in extension. These single-molecule experiments reveal ture. To address how proteins fold, how misfolding occurs, and how potential the folding dynamics at a level of detail not possible by traditional ensemble misfolding is avoided by natural proteins, we study the helical colicin immunity methods, since we can directly observe folding intermediates and off- protein 9 (Im9) as a model system. Im9 has been characterized extensively by pathway states. We find that domain proximity, in this case determined by biochemical and molecular simulation techniques. Here, we employ a system- the length of a bridging helix, impacts folding and unfolding cooperativity atic computational approach to estimate the free energies of native and non- even though the proteins share folding motifs. Studying these folding mecha- native arrangements of Im9 helices. We find that the two helical pairs that nisms allows us to obtain a better understanding of how domains communicate form the largest contact surfaces in the native state (helices 1 and 2, and 1 with each other and how tertiary contacts affect protein stability, while prompt- and 4) both associate in moderately non-native configurations in isolation. ing further studies to examine their structure-function relationship. Our results suggest that helix 3 can assist the native docking of helix 1, and that non-helical residues on either end of helix 4 can do so even more exten- 2603-Pos Board B33 sively. Our results also indicate that these non-native orientations of helix 1 The Computational Studies of Co-Translational Protein Folding can rapidly relax into the native state after the native assembly of other struc- 1 1 1 2 Tomasz Wlodarski , Chris Waudby , Chan Sammy , Michele Vendruscolo , tural components. Notably, the quantitative atomistic descriptions of transient John Christodoulou1. 1 2 non-native interactions derived from our studies point us beyond the simple University College London, London, United Kingdom, University of notion that pairs of nonpolar residues can always form strong contacts, and Cambridge, Cambridge, United Kingdom. emphasize the dependence of contact stability on the local and global environ- Protein synthesis in all kingdoms of life occurs on the ribosome, a macromo- mental context. lecular machine of >2.5 MDa. Most of the current knowledge about the crucial process of protein folding is based on in vitro investigation of isolated polypep- 2606-Pos Board B36 tide chains, which typically consider the refolding of full length proteins previ- Chemical Regulation of Disulfide Coupled Folding of Disulfide Rich ously denaturated by various chemical or thermal conditions. However, in vitro Peptide, Hepcidin, and its Precursor Protein folding is likely to differ from in vivo, as in the latter proteins start to fold while Yuji Hidaka, Kana Ohshige, Takeyoshi Nakanishi, Shigeru Shimamoto. they are still gradually emerging through the ribosomal exit tunnel. The avail- Kinki University, Higashi-Osaka, Japan. able conformational space to the nascent polypeptide chain is therefore Multi disulfide-containing-proteins are folded into the native conformations via different from that of the full-length protein. the assistance of redox molecules, such as glutathione and cysteine in vivo. Excellent system to study impact of protein vectorial synthesis on protein struc- Because of this, the glutathione redox system is also generally used in in vitro ture and dynamics are protein C-terminal truncations. Specifically, carried in folding studies [1]. However, in vitro disulfide-coupled peptide and protein our group NMR and computational investigations of C-terminal truncations folding are often problematic, since the folding intermediates are sometimes 516a Wednesday, February 11, 2015 prone to aggregation. In this study, to overcome this issue and chemically regu- R65S, AL-103 delP95aIns or kI O18/O8. Chondroitin sulfate A on the other late disulfide-coupled peptide and protein folding, a series of chemical reagents hand, shows a strong fibril formation inhibition for all proteins. was examined in the refolding of hepcidin and prohepcidin, as a model peptide We propose that Heparan sulfate facilitates the formation of transient amyloi- and protein, and their folding recoveries were estimated. dogenic conformations of AL light chains, hereby promoting amyloid forma- Hepcidin consists of 25 amino acid residues and four intra-molecular disulfide tion, whereas Chondroitin sulfate A kinetically traps partially unfolded bonds, which are absolutely required for its biological activity [2], not only for intermediates and further fibril elongation into fibrils is inhibited, resulting in iron homeostasis, but also for anti-microbial activity. To investigate structure- formation/accumulation of oligomeric/protofibrillar aggregates. function relationships, hepcidin was chemically synthesized. However, the yield of synthesized hepcidin was quite low under the typical folding condi- 2609-Pos Board B39 tions. The major problem in the disulfide-coupled folding of hepcidin is that Small Angle Neutron and X-Ray Scattering of Plasma Glycoprotein Inter- it undergoes aggregation during its folding reaction [3]. To solve this problem, actions with Lipid Membranes 1 2 3 4 several types of redox reagents and solvents were examined to improve the Luis A. Palacio , Christopher B. Stanley , Soenke Seifert , Ryan Lybarger , Horia I. Petrache4. folding efficiency of hepcidin [3]. However, all of the reagents resulted in quite 1 low yields for the disulfide-coupled folding of hepcidin. Therefore, to regulate Physics Department, Indiana University Purdue University Indianapolis, Indianapolis, IN, USA, 2Biology and Soft Matter Division, Oak Ridge the folding reaction of hepcidin and its precursor protein, we estimated the 3 folding conditions, such as pH values, salt concentrations, and a variety of National Laboratory, Oak Ridge, TN, USA, Chemical and Materials Science, X-ray Science Division, APS, Argonne National Laboratory, redox reagents. The results will be discussed in this paper. 4 [1] Chakravarthi, S.; Jessop, C.E.; Bulleid, N.J. EMBO reports 2006, 7, Lemont, IL, USA, Physics, Indiana University Purdue University 271-275. Indianapolis, Indianapolis, IN, USA. [2] Hocquellet, A.; le Senechal, C.; Garbay, B. Peptides 2012, 36, 303-307. Fibrinogen and Alpha 1-Antitrypsin (A1AT) are plasma glycoproteins with [3] Zhang, J.; Diamond, S.; Arvedson, T.; Sasu, BJ.; Miranda, LP. Biopolymers different, but specific functions. A1AT has been shown to have protective roles 2010, 94, 257-264. of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. Most known glycoproteins have been shown to play a role in 2607-Pos Board B37 cellular interactions but the exact role of the glycan chains is still under inves- Spectroscopic and SAXS Studies of Human Cystatin C Mutants - Early tigation. Previous electrophysiological measurements show that A1AT has a Stages of Amyloid Formation Process strong affinity to lipid bilayers, perturbing the function of ion channels present Maciej Kozak1, Zuzanna Pietralik1, Aneta Szymanska2, Micha1 Taube1. in the membrane. These observed protein-membrane and protein-protein inter- 1Macromolecular Physics, A. Mickiewicz University, Poznan, Poland, actions in solution were studied using contrast-matching small-angle neutron 2Gdansk University, Gdansk, Poland. scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light Human cystatin C (HCC) is a cysteine protease inhibitor. This protein in path- scattering (DLS). To establish a structural reference point for each protein in ological conditions, forms dimers via a ‘‘domain swapping’’ mechanism. HCC solution, a series of polyethelene glycol-1,500 MW (PEG-1500) induced os- is also associated with two types of amyloid deposition diseases - hereditary motic stress measurements were performed. The following D2O:H2O contrast amyloid angiopathy (related to the Leu68Gln mutation) and wild-type cystatin matches were used for SANS: 40% for proteins, 15% for lipids and 16% for C co-precipitation. PEG-1500. Radius of gyration (Rg) approximations using Guinier analysis of The aim of our studies was the characterisation of the self-assembling proper- A1AT in solution show a structural phase transition when the concentration ties of native and mutated (at positions 57 or 68) forms of human cystatin C in of PEG-1500 is between 33% and 36% by volume. Significant structural solution. The structure, overall conformation and secondary structure changes changes were also observed for fibrinogen when the concentration of PEG- in solution were studied by Fourier transformed infrared spectroscopy (FTIR), 1500 was above 40% by volume. These structural changes were compared circular dichroism spectroscopy (CD), dynamic light scattering (DLS) and time with changes observed when A1AT was in the presence of three different lipid resolved small angle scattering of synchrotron radiation (TR-SAXS). membranes: POPC, POPS and DLPC and when fibrinogen undergoes polymer- SAXS data for native and mutated HCC were subjected to analysis by using ization. Bragg peaks produced by lipid membranes show that A1AT interacts SVD and MCR-ALS methods as well as the low resolution structure determi- with unilamellar vesicles. DLS was used to find suitable concentrations of nation. Besides the monomeric forms of human cystatin C, also dimers and fibrinogen (4 mg/ml) and thrombin (0.01 units/ml) that would yield a signifi- higher oligomers were formed even after short (50-ms) exposure on synchro- cant signal-to-noise ratio for SANS experiments (or approximately 90 minutes tron radiation. In addition we observed for first time, formation of domain of reaction time). SANS time-resolved measurements show that fibrin structure swapped dimers of human cystatin C induced by irradiation. The spectroscopic is affected by its polymerization rate. studies confirmed conformational changes. Authors acknowledge also the partial support by HARMONIA3 grant (Project 2610-Pos Board B40 No. 2012/06/M/ST4/00036) from National Science Centre (Poland). Analytical Characterization of FGF Signaling Complex Jason N. Payne, Tulsi Modi, Oluwadamilola Filani, Raja Murthy. 2608-Pos Board B38 Chemistry, Western Kentucky University, Bowling Green, KY, USA. Differential Effects on Light Chain Amyloid Formation Depend on Muta- Fibroblast growth factors (FGF) are a family of growth hormones that act tions and Type of Glycosaminoglycans as regulators in key biological processes such as angiogenesis, wound heal- Luis M. Blancas-Mejia1, Jared A. Hammernik2, Marta Marin-Argany1, ing, embryonic development and select endocrine signaling pathways. It is Marina Ramirez-Alvarado1. currently theorized that the mechanism of the interaction of FGF to their recep- 1Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, tors, fibroblast growth factor receptors (FGFR) is greatly reliant upon the bind- 2School of Molecular and Cellular Biology, University of Illinois, Urbana, ing of heparin to FGFs. It is theorized that the D2 domain is the primary site for IL, USA. the FGF bind and interaction. The objective of this study is to characterize the Light chain (AL) amyloidosis is a protein misfolding disease where immuno- structure of the FGF-D2 domain. A structural analog of heparin, sucrose octa- globulin light chains sample partially folded states that lead to misfolding sulfate (SOS), is employed in this study to determine the role of heparin in the and amyloid formation, resulting in organ dysfunction and death. In vivo, am- formation of the FGF-FGFR complex. Isothermal titration calorimetry experi- yloid deposits are found in the extracellular space and involve a variety of ments were preformed to assess the binding affinity of FGF and the D2 domain, accessory molecules such as glycosaminoglycans, one of the main components these experiments indicate that human acidic FGF (FGF-1) binds to the D2 of the extracellular matrix. Glycosaminoglycans are a group of negatively domain with high affinity in both the presence and absence of SOS (Kd(appa) 7 8 charged, heteropolysaccharides composed of repeating disaccharide units. In ~10 M and Kd(appa) ~10 M respectively). Thermal denaturation experi- this study, we investigated the effect of glycosaminoglycans on the kinetics ments, monitored by far-UV CD, indicate that both FGF-1 and D2 domain un- of amyloid fibril formation of three AL cardiac amyloidosis light chains. These dergo slight conformational changes as a result of binding and elucidates the proteins have similar thermodynamic stability but exhibit different kinetics of presence of SOS stabilizes a 1:1 FGF-D2 domain complex. The FGF signaling fibril formation. We also studied single restorative and reciprocal mutants and complex, comprised of the FGF-1, the D2 domain, and SOS, structure eluci- wild type germline control protein. dated by X-ray crystallography indicated that the complex exists as a 2:2:2 We found that the type of glycosaminoglycan has different effect on the ki- symmetrical conjugation. Analysis via 2D 1H and 15N NMR chemical shift netics of fibril formation, and this effect seems to be associated with the natural perturbation (CSP) has allowed for the accurate mapping of the SOS and propensity of each AL protein to form fibrils. Heparan sulfate accelerated FGF binding sites of the D2 domain. The data resulting from this study suggest AL-12, AL-09, kI Y87H, and AL-103 H92D fibril formation, it delayed fibril that the primary role of heparin in the FGF signaling process is limited to sta- formation for AL-103 and did not promote any fibril formation for AL-12 bilizing the FGF-FGFR complex. Wednesday, February 11, 2015 517a

2611-Pos Board B41 characteristics of the protein. A detailed analysis has been performed in Interactions between Pairs of Charges Buried in the Hydrophobic Interior response to a concern that the use of conventional gamma irradiation may dam- of a Protein are Unexpectedly Weak age biologically active proteins. The results demonstrate that although gamma Aaron Robinson1, Andrea Theodoru1, Jamie Schlessman2, irradiation of the IgG may have some impact on protein structure, the damage Bertrand E. Garcia-Moreno1. can be reduced or even prevented by appropriate irradiation conditions. At the 1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2Chemistry, virucidal dose of gamma irradiation (50 kGy) and a temperature of 80oC, the US Naval Academy, Annapolis, MD, USA. integrity of the polypeptide chain of immunoglobulin and the secondary struc- Internal ion pairs buried in the hydrophobic protein interior are essential for ture of IgG can be completely protected, while conformational changes in ter- many important biochemical processes, including Hþ transport, e- transfer, tiary structure are significantly minimized to a level that preserves functional ion homeostasis, and catalysis. Despite the importance of these pairs, their activity. The irradiated IgG retains specific antigen-binding properties and properties remain poorly understood. It has been suggested that, for some sys- Fc-binding activity, indicating that the conformational integrity of the most tems, medium or long-range Coulomb interactions between buried groups important structural regions is not affected by gamma-irradiation. These results could play a role in determining biological function. In principle, this should present strong evidence that gamma irradiation treatment can be effectively im- only be possible if the protein interior behaves like a medium of low dielectric plemented for inactivation of pathogens in IgG solutions that are used for intra- constant, as assumed in most electrostatics models. These motifs are then of venous injection. special interest not only for their functional roles, but also as probes to examine the balance between Coulomb and hydration energies experienced by buried 2614-Pos Board B44 charges. As the distance between the internal charges increases, the balance Photoacoustic Calorimetry Studies of Ferric Cytochrome-C Folding using of these energies will disfavor burying the groups in the charged state, elimi- an No Photo-Trigger nating any Coulomb interaction between them. To examine the balance Tarah A. Word, Randy W. Larsen. between Coulomb and dehydration energies in the protein interior we engi- Chemistry, University of South Florida, Tampa, FL, USA. neered a series of double variants in a highly stable variant of staphylococcal In this study, NO is utilized as a photo-trigger together with photoacoustic calo- nuclease. Each variant included an internal histidine paired with either an rimetry to probe the kinetics, enthalpy and molar volume changes associated þ aspartate or glutamate buried at various internal positions throughout the with the earliest folding events in ferric Cytochrome-c (Cc3 ). The ferric protein interior. This set of proteins used to probe the distance dependence heme protein was examined under different denaturing conditions including of potential Coulomb interactions. No significant electrostatic coupling was guanidine hydrochloride (2.8M GdnHCl) and Sodium dodecyl sulfate (SDS observed for pairs that interacted through medium- or long-range Coulomb in- 0.4mM) (both in 50 mM Hepes buffer, pH ~7.5) along with NO resulting in teractions. When the distance between the groups was short, favorable coupling the disruption of the axial heme Methionine-80 heme bond, triggering the par- energies were only observed when the side chains of the pair could achieve a tial unfolding of the complex. Under these conditions photo-dissociation of NO geometry favorable for a H-bond; spatial proximity alone was insufficient to leaves the protein in a conformational state that favors refolding of the protein. create favorable Coulomb interactions. Simple electrostatics models that The PAC data reveals three kinetic phases taking place subsequent to photol- describe ionized states as point charges interacting through space are unlikely ysis regardless of the denaturant environment. Specifically, in the presence to be able to reproduce these data. of 2.8 M GdnHCl photolysis gave rise to kinetic events with lifetimes of <20 ns, ~860 ns, and ~6 ms that were associated with DH/DV ¼25 kcal 2612-Pos Board B42 mol-1/-9 mL mol-1, 26 kcal mol-1/15mL mol-1, and 9 kcal mol-1/-26 mL A Reason for Long Tales mol-1, respectively. In the presence of 0.4 mM SDS, kinetic events were < Kathryn R. Geiger, Doug Barrick. observed with lifetimes of 20 ns, ~640 ns, and ~8 ms with corresponding Johns Hopkins University, Baltimore, MD, USA. DH/DVof 11 kcal mol-1/-1 mL mol-1, 5 kcal mol-1/3 mL mol-1, and 30 Transcription Activator-Like Effectors (TALEs) are bacterial virulence factors kcal mol-1/-9 mL mol-1, respectively. The data suggests a uniform mechanism þ containing a domain of repeats that recognize specific DNA sequences and for the early folding events occurring in the folding of Cc3 complex subse- reprogram transcription activation of invaded plant cells. TALE genes encode quent to NO photo-dissociation which are attributed to NO dissociation from 5 to 30 repeats with average pairwise repeat identities greater than 91%. Most the heme, followed by reorganization of the distal pocket (i.e hydrogen bond variability arises from only two positions termed Repeat Variable Diresidues formation/breakage, NO solvation, etc.) and potentially a intermolecular bind- (RVDs), which confer DNA binding specificity. Crystal structures of free ing of Methionine (80 or 65) or Histidine (23 or 33) to the heme iron upon and DNA-bound TALEs (Deng et al. Science 2012) show a large conforma- folding. tional change upon DNA binding. Thus, DNA binding is likely coupled to 2615-Pos Board B45 the free energy of tertiary structural change between the TALE repeats. We Simulation of Pressure-Induced and Temperature-Induced Denaturation are interested in quantifying this relationship and relating it to folding cooper- of Phosphoglycerate Kinase ativity using nearest-neighbor (‘‘Ising’’) models. Jianfa Chen1,2, Margaret S. Cheung1,2. To investigate the length dependence of folding, we created a set of consensus 1Department of Physics, University of Houston, Houston, TX, USA, 2Center TALE constructs of varying length. Solubilizing N- and C-terminal caps are for Theoretical Biological Physics, Rice University, Houston, TX, USA. needed to favor monomeric protein in solution as detected by sedimentation ve- Phosphoglycerate kinase (PGK) is a 415-residue protein composed of two do- locity analytical ultracentrifugation. Capped consensus TALE repeat constructs mains of almost equal size. Under the heat-induced denaturation, folding and have alpha-helical secondary structure as measured by farUV CD. Urea- unfolding of PGK is a two-state process. However, folding and unfolding of induced unfolding transitions of TALE repeat arrays were measured and PGK under pressure-induced denaturation,is a three-state process. How the show cooperative unfolding transitions as well as increases in stability with temperature and pressure affect the denaturation of PGK is still unknown. length. These data are well-fitted by an Ising model, which separates the con- We aim to provide a molecular explanation for thermal and pressure denatur- tributions of intrinsic and interfacial free energies. TALE repeats have an ation by using coarse-grained molecular simulations where the interactions unfavorable intrinsic folding free energy of 5.3 kcal/mol and a favorable inter- of residues experience expulsion of water molecules. facial free energy of 6.8 kcal/mol. Using these values, the length dependence of TALE stability can be modeled, and shows TALEs under 5 repeats to be 2616-Pos Board B46 unfolded. Using the Ising parameters, we find that partially folded states with Intermolecular Interactions in Highly Concentrated Protein Solutions a single repeat unfolded are energetically accessible. Population of these Upon Compression and the Role of the Solvent partially folded states may be important for DNA binding. Sebastian Grobelny1, Mirko Erlkamp1, Johannes Mo¨ller2, Metin Tolan2, Roland Winter1. 2613-Pos Board B43 1Physical Chemistry I, TU Dortmund University, Dortmund, Germany, Effect of Gamma Radiation on the Structural and Functional Integrity 2Experimentelle Physik I, TU Dortmund University, Dortmund, Germany. of IgG The influence of high hydrostatic pressure on the structure and protein-protein Claudia C. Smeltzer1, Nina N. Lukinova1, Nicole D. Towcimak1, interaction potential of highly concentrated lysozyme solutions up to about 370 David Mann1, William N. Drohan1, Yuri V. Griko2. mg mL 1 was studied and analyzed using small-angle X-ray scattering (SAXS) 1Clearant Inc., Gaithersburg, MD, USA, 2Space Biosciences, NASA Ames in combination with a liquid-state theoretical approach. In the concentration Research Center, Mountain View, CA, USA. region below 200 mg mL1, the interaction parameters of lysozyme solutions Plasma-originated commercial intravenous immunoglobulin, which is used are affected by pressure in a nonlinear way, which is probably due to significant for a variety of clinical purposes, has been studied to determine the effect of changes in the structural properties of bulk water, i.e., due to a solvent- virus-inactivating doses of gamma irradiation on the structural and functional mediated effect. Conversely, for higher concentrated protein solutions, where 518a Wednesday, February 11, 2015 hydration layers below ~4 water molecules are reached, the interaction poten- includes explicit treatment of electronic polarization based on the classical tial turns rather insensitive to compression. The onset of transient (dynamic) Drude oscillator model.[1] The Drude force field was found to maintain protein clustering is envisaged in this concentration range. Our results also show that native structures during microsecond molecular dynamics simulations of multi- pressure suppresses protein nucleation, aggregation and finally crystallization ple folded proteins, and leads to significant variability of backbone and side in supersaturated condensed protein solutions. These findings are of importance chain dipole moments as a function of environment.[2] Here we report replica for controlling and fine-tuning protein crystallization. Moreover, these results exchange simulations of the helix-forming (AAQAA)3 peptide and the b- are also important for understanding the high stability of highly concentrated sheet-forming GB1 hairpin using this fully polarizable model. protein solutions (as they occur intracellularly) in organisms thriving under hy- Polarizable simulations of (AAQAA)3 reveal the presence of folding coopera- drostatic pressure conditions such as in the deep sea, where pressures up to the tivity consistent with experimental observations. The cooperativity is signifi- kbar-level are reached. cantly larger than that modeled by currently available non-polarizable force fields and is shown to be directly associated with enhanced dipole moments 2617-Pos Board B47 of the peptide backbone upon helix formation.[3] The GB1 hairpin is found Atomistic and Coarse-Grained MD Simulations of the Intrinsically Disor- to be less stable with the Drude force field compared to the experimental obser- dered Bacillus Subtilis Ribonuclease P Protein vation. Results from these extensive condensed phase simulations of peptide Cecilia G. Rambarat. folding will be utilized, together with QM calculations of model alanine poly- Wake Forest University, Winston-Salem, NC, USA. peptide systems, to further refine the backbone parameters in the Drude protein The Bacillus subtilis Ribonuclease-P (RNase P) holoenzyme is a protein-RNA force field. In summary, our results demonstrate that the inclusion of explicit complex; however, the protein becomes disordered in the absence of its RNA electronic polarizability leads to a fundamentally improved model of the phys- binding partner. To identify the main determinants of why the RNase P protein ical forces dictating the structure and dynamics of polypeptides. becomes disordered, we performed (i) a statistical analysis of its interactions to [1] P. Lopes, J. Huang, J. Shim, Y. Luo, H. Li, B. Roux, and A.D. MacKerell, J. identify the critical minimally frustrated residues that are important for struc- Chem. Theo. Comput., 2013, 9, 5430. tural stabilization, (ii) atomistic MD simulations in the presence and absence [2] J. Huang, P. Lopes, B. Roux, and A.D. MacKerell, J. Phys. Chem. Lett., of the osmolyte Trimethylamine N-Oxide (TMAO), which has been shown 2014, 5, 3144. experimentally to stabilize its native structure even without its RNA binding [3] J. Huang and A.D. MacKerell, Biophys. J., 2014, 107, 991. partner, and (iii) coarse-grained Go-type MD simulations to determine the folding/unfolding mechanism. We observed an intermediate in our coarse- 2620-Pos Board B50 grained MD simulations that we propose is the partially disordered state Dependence of Internal Friction on Native Topology in the absence of its RNA binding partner and the intermediate seen experi- Wenwei Zheng1, David de Sancho2, Travis Hoppe1, Robert B. Best1. mentally in TMAO-induced folding experiments. We also identified critical 1Laboratory of Chemical Physics, NIDDK, NIH, Bethesda, MD, USA, residues for stabilization that can be tested experimentally using standard muta- 2Department of Chemistry, University of Cambridge, Cambridge, United genesis folding kinetic experiments. Kingdom. 2618-Pos Board B48 Apparent internal friction has been identified in the folding rates of several pro- The Dock-and-Coalesce Mechanism for the Association of Intrinsically teins and in the dynamics of unfolded proteins, via their dependence on solvent Disordered WASP with the Cdc42 GTPase viscosity. Theoretical and computational studies have attributed this phenome- Li Ou1, Megan Matthews2, Xiaodong Pang1, Huan-Xaing Zhou1. non, at least in part, to local barrier crossing events, in particular torsion angle 1Institute Of Molecular Biophysics, Florida State University, Tallahassee, isomerizations. However, there are still many systems for which no internal FL, USA, 2Florida State University, Tallahassee, FL, USA. friction has been observed in experiment, and others for which the effect is Intrinsically disordered proteins (IDPs) play key roles in signaling and regu- only weak. Here, we elucidate the origin of this discrepancy by studying a lation. Many IDPs undergo folding upon binding to their targets. We have model system whose native state can be either an a-helix or a b-hairpin. proposed that coupled folding and binding of IDPs generally follow the Remarkably, we find clear apparent internal friction for the helix, but no evi- dock-and-coalesce mechanism, whereby a segment of the IDP through diffu- dence of it for the hairpin. These results are consistent with the observation sion docks to its cognate subsite and subsequently the remaining segments coa- that proteins whose folding rates exhibit internal friction tend to be a-helical. lesce around their subsites [PCCP 14:10466(2012)]. Here we tested the validity We can explain the difference by examining the occurrence of torsional transi- of this mechanism on the association between the intrinsically disordered tions along the folding coordinate of the hairpin and helix. Finally, we consider GTPase binding domain (GBD) of the Wiskott-Aldrich Syndrome protein the folding of the Trp cage mini protein, whose structure includes both helical (WASP) and the Cdc42 GTPase, by both experiment and computation. The as- and hairpin elements. We obtain two barriers – the major barrier for formation sociation rate constants (ka) were measured by stop-flow fluorescence under of the hairpin and a minor barrier for initial helix formation, allowing us to various solvent conditions and temperatures. ka reaches 107 M-1s-1 at physio- dissect the contribution of internal friction to different parts of the energy land- logical ionic strength and has a strong salt dependence, suggesting that an elec- scape. We find a clear signature of internal friction for the helical barrier and, trostatically enhanced, diffusion-controlled docking step is rate-limiting. within the uncertainty of our calculation, little internal friction for the hairpin Diffusion control is supported by an inversely proportional relation between barrier. Our results show how variation of solvent viscosity can be used to ka and the solvent viscosity with glucose as the viscogen. ka increases with probe features of folding mechanisms. increasing temperature; though the increase is larger than expected from the ef- fect of temperature on the protein diffusion constants, the discrepancy may be 2621-Pos Board B51 accounted for by the effect of temperature in decreasing the solvent dielectric Thermodynamics of b-Structures from Molecular Dynamics Simulations constant, leading to stronger electrostatic rate enhancement. Similarly, a Anthony Hazel, James C. Gumbart. modest decrease in ka by urea may be rationalized by the latter’s effect in Georgia Institute of Technology, Atlanta, GA, USA. increasing the solvent dielectric constant. Our computation, based on the b-Sheets are some of the most common secondary structure motifs in proteins, transient-complex theory [Structure 19:1744(2011)], identified the N-terminal and are important for mediating protein-protein interactions through their asso- basic region of the GBD as the docking segment, which has strong electrostatic ciation. This association can also lead to the aggregation of misfolded proteins complementarity with the cognate subsite. Our study suggests that the dock- into b-pleated-sheets in neurodegenerative disorders like amyloidosis. The and-coalesce mechanism allows WASP and other IDPs to code electrostatic folding pathway from random coil to b-sheet usually involves two competing complementarity into the docking segment to gain binding speed and use addi- process: (1) the collapse of a hydrophobic core, and (2) the formation of tional interactions formed by the coalescing segments to reinforce binding intrapeptide hydrogen bonds. It has been proposed, and shown computation- affinity. ally, that the hydrophobic core collapse precedes hydrogen bond formation. In this study we examine the thermodynamics of b-hairpin formation for the 2619-Pos Board B49 GB1 domain of protein G with molecular dynamics simulations by calculating Cooperative Helix Formation in the (AAQAA)3 Peptide Obtained with the a two-dimensional free energy surface in both vacuum and explicit water Drude Polarizable Force Field using as our reaction coordinates (1) the radius of gyration of the hydrophobic Jing Huang, Alexander D. MacKerell. core and (2) the number of native hydrogen bonds, corresponding to the two Department of Pharmaceutical Science, University of Maryland, Baltimore, aforementioned folding processes, respectively. We also compare the results Baltimore, MD, USA. of different versions of the CHARMM force field, namely CHARMM22, Molecular simulations can provide atomic-level details of protein folding. How- CHARMM22/CMAP, CHARMM22* and CHARMM36. Finally, we show ever, their accuracy is limited by approximations made in the underlying empir- how these methods can be applied to other b-structures in vivo, namely b-helix ical force fields. Recently we presented a force field for peptides and proteins that structures in the outer membrane of Gram-negative bacteria. Wednesday, February 11, 2015 519a

2622-Pos Board B52 pretation of the data to make this useful experimental technique accessible to Using Kinetic Network Models to Understand Folding Mechanisms of GB1 naı¨ve users, as well as providing a starting point for detailed analyses by Hairpin and its Trpzip Variants more experienced users. Vincent A. Voelz, Asghar Razavi. Department of Chemistry, Temple University, Philadelphia, PA, USA. 2626-Pos Board B56 We used Markov State Model (MSM) approaches to analyze over 9 ms of Physics Based Structure Refinement in Casp11 using Geometric Unfolding explicit-solvent simulation trajectories for GB1 hairpin (the 16-residue C-ter- and Hierarchically Restrained Replica Exchange Molecular Dynamics minal domain of protein G) and its three mutants (trpzip4, trpzip5, and trpzip6) Avishek Kumar, Paul Campitelli, Sefika Banu Ozkan, Michael F. Thorpe. at multiple temperatures, to investigate folding thermodynamics, kinetics, and Physics, Arizona State University, Tempe, AZ, USA. mechanisms. Our MSM results show predicted folding rates and equilibrium We have developed a new physics based approach to the protein refinement populations that agree well with experimental data. Furthermore, we show problem by mimicking the mechanism of chaperons that rehabilitate proteins. how MSMs constructed from combined datasets reveal mechanistic differences The template structure is unfolded by selectively pulling on different portions resulting from tryptophan mutations. While wild type and trpzip4 hairpins are of the protein using the geometric based technique FRODA, and refolding the predicted to be two-state folders, our MSMs predict more complicated kinetics protein using hierarchically restrained replica exchange molecular dynamics. for trpzip5 and trpzip6 due to the presence of non-native traps. We find that FRODA unfolding is used to create a diverse set of topologies for surveying changes in the folding landscape can also be revealed by analyzing MSM near the native like structures from a template. The unfolding trajectories are rate perturbations, provided that metastable states are conserved. then used to find energetic restraints to enforce contacts and dihedral restraints. An REMD simulation is performed for the entire ensemble using consensus and 2623-Pos Board B53 reservoir techniques, which allow multiple structural candidates to ‘‘swap’’ into Influence of Zinc-Binding on Folding and Dynamics of Zinc Finger Pro- the replica cascade at the highest temperature replica and the most favorable teins: In Silico folds to propagate to the lowest temperature replica. The restraints are added Ryan Godwin1, William Gmeiner2, Freddie Salsbury1. in a hierarchical fashion where local contacts are restrained first followed by 1Physics, Wake Forest University, Winston Salem, NC, USA, 2Cancer the addition of non-local restraints to narrow the conformational search toward Biology, Wake Forest University Health Sciences, Winston Salem, NC, USA. the native state. After REMD structures are clustered, refined structures are We present results of microsecond, all atom molecular dynamics simulations selected based on the highest populated cluster, RMSD and DFIRE score. for various proteins including the zinc binding domains of NEMO and XIAP. Each zinc finger was simulated with and without zinc and with proton- 2627-Pos Board B57 ated and deprotonated zinc-binding cysteines. Simulation analyses suggest the Role of Side Chain Size in the Formation of Secondary Structures in Model bound zinc increases stabilization of the protein structure and causes changes to Peptides protein dynamics. In the absence of a bound zinc ion, proteins have a tendency Farbod Mahmoudinobar1, Cristiano L. Dias1, Ronen Zangi2. 1 2 to at least partially unfold and some simulations show complete loss of second- Physics, New Jersey Institute of Technology, Newark, NJ, USA, Organic ary structure. Differences in free energy of the four structural cases suggest that Chemistry I, University of the Basque Country UPV/EHU, San Sebastian, binding the zinc-ion is energetically inexpensive, where the (de)-protonation Spain. of the cysteines is the more significant contributor to free energy differences To study the role played by side chain interactions in -helix formation, we between the four structures. perform extensive all-atom molecular dynamics simulations of modified poly-alanine peptides in explicit TIP4P water. Our model systems include 2624-Pos Board B54 two poly-alanine peptide lengths (nine-mer and twelve-mer) described by the Folding Mechanism of Proteins IM7 and IM9, from Computer Simulations OPLS force-field in which we change systematically the value of the in a Realistic Atomistic Force Field Lennard-Jones diameter, s, and the well-depth, ε,ofCb atoms. We identify Fang Wang1, Giorgia Cazzolli2,3, Patrick Wintrode1, Pietro Faccioli2,3. characteristic length-scales that promote -helices formation. To rationalize var- 1 2 3 UMD, Baltimore, MD, USA, University of Trento, Trento, Italy, Trento iations in -helix content observed in our simulations we computed effective in- Institute for Fundamental Physics and Applications (TIFPA), Trento, Italy. teractions, i.e., potential of mean force (PMF) between methane-like particles IM7 and IM9 are small evolutionarily related proteins which fold according to that resemble side chains in our modified poly-alanine peptides. Contact- different kinetics, in spite of their remarkable structural homology. While the minimum, desolvation barrier, and solvent-separated-minimum of computed former chain clearly folds according to three-state kinetics, the evidence for PMF(s) when superposed to distances between i-iþ1, i-iþ3 and i-iþ4 neigh- an on-pathway intermediate in the folding of IM9 is much more elusive. bors are consistent in explaining qualitatively -helix content in our simulations. This observation has triggered considerable theoretical and experimental effort, Implications of these results to the role of pressure in secondary structure for- aiming to characterize the folding pathways of these chains and clarify the mation are discussed. physical origin of the observed differences. In this work, we use the Dominant Reaction Pathway (DRP) method to efficiently generate many folding trajec- 2628-Pos Board B58 tories for these proteins, from a realistic atomistic force field. Overall, our re- The Association Landscape of Ubiquitin Dimerization sults are found to be in good agreement with with experimental 4-values and Haiqing Zhao1, David Fushman2, Garegin A. Papoian2. with the result of 4-value-restrained Molecular Dynamics (MD) simulation, 1Biophysics Program, Institute for Physical Science and Technology, and suggest that the differences in the folding pathways and kinetics is largely University Of Maryland, College Park, MD, USA, 2Department of Chemistry influenced by the chains’ native topology. On the other hand,by performing and Biochemistry, University Of Maryland, College Park, MD, USA. MD simulations starting from the calculated on-pathway intermediates we Ubiquitin is a highly conserved regulatory protein that acts as a post- argue that the difference in the life-times of the two on-pathway intermediates translational modifier of various proteins in eukaryotes. For this, the C-terminus is due to non-native electrostatic interactions between specific residues and the of ubiquitin is covalently linked to a lysine side chain of the target protein. solvent. Furthermore, ubiquitin can also form isopeptide-linked polymers, called polyubiquitin chains, which serve as versatile molecular signals regulating a 2625-Pos Board B55 vast range of cellular processes. Using molecular dynamics (MD) simulations Meltdown - a Tool for Classification and Analysis of DSF Data and the Associated memory, Water mediated, Structure and Energy Model 1 2 2 2 Michael Jayne , Marko Ristic , Nicholas Rosa , Shane A. Seabrook , (AWSEM) coarse-grained model, we predicted the dimerization interface of Janet Newman2, David Lovell3, Del Lucent1. 1 two unconjugated ubiquitin monomers. Surprisingly, even without any cova- Department of Electrical Engineering and Physics, Wilkes University, lent linkage ubiquitins recognized each other and formed a stable dimer. The Wilkes Barre, PA, USA, 2Manufacturing Flagship, CSIRO, Parkville, 3 obtained results are in strong agreement with experimental NMR data and pro- Victoria, Australia, Digital Productivity and Services Flagship, CSIRO, vide detailed insights into the nature of ubiquitin’s recognition and association. Acton, ACT, Australia. An application that translates raw thermal melt curve data into more easily 2629-Pos Board B59 assimilated knowledge is described. This program, called ‘Meltdown’, per- Roles of Urea and TMAO on the Interaction between Extended Non-Polar forms a number of data remediation steps before classifying melt curves Peptides and estimating melting temperatures. The final output is a report that summa- Zhaoqian Su, Jampanis R. Srinivasa, Cristiano L. Dias. rizes the results of a differential scanning fluorimetry experiment. Meltdown New Jersey institute of Technology, Newark, NJ, USA. uses a Bayesian classification scheme, enabling reproducible identification of In this study, we investigate the role played by urea and trimethylamine n-ox- various trends commonly found in DSF datasets. The goal of Meltdown is ide (TMAO) in the stability of extended poly-alanine and poly-leucine dimers not to replace human analysis of the raw data, but to provide a sensible inter- using all-atom molecular dynamics simulations and the explicit TIP3P water 520a Wednesday, February 11, 2015 model. An umbrella sampling protocol is used to compute the potential of have first implemented an enhanced-sampling algorithm which allows us mean force (PMF) describing the interaction between these dimers at different to explore the proteins’ free-energy landscape over a wide range of temper- concentrations of urea and TMAO. We show that while urea has a destabiliz- atures, and we have used constant-force steered MD to investigate the effect ing effect on the interaction between poly-leucine chains, TMAO has an of mechanical force. Our results suggest that thermal stability does not guar- opposite effect. This is consistent with the use of urea as a denaturant and antee a mechanical one and that the associated unfolding mechanisms, of TMAO as an osmolyte stabilizing native structures of proteins. However, which our simulations provide extensive details, are very different. We both cosolvents have no significant effect on the interaction between poly- also aim at identifying the weak-points of the protein mechanical stability alanine chains. To unravel the molecular mechanisms of TMAO and urea, and how more stable mutants can be designed. If such mutants also exhibit we study enthalpic components of the energy, i.e., Lennard-Jones and electro- increased thermal stability, our approach could prove useful in numerous static energies. No clear correlation is observed between changes in the applications. enthalpy and the PMF, suggesting that an indirect mechanism mediated by water molecules could be responsible for the role of urea and TMAO. To pro- 2632-Pos Board B62 vide insights into the interaction of urea and TMAO with the peptide dimer, Thermal Stability of a B-Type Heme Peroxidase from the Psychrophilic we compute preferential interaction coefficients and spatial distributions of Diatom Fragilariopsis Cylindrus cosolvent and water around poly-alanine and poly-leucine. Urea and TMAO Thuy Duong Nguyen, Katherine Frato. have a distinct distribution around the peptide with urea preferring to be Chemistry, Seattle University, Seattle, WA, USA. located in its vicinity. The eukaryotic, unicellular diatom Fragilariopsis cylindrus thrives in marine environments at temperatures near the freezing point of water. As a result, crit- 2630-Pos Board B60 ical enzymes of F. cylindrus must have specifically evolved to maintain flexi- Probing the Downhill Folding Kinetics of Lambda{6-85} Mutants with bility and activity at very low temperatures. Like all photosynthetic organisms, Optical Tweezers F. cylindrus must also have robust pathways, possibly including peroxidases, to Ann Mukhortava, Andreas Hartmann, Michael Schlierf. protect its cellular components against oxidative damage. In this study, we B CUBE – Center for Molecular Bioengineering, TU Dresden, Dresden, report the recombinant expression and purification of a b-type heme peroxi- Germany. dase, Fc 209911, identified from the published genome of F. cylindrus. The Protein folding is a process of molecular self-assembly during which a disor- enzyme is expressed in the apo form in Escherichia coli, and forms an active dered polypeptide chain collapses to form a compact and well-defined three- holo-enzyme upon reconstitution with hemin. The purified enzyme turns dimensional structure. The process of folding is described as a path on a over hydrogen peroxide in the presence of ABTS with modest turnover rates multi-dimensional energy landscape. If at each folding step along the path a (20 min-1), yet binds hydrogen peroxide with a very high affinity (Km < decrease of entropy is nearly compensated by a decrease of enthalpy, these pro- 100 nM). As predicted, mutation of the conserved histidine H73, which pre- teins are termed downhill folders and display ultrafast kinetics. Studying down- sumably forms a hydrogen bond donor/acceptor in the proximal heme pocket, hill folders allows understanding of fundamental principles of folding by to alanine decreases hydrogen peroxide binding affinity (~ 10,000x increase in resolving intramolecular dynamics in a step-by-step manner. However, there Km), suggesting that the enzyme has a typical peroxidase fold and mechanism. is still lack of knowledge about details of downhill energy landscapes, as the Thermal unfolding monitored by the intensity of the Soret absorbance band direct time-resolving experimental studies of ultrafast processes has been shows a single unfolding transition around 295 K. Analysis of the rate of limited to a few ensemble methods. hydrogen peroxide turnover at a range of temperatures shows maximal activity Here, we present a comparative study of single-molecule protein folding around 303 K, with a steep decrease in activity as temperature is raised further. using optical tweezers that provide the possibility to measure structural The temperature dependence of folding and peroxidase activity confirms that dynamics with sub-millisecond and nanometer resolution. We charac- Fc 209911 is optimized to function at low temperatures. Experiments are terize the folding dynamics of three different lambda repressor variants: currently underway to characterize the van’t Hoff enthalpy of the unfolding a two-state folder LambdaWT* (Y22W) and two downhill folding vari- transition. Future experiments will investigate the basis of the thermal insta- ants, LambdaYA (Y22W/Q33Y/G46,48A) and LambdaHA (Y22W/Q33H/ bility of Fc 209911 by designing site-directed mutants to increase the melting G46,48A). We show that force perturbation of their energy landscape temperature. slowed down the ultrafast kinetics of downhill folders, making it accessible to single-molecule studies. Interestingly, the downhill variants of lambda 2633-Pos Board B63 repressor appeared as two-state folders under load with significantly Understanding the Role of Glycosylation in the Thermostability of CEL7A different folding kinetics and force dependence. A comparison between Jodi A. Hadden. these variants allowed us to extract fine details of their underlying energy Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, landscape. IL, USA. Many industrially important cellulolytic enzymes, such as CEL7A (also known as cellobiohydrolase 1), natively exhibit numerous glycosylation sites for cova- Protein Stability lent attachment of N- and O-linked glycans. The presence of such glycans, as well as variations in their chemical structures, can influence enzyme properties 2631-Pos Board B61 such as substrate binding, catalytic efficiency, and thermostability. While the Mechanical Aspects of Protein Thermostability CEL7A sequence manifests four potential N-glycosylation sites, only three Guillaume Stirnemann, Maria Kalimeri, Fabio Sterpone. of which are typically glycosylated, the extent of glycosylation at each site Laboratoire de Biochimie Theorique, Paris, France. varies with expression host. For example, yeast systems generally express cel- Because thermophilic proteins are stable and functional at high temperature but lulases that are hyperglycosylated compared to fungi. Both hyperglycosylation generally lack activity under ambient conditions, it was suggested that thermal and the presence of glycans at particular glycosylation sites have been shown to stability can correspond to enhanced mechanical rigidity. While this concept negatively impact catalytic activity, yet the presence of glycans at alternative has initially found wide experimentally support, its generality has been later sites, including those engineered into the enzymes, have been shown to enhance questioned. It is then natural to inquire the correlation between protein thermal activity. Furthermore, studies suggest that glycosylation contributes structural and mechanical stabilities using single-molecule force-spectroscopy experi- integrity that facilitates the functioning of cellulases under harsh reaction con- ments. Here, we will present results of a combination of computational tech- ditions, such as high temperature or pressure, that are often preferred during the niques, mainly based on Molecular Dynamics simulations, to tackle these industrial production of bioethanol. Despite the relationship between enzyme problems for several mesophilic/thermophilic homologues of different molec- glycosylation and demonstrated cellulolytic functionality, the structural mech- ular complexity. anisms by which glycosylation alters enzyme properties remain largely By applying innovative network analysis to map the protein conformational undetermined. landscape, we first question the universality of the rigidity paradigm. In fact Molecular dynamics simulations provide a powerful solution for investigating we show that for several paris of homologues, thermophiles can be as flexible structure/function relationships in biomolecular systems. Here, a range of as their mesophilic variant with important effects coming from the local parti- CEL7A glycoforms are simulated at high temperature/pressure in order to tion of rigid/flexible regions, protein oligomerization state, or the time and elucidate the structural role by which glycosylation enhances thermodynamic length scales considered. integrity. Findings are supported by analogous experimental studies of We will then focus on the correlation among thermostability and mechanical CEL7A under thermal/baric stress. Combining theory with experiment to resistance. The object under investigation is a pair of mesophilic/thermo- develop a more comprehensive understanding of glycosylation as a factor con- philic cold-shock proteins for which experimental data are available. We trolling cellulolytic enzyme functionality will ultimately enable researchers to Wednesday, February 11, 2015 521a exploit glycosylation as a strategy for tuning cellulase efficiency and stability, of actin assemblies commences already, in agreement with in vivo observa- which can serve to enhance the cost-competitiveness of second-generation tions. The limited pressure stability of the monomeric building block seems biofuels. to be responsible for the suppression of actin assembly in the kbar pressure range. 2634-Pos Board B64 Single-Molecule Studies of Multidomain Protein Folding 2637-Pos Board B67 Kaixian Liu, Christian Kaiser. Thermal Stabilization of Adenylate Kinases by Optimizing Local Struc- BIOLOGY, Johns Hopkins University, Baltimore, MD, USA. tural Entropy Proteins are synthesized as linear polymers that must fold into specific three- Sojin Moon, Euiyoung Bae. dimensional structures to become functionally active. How proteins find their Seoul National University, Seoul, Korea, Republic of. native structures is still not well understood, particularly for multi-domain pro- Local structural entropy (LSE) is an empirical descriptor for the extent of teins that account for a large fraction of proteomes. It has become evident that conformational variability in short protein sequences, computed from struc- the way proteins are synthesized directly affects their folding: The ribosome tural information derived from structures in the . Reducing synthesizes proteins through the addition of single amino acids to the C-termi- the LSE of a protein sequence by mutating amino acid residues can result in nus of the growing nascent chain. Folding can begin while elongation is still fewer conformational states and thus a more stable structure. Here, we report proceeding. Thus, folding and synthesis are kinetically coupled, and modula- a series of LSE optimization experiments designed to stabilize mesophilic and tions in elongation rates can affect the folding outcome. Molecular chaperones thermophilic adenylate kinases (AKs). The crystal structures of the LSE- interact with nascent polypeptides and begin to guide their folding as soon as optimized AK variants reveal that stabilization by LSE reduction may result they emerge on the ribosomal surface. We are using single-molecule force from the optimization of local hydrophobic contacts. The limitations of the spectroscopy with optical tweezers to study the folding of multi-domain pro- LSE optimization are likely due to ignorance of interactions connecting teins in the context of the translation machinery and molecular chaperones. distant regions of the polypeptide. Our results illustrate the effectiveness Our measurements are revealing how the process of translation and interactions and limitations of LSE optimization as a protein stabilization method and with molecular chaperones shape the folding energy landscapes of nascent pro- highlight the complementarity of local and global structural features in protein teins and efficiently guide newly synthesized proteins toward their native thermal stability. structures. 2638-Pos Board B68 2635-Pos Board B65 Mos1 Transposase Thermodynamic Stability and Flexibility Folding and Unfolding Dynamics of Titin Immunoglobulin Domain Under Daniel P. Godfrey, Christopher M. Singer, Donald J. Jacobs, Constant Forces Irina V. Nesmelova. Hu Chen1,2, Guohua Yuan1,2, Jie Yan2. Department of Physics and Optical Science, UNC Charlotte, Charlotte, 1Department of Physics, Xiamen University, Xiamen, China, NC, USA. 2Mechanobiology Institute, National University of Singapore, Singapore, DNA transposons are mobile DNA elements that can move (or transpose) Singapore. from one DNA molecule to another and thereby deliver genetic information The mechanical stability of force-bearing proteins is crucial for their functions. into human in order to confer a new function or replace a However, slow transition rates of complex protein domains have made it chal- defective gene. This process is catalyzed by a transposase enzyme. The reac- lenging to investigate their equilibrium force-dependent structural transitions. tion of transposition occurs in several steps, during which two or more trans- Using ultra-stable magnetic tweezers, we report the first equilibrium single- posase enzymes bind to the terminal inverted repeats on the transposon DNA, molecule force manipulation study of the classic titin I27 immunoglobulin bring them together to form a synaptic complex, excise the gene flanked by domain. We found that individual I27 in a tandem repeat unfold/fold indepen- the terminal inverted repeats, and catalyze strand transfer to insert the excised dently. We obtained the force-dependent free energy difference between gene at a new location. Thus, transposases must be sufficiently flexible to unfolded and folded I27, and determined the critical force of ~ 5.2 pN at which allow conformational rearrangements of their domains to bind the transposon unfolding and folding have equal probability. We also determined the force- DNA and to supply a catalytic site during each step of transposition. Here, dependent free energy landscape of unfolding/folding transitions based on mea- we investigate the dynamics, thermodynamic stability, and flexibility of surement of the free energy cost of unfolding. Mos1 transposase, a member of the Tc1/mariner family of transposases. Our results suggest that the conformations of titin immunoglobulin domains We use a computational model called the minimum Distance Constraint can be significantly altered during low force, long duration muscle stretching. Model (mDCM) and the analysis of quantitative stability/flexibility relation- The ultra-stable magnetic tweezers can be used to study the folding/unfolding ships (QSFR). With these tools, we determine the free energy landscape and dynamics of other complex proteins. the flexibility and mechanical coupling of secondary structure elements or residues in Mos1. Our data provide an insight into how Mos1 is structured 2636-Pos Board B66 and how it functions and are applicable to Tc1/mariner transposases in Exploring the Stability Limits of Actin and its Suprastructures general. Christopher Rosin1, Mirko Erlkamp1, Julian von der Ecken2, Stefan Raunser2, Roland Winter1. 2639-Pos Board B69 1Chemistry & Chemical Biology, TU Dortmund, Dortmund, Germany, Stabilization and De-Stabilization of (Membrane-)Proteins by Microbial 2Structural Biochemistry, Max Planck Institute of Molecular Physiology, Glycolipid and Lipopeptide Biosurfactants - in-vivo Relevance and Indus- Dortmund, Germany. trial Applications Actin is the main component of the microfilament system in eukaryotic Kell K. Andersen1, Brian S. Vad1, Marcel Stenvang1, Morten S. Dueholm2, cells and can be found in distinct morphological states. G-actin is able to Per H. Nielsen2, Daniel Otzen1. assemble into highly organized, supramolecular cellular structures, fila- 1Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, mentous (F) and bundled (B) actin. To evaluate the structure and establish Denmark, 2Department of Biotechnology, Chemistry and Environmental the stability of G-, F- and B- actin over a wide range of temperatures and Engineering, Aalborg University, Aalborg, Denmark. pressures, FTIR spectroscopy in combination with differential scanning Glycolipid and lipopeptide biosurfactants are secondary metabolites which are and pressure perturbation calorimetry, small-angle X-ray scattering, laser produced and secreted by a wide range of both pathogenic and non-pathogenic confocal scanning and transmission electron microscopy was used. On the microorganisms. Under optimal conditions production of biosurfactants can one hand, our analysis was designed to provide new insights into the stabi- reach 400 g/L. In-vivo, biosurfactants interact with a wide range of functional lizing forces of actin self-assembly and to reveal the stability of the actin proteins and biosurfactants also has the potential to substitute non-sustainable polymorphs, including conditions encountered in extreme environments. chemical surfactants in detergents where they interact with enzymes. It is there- On the other hand, our data help understand the limited pressure stability fore relevant to understand how biosurfactants in-vivo interact with functional of actin self-assembly observed in vivo. G-actin is not only the least extracellular proteins and also how biosurfactants interact with industrial deter- temperature-stable, but also the least pressure-stable actin species. At abys- gent enzymes. sal conditions, where temperatures as low as 1-4Candpressuresupto1 Biosurfactants can denature globular proteins such as myoglobin and a-lactal- kbar are reached, G-actin is hardly stable anymore. The supramolecular as- bumin. Denaturation kinetics is however an order slower when compared to semblies of actin are stable enough to withstand the extreme conditions usu- classical chemical surfactants such as SDS. Commercial detergent enzymes ally encountered on Earth, however. Beyond ~3-4 kbar, filamentous are not denatured and inactivated by biosurfactants. This is in contrast to chem- structures disassemble, and beyond ~4 kbar, complete dissociation of F- ical surfactants such as SDS. Biosurfactants are thus promising eco-friendly al- actin structures is observed. Between about 1 and 2 kbar, some disordering ternatives to chemical surfactants. 522a Wednesday, February 11, 2015

Biosurfactants can promote and stabilize protein structure. Biosurfactants fold 2642-Pos Board B72 and stabilize outer membrane proteins. The thermal stability is however per- Theoretical Prediction of Thermal-Stability Changes upon Mutations of a turbed in anionic biosurfactants compare to non-ionic dodecyl maltoside which Protein is often used for membrane protein stabilization. Shota Murakami1, Hiraku Oshima2, Tomohiko Hayashi2, Upon infections, human proteins may be exposed to secreted microbial bio- Masahiro Kinoshita2. surfactants. Lysozyme, a defensive and antimicrobial human enzyme, is highly 1Kyoto University, Graduate School of Energy Science, Uji, Japan, 2Kyoto protease resistant. However in the presence of anionic rhamnolipid, produced University, Institute of Advanced Energy, Uji, Japan. by the opportunistic pathogen Pseudomonas Aeruginosa, the positive surface Maintaining the structure and function of a protein at a temperature above the potential is neutralized and lysozyme becomes easily susceptible for both hu- denaturation temperature of its native state is a principal objective of re- man and bacterial proteases. Rhamnolipid also promotes fibrillation of FapC, searchers in a variety of fields. One of the methods of enhancing the thermal the main protein in Pseudomonas Aeruginosa functional amyloids. Rhamnoli- stability is the mutation. In the present study, we report the results of the first pid induces instant fibrillation and also morphological changes in FapC fibrils. attempt to develop a theoretical approach for predicting the thermal-stability Biosurfactants may thus play an important role in microbial biofilms and changes upon mutations of a protein without using any parameters fitted infections. to the experimental data. The approach is based on our recently developed In general, biosurfactants can interact with proteins in multifarious ways with free-energy function wherein the water-entropy effect is treated as an essential fundamental, medical and industrial relevance. factor. Protein folding is driven by a large gain of water entropy. The gain is ascribed to an increase in the number of accessible configurations of water 2640-Pos Board B70 which is brought primarily by an increase in the total volume available to the Stability Analysis of CFTR via Tryptic Digestion translational displacement of the water molecules in the bulk. This water- Christopher Holmes1,2. 1 2 entropy effect can be characterized by the following: It is reasonably taken University of Mount Union, Alliance, OH, USA, University of North into account only by a molecular model for water; not only the water Carolina at Chapel Hill, Chapel Hill, OH, USA. near the protein surface but also the water in the bulk makes a substantial Cystic fibrosis (CF) is caused by malfunctioning cystic fibrosis transmem- contribution to the effect (i.e., the effect cannot be considered in terms of brane conductance regulator (CFTR). When the flow of chloride ions the water-accessible surface area alone); and the protein-water-water triplet through the CFTR channel becomes hindered or ceases, mucus begins to and higher-order correlations play critical roles. Our theoretical approach can accumulate within the body causing a plethora of respiratory, digestive, also be distinguished from the previously reported approaches in the respect and reproductive complications. From the 1,500 different CFTR mutations that the water-entropy effect is taken into account to its full extent. Its perfor- that exist, a genetic mutation resulting from the deletion of a phenylalanine mance is compared with that of FOLD-X, one of the most popular approaches residue at the 508 position (DF508) has been identified as the most common. using the fitting parameters, for ten proteins and single and multiple mutations. For that reason, the DF508 mutation has become a target for initial treatment Ours is shown to be superior to FOLD-X. options. Due to the transmembrane nature of CFTR and a length of 1480 residues, the crystal structure and consequently the functionality of CFTR, 2643-Pos Board B73 are still vastly unknown. The objective of this research is to more thor- Tuning of Alpha-Synuclein Aggregation by Small Molecules and Bacterial oughly understand the relative stability of CFTR, the DF508 mutant and Proteins the treated DF508 mutant via limited tryptic digestion and the quantification Pernilla Wittung-stafshede. of western blots. The DF508 mutant was treated with an energy source in chemistry, Umea˚ Univeristy, umea˚, Sweden. which it typically binds (ATP), a non-hydrolysable ATP analogue (AMP- Parkinson’s disease affects a growing number of the population and involves PNP), and with an available cystic fibrosis treatment option (lumacaftor/ motor complications due to the death of dopamine neurons. Cytosolic inclu- VX-809). To adequately represent both of the nucleotide binding domains sions containing amyloid fibrils of a-synuclein are a hallmark of the disease (NBDs) in CFTR, antibodies 660 (IGg1) and 769 (IGg2b) were used. The and it is believed that the aggregation process (going from monomers to quantification of results indicated that the induced stability of the AMP- amyloid fibers) of alpha-synculein somehow causes neurodegeneration. The PNP treated and ATP treated DF508 CFTR were significantly greater than synuclein-rich inclusions share structural characteristics with amyloid fibers the DF508 mutant alone, but not significantly greater than the VX-809 found in many other neurodegenerative disorders. In addition, many organisms treated CFTR. employ amyloid structures for mechanical or biological functions; for example, amyloid fibers are the major component of microbial biofilms. Mature amyloid 2641-Pos Board B71 fibers of alpha-synculein may not be the source of cytotoxicity; instead, tran- Modulation of Cytochrome C Stability during Chemical and Thermal sient oligomeric structures may be most dangerous to the neuronal cells. To Denaturization by Addition of Hofmeister Ions investigate molecular pathways leading to alpha-synculein amyloid fibers, Eric S. Peterson, Mikayla J. Freese, Sean J. Steinke. and thereby get hints for how to combat Parkinson’s disease in vivo, we Chemistry and Biochemistry, University of Northern Iowa, Cedar Falls, have taken a unique approach that involves purified proteins, biophysical IA, USA. experiments in vitro, and small-molecule tools. We have found that strategic The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of ring-fused 2-pyridone compounds (mimics of small peptides), can tune ions from the Hofmeister series is examined. Unfolding was initiated both alpha-synuclein aggregation such that either inhibitory or templating oligomers thermally and with chemical denaturants. Hofmeister ions were added singly accumulate. Moreover, a fine balance between templation and inhibition and in pairs to alter the stability of the native folded state, the unfolded state, processes is evident since one particular 2-pyridone inhibits bacterial amyloid and two partially folded intermediates. Protein stability was characterized by formation but promotes alpha-synuclein amyloid fibers. In analogy with the either the midpoint of the chemical denaturization curve or by the melting small molecule tools, we found that bacterial proteins can cross-react with temperature in the thermal studies. UV/VIS absorption spectroscopy and a alpha-synculein and inhibit as well as promote amyloid fiber formation at basis spectra fitting analysis were used to determine the populations of each sub-stoichiometric levels. Direct interactions of alpha-synculein with bacterial protein conformation along the folding pathway. These species can be differ- proteins and/or natural metabolites may play a role in controlling Parkinson’s entiated by their axial heme ligands. Four species exist in solution: the native disease in humans. HM state (His18/Met80), the partially folded HW (His18/water) and HH (His18/His33) intermediates, and the 5C (water) unfolded state. The results 2644-Pos Board B74 indicate that the thermal and chemical denaturization pathways are not the Ezrin is a Potent Modulator of Alpha-Synuclein Oligomerization same and that both involve significant backbone rearrangement. The relative Timea E. Illes-Toth1, Maliha Shah2, Nelson Wu1, Silvia Verzini3, populations of the conformational states depends on how the protein is dena- Philipp Selenko3, Erich Wanker2. tured. Additionally, it was found that addition of multiple ions changed the 1Biomedical Engineering, Washington University in St Louis, St Louis, MO, protein’s stability in an additive manner. For example addition of guanidi- USA, 2Dept of Neuroproteomics, Max-Delbrueck Centrum, Berlin, nium, which destabilizes folded structure, can be countered by addition of Germany, 3Leibniz Institute of Molecular Pharmacology (FMP), Berlin, phosphate, which stabilizes folded structure. Both the (un)folding kinetics Germany. and the accessible conformations were found to depend on the identity of Alpha-synuclein oligomers are associated with toxicity in Parkinson’s disease. ions present. These results are discussed in terms of the hydrophobic effect, Different forms of alpha-synuclein (aS) have been described, some of which partitioning of the ions to the protein surface, and an altered water structure can destabilize lipid bilayers, and seed the formation of fibrillar assemblies. around the protein. Therapeutic interventions such as molecular chaperones and small molecule Wednesday, February 11, 2015 523a

inhibitors are sought to depopulate these transient intermediates and thus Glun structures along MD trajectories that show evidence of sidechain ordering diminish their toxicity. in qualitative agreement with experiment. We have identified ezrin, a microtubule-associated protein that belongs to the ezrin radixin moesin family, as a potent inhibitor of aS aggregation at sub- stoichiometric concentrations. Evidence showed that binding of ezrin to aS Protein Assemblies II causes conformational rearrangements and sequesters the protein in amorphous aggregates thus preventing fibril formation and rescuing toxicity in primary 2647-Pos Board B77 neurons and yeast cells, thereby acting as a non-conventional chaperone. Assembly of the RIG-I 2CARD Signaling Complex We have produced and characterized alternate aS oligomers by size exclusion David Mouser. chromatography, circular dichroism, TEM, dot blotting and ThT fluorescence. Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA. One group of oligomers is associated with a higher b-sheet content and can be The innate immune system of mammals utilizes pattern-recognition receptors internalized in human neuronal model cells. Following treatment with these to differentiate between pathogen associated molecular patterns and host mol- oligomers prepared with incorporation of fluorescently labeled aS, we observed ecules. The retinoic acid inducible-gene-I (RIG-I) like receptors (RLRs) bind their uptake into human SH-EP cells. The other group of oligomers is A11 anti- viral dsRNAs, triggering an innate immune response. RLRs contain two N-ter- body reactive, mainly unstructured and result in significant cell death. minal caspase activation and recruitment domains (2CARD), a DExD/H-box We hypothesize that binding of ezrin may alter the biophysical, biological char- helicase-like domain, and a C-terminal regulatory domain. In the absence of acteristics and aggregation properties of aS oligomers. Current efforts are RNA, the 2CARD region is sequestered by the helicase domains in an autoin- 0 concentrating on investigating the interaction of ezrin with these alternate olig- hibited state. Upon binding to the 5 -ends of activating viral dsRNAs a confor- omeric species. mational change exposes 2CARD. The CARD domains then interact with MAVS, a mitochondrial outer membrane protein, leading to downstream 2645-Pos Board B75 signaling and interferon expression. Signaling is modulated by K63-linked Electrostatics Promotes Molecular Crowding and Selects the Fibrillation polyubiquitin chains (polyUb). It has been reported that polyUb binding to Pathway in Fibril-Forming Protein Solutions 2CARD induces formation of 2CARD tetramer. However, the nature of the as- Samuele Raccosta, Vincenzo Martorana, Mauro Manno. sembly process is not well understood and we are investigating the thermody- Institute of Biophysics, National Research Council of Italy, Palermo, Italy. namic linkage between polyUb binding and 2CARD oligomerization using The role of intermolecular interaction in fibril-forming protein solutions and its sedimentation velocity analytical ultracentrifugation. 2CARD alone exists as relation with molecular conformation is a crucial aspect for the control and in- a monomer; however, in the presence of polyUb several high molecular weight hibition of amyloid structures. Here, we use optical spectroscopies, x-ray and complexes are formed. The assembly process is dependent on salt, polyUb con- light scattering to study the fibril formation and the protein-protein interactions centration, and polyUb chain length. of different model proteins. In the case of lysozyme, the monomeric solution is kept in a thermodynamically metastable state by strong electrostatic repulsion, 2648-Pos Board B78 even in denaturing conditions. At high temperature proteins are driven out of Characterization of the MDA5 2CARD Signaling Complex metastability through conformational sub-states, which are kinetically popu- Cassie Zerbe, James Cole. lated and experience lower activation energy for fibril formation. This explains University of Connecticut, Storrs, CT, USA. how electrostatic repulsion may act as a gatekeeper in selecting the appropriate The mammalian innate immune system is the first line of defense against pathway to fibrillation. The protein density fluctuations are measured by light invading pathogens. The RIG-I like receptors (RLRs), RIG-I and MDA5 are scattering and exhibit a regime dominated by protein concentration. Highly critical for host recognition of viral RNAs. These receptors contain a charged colloidal solutions are characterized by damped density fluctuations helicase-like core domain, two caspase activation and recruitment domains at and mimics locally a crowded environment. Further, by photon correlation the N-terminus (2CARD) and a C-terminal regulatory domain. In RIG-I, spectroscopy (PCS) and fluorescence correlation spectroscopy (FCS) we mea- RNA binding leads to exposure of the 2CARD domain and 2CARD oligomer- sure protein collective and self-diffusion. Both PCS and FCS evidence how an ization. The 2CARD signaling domain associates with the CARD domain of increase of concentration hinders protein self-diffusion, likely due to both hy- MAVS, a mitochondrial outer membrane protein, leading to oligomerization drodynamics and crowding effect. of MAVS, downstream signaling and interferon induction. K63-linked polyu- biquitin chains (polyUb) play a role in RLR signaling by interacting with the 2646-Pos Board B76 2CARD domain. PolyUb binding to RIG-I 2CARD induces formation of Role of Side-Chains in Forming Peptide Aggregates and Fibrils. IR and 2CARD tetramer. However, the nature of the signaling complex of MDA5 VCD Spectroscopic Studies. Theory and Experiment and the role of polyUb binding in MDA5 activation are not well understood. 1 1  2 3 2 Heng Chi , Fernando Tobias ,Jirı´ Kessler , Jan Kubelka , Petr Bour , We have used sedimentation velocity analytical ultracentrifugation to charac- Timothy A. Keiderling1. terize MDA5 2CARD oligomerization in absence and presence of polyUb. 1 Department of Chemistry, University of Illinois at Chicago, Chicago, IL, MDA5 2CARD forms a broad distribution of oligomers. The size distribution 2 USA, Institute of Organic Chemistry and Biochemistry, Academy of is concentration dependent, indicating reversibility. MDA5 2CARD forms 3 Sciences of the Czech Republic, Prague, Czech Republic, Department of complexes with polyUb, and complex formation is highly dependent on Chemistry, University of Wyoming, Laramie, WY, USA. NaCl concentration. Multisignal sedimentation velocity experiments will probe Formation of peptide aggregates and fibrils must ultimately be driven by inter- the nature of the polyUb-2CARD complexes. actions of side chains. Typically the lower solubility of hydrophobic sequences favors desolvation by formation of interchain contacts which are then stabilized 2649-Pos Board B79 further by various means. One way is development of ordered interdigitization Detailed Mechanism of Rapid Amyloid Fibril Self-Assembly Due to Sur- of the side chains, which is particularly evident in poly-glutamic acid at low face Diffusion pH. The Glu side chain -COOH groups form bifurcated H-bonds with the amide Yi-Chih Lin, E. James Petersson, Zahra Fakhraai. C¼O groups that are cross-strand H-bonded in what is termed a b2 sheet. This Chemistry, University of Pennsylvania, Philadelphia, PA, USA. extra H-bond interaction leads to large shifts of the amide I vibrational fre- Surface-mediated fibrillization has been considered as an alternative pathway quencies and formation of stacked arrays of b-sheets. In other systems, hydro- to form amyloid fibrils under physiological conditions. In order to develop phobic interactions can results in similar stacking interactions. Vibrational new probes and therapies for neurodegenerative diseases, such as Alzheimer’s spectroscopic methods have been used to establish b-sheet structures underly- and Parkinson’s diseases, it’s important to explore the detailed mechanism ing many of these and isotope labeling has been used to differentiate parallel vs. of the surface effects. However, the surface morphologies change with time antiparallel forms. Our DFT-based calculational model of b-sheet vibrational and are hard to image without staining or labeling, which may alter the spectra was originally based on Ala-oligopeptides, forming normal b1 sheets, process of self-assembly. In recent work, we demonstrated a label-free proce- and focused on IR, VCD, and Raman spectra for isolated and stacked sheet dure via controlling incubation time and the drying process to monitor the ki- structures, with variations in structure, alignment of strands, isotope labeling, netics of fibril growth on surfaces across many length scales. We propose twisting of single sheets and relative rotation of multiple sheets in the stack. an anisotropic growth model to fit the time-evolution of morphologies of In particular, our analyses of the spectra of Glu10 based peptides that form amyloid fibrils forming near a surface as measured using Atomic force micro- b2 sheets, are shown to be antiparallel but out of register by one residue, scopy (AFM). Two important variables, the deposition rate and the two- most likely in a step-wise pattern. Alternate (Glu-Aaa)n variants, where Aaa dimensional diffusion coefficient, can be used as fitting variables and can is a hydrophobic residue, can also show this pattern of intersheet side-chain be characterized by the combination of experimental data and the theoretical intercalation. We extended these calculations by computing IR and VCD for approach presented here. 524a Wednesday, February 11, 2015

2650-Pos Board B80 Florida, Orlando, FL, USA, 4Department of Physics, University of Central Co-Aggregation of Alpha-Synuclein with Amylin(HIAPP) Leads to an Florida, Orlando, FL, USA. Increased Risk in Type ii Diabetes Patients for Developing Parkinson’s Amyloid beta-peptide (Abeta) forms cytotoxic assemblies that contribute to Disease Alzheimer’s disease. Recent evidence indicates that prefibrillar aggregates Yoav Atsmon-Raz, Yifat Miller. and not the fibrillar deposits exert the main toxic effect. In addition, naturally Ben Gurion University in the Negev, Beer Sheva, Israel. occurring N-terminally truncated and pyroglutamylated peptide (pE-Abeta) Clinical studies have reported that Type II diabetes patients (T2D) are more displays augmented cytotoxicity by an unknown mechanism. This study exam- likely to develop Parkinson’s disease (PD). However, the mechanisms which ines the conformational changes in both unmodified Abeta and pE-Abeta upon link these two diseases have remained elusive. In T2D, Amylin forms aggre- exposure to an aqueous environment. FTIR and circular dichroism were used to gates in the pancreas. However, it can also form neurotoxic oligomers in the identify alpha-helix-to-beta-sheet conformational transitions of both peptides brain. In PD, a-synuclein also forms neurotoxic amyloid aggregates. This during aggregation. To gain site-specific structural information, the peptides work hypothesizes a new mechanism in which Amylin and a-synuclein may were 13C,15N-labeled at residues 16-18 (KLV) or 36-39 (VGGV), followed co-aggregate together and thus patients with T2D have an increased risk to by FTIR analysis. The peptides dried from hexafluoroisopropanol were develop PD. Since the non-amyloidogenic component (NAC) domain plays a alpha-helical (amide I peak at 1660-1657 cm-1) and showed negligible inten- major role in a-synuclein aggregation, we investigated the co-aggregation of sity of the labeled segments around 1615 cm-1, suggesting that in alpha- Amylin-NAC oligomers. helical conformation the labeled amide groups behave like isolated oscillators. In this study, we have constructed four different Amylin oligomeric structures Upon addition of aqueous buffer (pH 7.2) both peptides rapidly adopted beta- and one NAC oligomeric structure, based on solid state NMR (ssNMR). We sheet structure (amide I peak at 1637-1629 cm-1), with disproportionally prom- then constructed 12 Amylin-NAC oligomeric complexes while taking into inent components around 1604-1597 cm-1 generated by the labeled segments. consideration both single and double layered conformations. We then applied The intensity and the frequency of the amide I mode of the isotope-labeled seg- molecular dynamics simulations to investigate the stability of these structures. ments suggest 12C-13C vibrational coupling, consistent with formation of anti- Our study has revealed three conclusions: 1) Amylin-NAC oligomers demon- parallel beta-sheet structures. Moreover, the amide I contours of the peptides strate polymorphic states with a preference towards two of the four Amylin under near-physiological and low ionic strength conditions were significantly oligomeric structures that co-aggregate with NAC.; 2) The Amylin-NAC olig- different; both peptides exhibited an increased alpha-helical and decreased omers’ interactions at an atomistic level have been identified for the first time; beta-sheet propensity under low ionic strength conditions, indicating a strong 3) Amylin prefers to form single layer conformations with NAC over double influence of the ionic strength on the aggregation kinetics and accompanying layered conformations. structural changes. Ongoing studies focus on structural differences between the unmodified Abeta and pE-Abeta peptides as well as their mutual structural 2651-Pos Board B81 effects when combined at various molar ratios, in an attempt to understand the Insight into the Metal Binding Sites in Amylin Aggregates structural basis of the elevated cytotoxicity of pE-Abeta. Vered Wineman-Fisher, Yifat Miller. Department of Chemistry, Ben-Gurion University, Beer-Sheva, Israel. 2654-Pos Board B84 Amylin peptide consists of 37 residues. The aggregation of Amylin is one of the Preparation Protocols of Beta-Amyloid (1-40) Promote the Formation of symptoms of type 2 diabetes (T2D). Amylin’s oligomers that are toxic lead to Polymorphic Aggregates and Altered Interactions with Lipid Bilayers b-cells death and thus to decreasing of insulin’s release to the blood and to pro- Elizabeth A. Yates1,2, Justin Legleiter2. gressing of T2D. The factors that affect Amylin aggregation are elusive, how- 1Chemistry, United States Naval Academy, Annapolis, MD, USA, ever it is known that Amylin peptides are found with insulin and zinc ions in the 2Chemistry, West Virginia University, Morgantown, WV, USA. pancreatic b-cells and that zinc ions bind to Amylin oligomers and may inhibit The appearance of neuritic amyloid plaques comprised of b-amyloid peptide Amylin aggregation. So far, it is unknown how zinc ions bind Amylin oligo- (Ab) in the brain is a predominant feature in Alzheimer’s disease (AD). In mers at the atomic resolution. Understanding the mechanism of zinc-binding the aggregation process, Ab samples a variety of potentially toxic aggregate sites in amylin oligomers is important for effective drug design to prevent species, ranging from small oligomers to fibrils. Ab has the ability to form a and alleviate aggregation. We constructed Amylin oligomers based on ssNMR variety of morphologically distinct and stable amyloid fibrils. Commonly and x-ray crystallography. These experimental studies illustrate four different referred to as polymorphs, such distinct aggregate species may play a role in Amylin oligomeric models, which differ in the orientation of His18 in accor- variations of AD pathology. It has been well documented that polymorphic ag- dance of the core domain of Amylin. Other ssNMR study proposed that the gregates of Ab can be produced by changes in the chemical environment and binding site of zinc ions is His18. We applied molecular dynamics simulations peptide preparations. As Ab and several of its aggregated forms are known to examine our constructed models. Two main conclusions had been obtained to interact directly with lipid membranes and this interaction may play a role from our simulations. First, the binding site of zinc in Amylin is His18 which is in a variety of potential toxic mechanisms associated with AD, we determine located outside the core domain. Second, the zinc:Amylin ratio is 1:2. how different Ab(1-40) preparation protocols that lead to distinct polymorphic fibril aggregates influence the interaction of Ab(1-40) with model lipid mem- 2652-Pos Board B82 branes. Using three distinct protocols for preparing Ab(1-40), the aggregate Characterizing Kinetic Intermediate in Amyloid Self-Assembly species formed in the absence and presence of a lipid bilayer were investigated Chen Liang. using a variety of scanning probe microscopy techniques. The three prepara- Chemistry, Emory university, Atlanta, GA, USA. tions of Ab(1-40) promoted distinct oligomeric and fibrillar aggregates in the Amyloid self-assembly is tightly associated with protein misfolding diseases absence of bilayers that formed at different rates. Despite these differences such as Alzheimer’s disease, but the pathway of self-assembly as well as in aggregation properties, all Ab(1-40) preparations were able to disrupt sup- the genesis of amyloid polymorphism remains unclear. Combining isotope- ported total brain lipid extract (TBLE) bilayers, altering the bilayer’s morpho- edited IR and solid state NMR, we experimentally demonstrated that the logical and mechanical properties. nucleating core of Dutch mutant, Ab (16-22) E22Q or Ac-16KLVFFA22Q- NH2 assembles into a kinetic intermediate of anti-parallel b-sheet that later 2655-Pos Board B85 transition automatically into parallel arrays as thermodynamically stable con- Thermodynamics of Abeta(16-21) Dissociation from an Amyloid Fibril formation. Additionally, we developed a method of quantifying such transition Cristiano L. Dias, Srinivasa Rao Jampani. using IR spectroscopy. Our findings reveal that the process of amyloid self- Physics, New Jersey Institute of Technology, Newark, NJ, USA. assembly is subject to both kinetic and thermodynamic control and that Here, we study the thermodynamic properties of Ab16-21 dissociation from an the actually mechanism of self-assembly could be far more complicated than amyloid fibril using all-atom molecular dynamics simulations and TIP4P water. currently expected. An umbrella sampling protocol is used to compute potentials of mean force (PMF) of peptide dissociation at five temperatures as well as changes in 2653-Pos Board B83 enthalpy, entropy and heat capacity upon dissociation. We find that similarly Site-Specific Structural Changes in Unmodified and Pyroglutamylated to protein unfolding, Ab16-21 dissociation is characterized by an unfavorable Amyloid Beta Peptide by Isotope-Edited Ftir change in the enthalpy (DH>0), a favorable entropic energy (-TDS<0), and 1 2 3 3 Greg Goldblatt , Jason O. Matos , Jeremy Gornto , Laura N. Puentes , an increase in the heat capacity (DCp>0). The exposure of non-polar residues Angel Docobo3, Suren A. Tatulian4. that are initially buried in the dry core of the fibril and become exposed to water 1Biomedical Sciences Graduate Program, University of Central Florida, as the peptide dissociates can be associated to the positive change in heat ca- Orlando, FL, USA, 2Biotechnology Graduate Program, University of Central pacity. The increased freedom of the backbone and the loss of native contacts Florida, Orlando, FL, USA, 3Undergraduate student, University of Central as the peptide dissociates from the fibril can explain the favorable entropy Wednesday, February 11, 2015 525a and unfavorable enthalpy, respectively. Furthermore, we characterize dock and 2658-Pos Board B88 lock states of the peptide based on the solvent accessible surface area. We Structural Variations of Amyloid b-Protein Fibrils Seeded with Extracted observe that the Lennard-Jones energy of the system increases continuously Fibrils from Brain Tissue of Alzheimer’s Disease Model Mice in lock and dock states as the peptide dissociates. The electrostatic energy in Hiroaki Komatsu, Paul H. Axelsen. the lock state increases as the peptide dissociates and inter-peptide hydrogen Pharmacology, University of Pennsylvania, Philadelphia, PA, USA. bonds are ruptured while it decreases in the dock state as new peptide-water In our previous studies by electron microscopy (EM) and two dimensional hydrogen bonds are formed. We also observe that before unbinding from the infrared spectroscopy of amyloid fibrils prepared in vitro from synthetic 40- fibril, the peptide has to overcome an enthalpic barrier of the order of 10 residue b-amyloid (Ab40) peptides, it was shown that the Ab40 fibril structure kJmol1. This barrier is associated to interactions between exposed phenylala- and its molecular structure are not uniquely determined by amino acid nine residues of the fibril and the peptide. Implication of these results to fibril sequence. Instead, the fibril structure is dependent upon the growth conditions. growth will be discussed. The molecular structures of b-amyloid fibrils that develop in Alzheimer’s dis- ease (AD) are therefore uncertain. In this study, fibrils extracted from brain 2656-Pos Board B86 tissue of AD model mice (three strains of aged transgenic mice with increased A Computational Study of Amyloid b-Protein Assembly in Crowded Envi- levels of human amyloid proteins, 3xTg-AD, J20Tg-AD and 5xFAD) were ronments used to seed the growth of synthetic Ab40 fibrils. Because amyloid fibril Matthew J. Voelker, Brigita Urbanc, Mark Betnel. structures propagate themselves via seeded growth, the structures of seeded Physics, Drexel University, Philadelphia, PA, USA. Ab40 fibrils likely reflect structures in AD brain. Negatively stained EM im- Alzheimer’s disease is strongly associated with aberrant amyloid b-protein ages indicate that seeded fibrils tend to appear twisted like a ribbon, with pe- (Ab) assembly into heterogeneous, metastable oligomeric assemblies with riodic narrowing or nodes. The distances between nodes (the ‘‘internodal’’ structures that have not been experimentally characterized yet. The 40 and distances) were relatively homogeneous distributions (~145 nm) in the 42 amino acids long Ab40 and Ab42 are the two predominant Ab alloforms 3xTg-AD and J20Tg-AD mice. In 5xFAD, on the other hand, fibrils having in the brain. Whereas Ab40 and Ab42 oligomer formation from monomeric short internodal distances (~30 nm) were observed in addition to the distribu- state is still inaccessible to fully atomistic explicit-solvent molecular dynamics, tions (~145 nm) which were measured in two other strains of mice. The mass- Ab40 and Ab42 oligomers were structurally characterized using discrete mo- per-length (MPL) evaluated from dark-field EM images indicates that the lecular dynamics (DMD) and an intermediate-resolution protein model within most prevalent numbers of filaments in fibrils are 3 and 4 but the dominant the DMD4B-HYDRA implicit solvent force field, and the corresponding olig- number of the filaments is dependent on the source of the fibril seeds. Overall, omer size distributions well matched the available in vitro data. In vivo, how- these results demonstrate that AD model mouse brain-derived fibrils have a ever, Ab coexists with other biomolecules in a rather crowded environment. To distinct fibril structures, and the model most relevant to human AD has yet understand the effect of crowding on Ab oligomer formation, we used the to be determined. DMD4B-HYDRA force field and added to an ensemble of 32 monomeric Ab40 or Ab42 peptides inert spherical ‘‘crowders’’ with a diameter of 0.5 2659-Pos Board B89 nm at various concentrations to examine their effect on Ab40 and Ab42 olig- Determination of Nucleation Mass for Amyloid-b Aggregation omerization pathways. Our results show that crowding shifts oligomer size dis- Preetam Ghosh1, Amit Kumar2, Vijayaraghavan Rangachari3, tributions towards smaller oligomer sizes and increases solubility of both Ashwin Vaidya4. peptides in a concentration-dependent way. The effect is stronger for Ab42, 1Computer Science, Virginia Commonwealth University, Richmond, VA, where crowding abolishes the multimodal character of the oligomer size distri- USA, 2Max Planck Institute, Tubingen, Germany, 3Chemistry and bution. Our structural analysis revealed that the stability of larger oligomers is Biochemistry, University of Southern Mississippi, Hattiesburg, MS, USA, compromised by effective osmotic pressure exerted by the crowders, resulting 4Mathematical Sciences, Montclair State University, Montclair, NJ, USA. in an increased rate of assembly breakage. While in vivo crowding agents are Protein misfolding and concomitant self-assembly towards ordered aggregates not inert as the crowders in our study, we here reveal that crowding-induced (amyloids) has emerged as an important event governing both functional and osmotic pressure strongly affects protein assembly dynamics, which is of sig- pathological events in cells. Both structurally and biophyiscally, amyloid for- nificance to the disease. mation is highly conserved involving the conversion of proteins (intrinsically disordered or globular) from their native monomeric states to well-organized, 2657-Pos Board B87 fibrillar aggregates in a nucleation-dependent manner. Although a plethora of Transition of Amyloid Oligomers to Mature Fibrils: Internal Conversion literature exists on modeling such aggregations, the molecular mechanisms Vs. Competing Assembly Pathways? are poorly understood, especially those leading up to nucleation. In our study Tatiana Miti, Mentor Mulaj, Martin Muschol. we use Ab as the model system to test our theoretical framework for amyloid physics, University of south Florida, tampa, FL, USA. aggregation. Specifically, we focus on nucleation, which we believe to be a Deposition of protein plaques, rich in long rigid fibrils with a characteristic critical gate-keeping event which controls the dynamics of the entire pathway cross-beta sheet structure, is the pathological marker for human disorders and determines the physiochemical and biochemical fate of the aggregates ranging from Alzheimer’s disease to type II diabetes and rheumatoid arthritis. formed. In this study we clarify the mechanics of aggregation leading to Significant evidence has implicated the formation of globular oligomeric am- nucleation, and how fibril morphology depends on size and conformation of yloids as the main pathogenic agent in amyloid diseases. At the same time, in the nucleus. The pre-nucleation dynamics are modeled by ODE simulations vitro experiments indicate that amyloid oligomers and rigid fibrils are formed based upon mass action kinetics and also supported by experimental data. along distinct assembly pathways with characteristic growth kinetics. This rai- An alternative, novel approach, based upon stability of the equilibria is uti- ses the questions how these early-stage oligomeric intermediates are con- lized to identify the optimal nucleation mass range and properties associated verted to the rigid fibrils dominating during the late-stages of most amyloid with the nucleus. diseases? We have investigated the transition from the formation of amyloid oligomers 2660-Pos Board B90 and their curvilinear polymers to the rigid late-stage fibrils using the model Morphology Selection through Geometric Frustration in Twisted Filament amyloid hen egg white lysozyme (hewL). We have shown that hewL oligo- Bundles and Fibers mers form a distinct aggregate phase with a well-defined transition boundary. Doug Hall1, Isaac R. Bruss1, Justin Barone2, Gregory M. Grason1. However these oligomers and their curvilinear fibrils are metastable against 1Polymer Science and Engineering, University of Massachusetts, Amherst, the formation of thermodynamically stable rigid fibrils. We therefore per- MA, USA, 2Biological Systems Engineering, Virginia Polytechnic Insitute, formed experiments to discern whether amyloid oligomer species were Blacksburg, VA, USA. directly converted into the stable rigid fibril conformation or whether rigid Rope-like assemblies of twisted protein filaments constitute a common fibril nucleation proceeded in parallel, i.e. in competition with, oligomer for- materials archetype appearing in a range of biological contexts from extra- mation. To do so, we monitored the rates of rigid fibril nucleation right cellular filament bundles to amyloid fibrils. Owing to the numerous distinc- outside and inside the transition boundary for oligomer formation. Our data tions in molecular structure and interactions underlying these diverse suggest that oligomer formation is in kinetic competition with rigid fibril assemblies, a common framework to predict and classify the basic mecha- nucleation for their monomeric growth substrate. Futhermore, we observed nisms of structure formation in twisted filament assemblies is still lacking. no signs that prior formation of oligomeric species accelerated the nucleation In this study, we exploit a recent and surprising connection between the as- of rigid fibrils. The latter would be expected for conformational conversion of sembly of self-twisting filaments and assembly on spherically-curved 2D oligomers into rigid fibrils. If anything, prior formation of oligomers retards surfaces to develop a universal theory of morphology selection in twisted the nucleation of rigid fibrils. fibers and bundles. This theory shows that the size and cross-sectional 526a Wednesday, February 11, 2015 shape of self-assembled fibers is determined by competition between the heterotetramers can modify water transport activity and pH sensitivity elastic costs of inter-filament frustration, bending deformation of constitu- by shifting the EC50 of the inhibitory response. Moreover, the first extracel- ent filaments and surface energy of fibers. We find that for sufficiently lular loop of PIP2 acts as a crucial structural element to achieve PIP large twist, isotropic (cylindrical) bundles are generically unstable to devel- heterotetramerization. oping anisotropic cross-sections (helical tapes). Critically, the anisotropy Thus, our findings throw light not only on PIP heterotetramerization as a novel of fiber cross-sections is found to give a direct measure of the anisotropy regulatory mechanism to adjust water transport across the plasma membrane of inter-filament vs. intra-filament elasticity. We corroborate the universal but also on the stoichiometry of PIP heterooligomers, issues that had been un- predictions of our theory with numerical simulations of self-twisting clear for many years in the biophysical field of plant aquaporins. fibers and compare the morphology diagram structural observations of anisotropy of micron-scale amyloid fibers assembled from hydrolyzed pro- 2663-Pos Board B93 tein fragments. Landscapes of Membrane Protein Interactions from High-Throughput MD Simulations using the Daft Approach 2661-Pos Board B91 Tsjerk A. Wassenaar1,2, Kristyna Pluhackova1, Anastassiia Moussatova3, Novel Single-Molecule Resolution Method for Spatio-Temporal Simula- Durba Sengupta4, Siewert J. Marrink2, D. Peter Tieleman3, tions of Protein Binding and Recruitment on the Membrane Rainer A. Bo¨ckmann1. Osman N. Yogurtcu, Margaret E. Johnson. 1Computational Biology, University of Erlangen-Nu¨rnberg, Erlangen, Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA. Germany, 2Molecular Dynamics, University of Groningen, Groningen, In the early stages of clathrin-mediated endocytosis (CME), a variety of Netherlands, 3Biocomputing, University of Calgary, Calgary, AB, Canada, distinct proteins can bind to the membrane and engage in further interac- 4Physical Chemistry, National Chemical Laboratory, Pune, India. tions with proteins on the membrane and in solution. Understanding the dy- Interactions between membrane proteins are key in many biological and namics of this process requires correctly accounting for the behavior of pathological processes and offer potential targets for pharmacological inter- protein interactions while restricted to the 2D membrane surface, as it is vention. Unfortunately, the complex environment makes it difficult to fundamentally distinct from binding in solution due to changes in the dy- explore these in high detail. In addition, the time scales of binding and un- namics of the proteins. Here we introduce the 2D Free-Propagator Re- binding pose difficulties for molecular simulations to probe such interac- weighting (2D-FPR) method that accurately models the spatial and temporal tions. Over the past few years, we have developed an approach using dynamics of proteins as they are recruited to the membrane surface and as large numbers of simulations, which avoids the problem of unbinding, allow- they interact with one another while bound to the membrane. In this method ing rapid building of a detailed map of the interaction landscape. The the position of each diffusing protein is tracked, and reactions between bind- method, called Docking Assay For Transmembrane components (DAFT), ing partners can occur upon collisions. Reaction probabilities are determined has to date been used to investigate a range of 23 Glycophorin A mutants, by the solution to the 2D Smoluchowski diffusion equation with reactive a set of 40 receptor tyrosine kinase (RTK) pairs, SNARE protein TM helices, boundary conditions, allowing us to take large time steps. Molecule posi- the DesK minimal thermosensor and GPCRs, accounting for >15M CPU tions are propagated by free diffusion, but by using a trajectory reweighting hours and representing a total simulation time of more than 30 milliseconds. approach we can recover the exact association rates for all reactive pairs. The results show that several hundreds of simulations are necessary for a This approach is uniquely able to capture the changes in protein binding dy- converged view and that the time scales required range from 300 ns per namics that can occur upon membrane binding because it accounts for both simulation for simple helices to microseconds for larger and more complex the diffusional motion of proteins and their binding reactions. These impor- systems. Yet the results also provide unique views on the convergence prop- tant details are absent from models that lack spatial resolution. We present erties of ensembles of simulations, yield detailed maps of interaction land- our simulation results on modeling adaptor protein interaction dynamics, scapes, and allow 2D-PMFs to be derived. Furthermore, the comparison of and discuss the effects of varying local protein concentration on both different members of the RTK family of or wild type proteins and mutants recruitment to the membrane and complex formation in the confined 2D gives insight in the mechanisms underlying the relative propensities to geometry. dimerize. 2662-Pos Board B92 2664-Pos Board B94 Stoichiometry as a Key Modulator of PIP Plant Aquaporins Biological Increased Signaling Efficiency of Conventional PKC through Self- Activity Assembled Clustering on the Plasma Membrane Cintia Jozefkowicz1, Lorena Sigaut2, Agustina Canessa Fortuna3, Xin Hui1, Mike Bonny2, Lars Kaestner1, Karsten Kruse2, Peter Lipp1. Florencia Scochera3, Luis Gonza´lez Flecha4, Lia I. Pietrasanta2, 1Institute for Molecular Cell Biology, Saarland University, Homburg/Saar, Nicola´s D. Ayub5, Gabriela Soto5, Gabriela Amodeo1, Karina Alleva3. Germany, 2Theoretical Physics, Saarland University, Saarbru¨cken, Germany. 1IBBEA (CONICET-UBA), CABA, Argentina, 2CMA, Dpto de Fı´sica, The Ca sensitive conventional Protein Kinases C (cPKCs) play critical roles FCEN, UBA; CONICET, CABA, Argentina, 3IBBEA (CONICET-UBA); in signaling processes. cPKCs are recruited to the plasma membrane by Ca Dpto. Fisicomatema´tica, FFyB, UBA, CABA, Argentina, 4IQUIFIB, UBA - binding to its . Diacylglycerol (DAG) at the membrane enforces CONICET, CABA, Argentina, 5Instituto de Gene´tica Ewald A. Favret cPKC-membrane binding via its C1 domain and activates the kinase. Phos- (CICVyA-INTA), CABA, Argentina. phorylation rates of cPKCs are around 6 per second. During the average The specific self-association of membrane channels to form oligomeric assem- Ca-dependent membrane residence time of some tens of milliseconds at blies is a biological relevant event, which usually confers functional advantages most one phosphorylation event may take place. How cells overcome this to biological systems. Evidences strongly support that plant plasma membrane apparent limitation is still unclear. We used quantitative video- and confocal aquaporin (PIP) can physically interact to form oligomers by combining PIP2 FRET measurements of PKCa-CFP and -eYFP and mathematical modeling and PIP1 monomers; however, the kind of oligomer and/or its stoichiometry of putative PKC-PKC interactions at the plasma membrane to address this. has not been experimentally elucidated yet. Following ATP stimulation, membrane recruitment of PKCa was accompa- Along this research, we aim at defining whether aquaporins are able to form nied by substantial increases in FRET. The FRET signal decayed significantly functional hetero-tetramers (different PIP monomers organized in a single slower than the underlying Ca transient. Sole increases in the intracellular tetramer) with a given stoichiometry that determines their activity and Ca concentration were also sufficient to induce long-lasting FRET signals regulation. resulting from membrane-bound PKCa. Since the FRET signal spontaneously To achieve this goal, we examine the functional properties of heterotetramers decayed at high Ca concentrations, we could exclude the possibility of molec- comprising different PIP2-PIP1 ratios. Our experimental approaches include: i) ular crowding as the underlying FRET mechanism. We defined a computa- designing mutants to alter PIP2-PIP1 interaction; ii) performing homo and het- tional model of Ca dependent PKCa attachment to the membrane, which erodimeric constructs made of either PIP1 or PIP2, as well as both subunits, iii) included formation of PKCa clusters. Assuming cooperative detachment analyzing PIP location by confocal fluorescence microscopy; iv) measuring from the membrane of PKCa in a cluster we could reproduce all experimental water transport in control and inhibited conditions (citosolic acidification); observations. Ca dependent formation of clusters on the membrane is not and v) studying PIPs in silico. limited to PKCa but appears as a universal property of C2-domain containing Results show that PIP heterotetramers with different stoichiometries can be proteins, including the sole C2-domain, PKCßII and -1. This functional in a heterologous cellular system since they are able to assemble mechanism leads to an increased membrane residence time of signalling pro- by expression of PIP2-PIP1 tandem dimers, and by co-expression of those di- teins and could thus represent a universal feature of cellular signaling. Sup- mers plus PIP2 or PIP1 monomers. Interestingly, the composition of these ported by the DFG (SFB1027). Wednesday, February 11, 2015 527a

2665-Pos Board B95 affected by incomplete cleavage CA from the Gag matrix (MA), and that Development of Tools to Study Retroviral Gag Assembly on Giant Unila- even a small percentage of uncleaved MA-CA would prevent the core from mellar Vesicles(GUV) rolling away. This prediction explains previous experimental results; only Yi Wen1, Robert Dick1, Volker Vogt1, Gerald Feigenson2. 4% of cleavage-resistant MA-CA is sufficient to cause a 50% reduction of 1Molecular Biology & Genetics, Cornell University, Ithaca, NY, USA, infectivity. 2Biophysics, Cornell University, Ithaca, NY, USA. The retroviral Gag polyprotein provides the principal driving force for virus as- 2667-Pos Board B97 sembly and budding from the cellular plasma membrane. The binding of Gag to Molecular Dynamics and Assembly Switch of FtsZ Filaments the plasma membrane (PM) is governed by several mechanisms, including Erney Ramı´rez-Aportela1,2, Jose´ Ramo´n Lo´pez-Blanco1, electrostatics, hydrophobics, Gag multimierization, and recognition of specific Jose´ Manuel Andreu2, Pablo Chacon1. 1 lipid head groups. To better understand how Gag interacts with the PM, control Department of Biological Physical Chemistry, Rocasolano Physical 2 proteins GFP-poly(K/R)n (n¼4/8/12 residues), were purified and used to Chemistry Institute.CSIC, Madrid, Spain, Department of Chemical and compare Gag membrane binding to an electrostatic membrane binding protein. Physical Biology, Biological Research Centre, CSIC, Madrid, Spain. Preliminary protein-liposome binding experiments suggest that the anionic The cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming lipids, PS and PI(4,5)P2, contribute to the recruitment of polycationic proteins dynamic filaments that are essential for bacterial cell division. Our studies as expected. Unlike previous results, which showed that HIV-1 Gag responded using unbiased molecular dynamics simulations from representative fila- strongly to cholesterol, GFP-poly(K/R)n responded weakly to cholesterol. ment crystal structures disclose different filament curvatures supported by PI(4,5)P2 enhances more liposome binding for retroviral Gag than for the con- nucleotide-regulated hinge-bending motions between consecutive FtsZ sub- trol protein GFP-poly(K/R)n. This study may shed light on how the retroviral units, in agreement with experimental observations, and unravel the natural Gag protein interacts electrostatically with membranes, and recognizes specific mechanism of the FtsZ assembly switch. Whereas GTP-FtsZ filaments lipids such as PI(4,5)P2. bend and twist in a preferred orientation, thereby occluding the nucleotide Membrane binding is not a prerequisite for Gag multimerization, however, binding site, the differently curved GDP-FtsZ filaments identified exhibit it might enhance the formation of Gag-Gag interactions. It is known that a heterogeneous distribution of open and closed interfaces between mono- assembly of Gag occurs on the PM, but it remains unclear when and where mers. The key stabilization component involved in closing the interfaces 2þ Gag multimerization takes place, and if membrane lipid composition influ- in GTP-FtsZ filaments is the coordinated Mg atom, which the opening ences Gag assembly. To visualize by fluorescence microscopy the interac- GDP filaments lack. We have monitored, for the first time, the relaxation tion of Gag with membranes and to detect Gag-Gag interactions, Rous of the assembly switch, from the initial open inter-domain cleft conformation Sarcoma Virus Gag is labeled with different Alexa-flurophores using Sfp in filaments to the closed-cleft inactive conformation in unassociated mono- synthase. Giant unilamellar vesicles (GUVs), composed of lipids similar mers. Integrating this assembly switch and the nucleotide-dependent interfa- to cellular inner leaflet lipid composition, were employed to study Gag cial filament stability, our work offers a detailed molecular interpretation assembly by measuring fluorescence resonance energy transfer (FRET). of the assembly-disassembly FtsZ cycle and its inhibition by antibiotic This study may provide a starting point for understanding where Gag multi- PC190723. merization happens as well as how membrane composition affects Gag assembly. 2668-Pos Board B98 Structural and Biophysical Comparison of UPEC and ETEC Adhesion 2666-Pos Board B96 Fimbriae Paradigm Shift in the Mechanism of HIV-1 Core Biogenesis Narges Mortezaei1, Bhupender Singh1, Esther Bullitt2, Johan Zakrisson1, Gabriel A. Frank1, Kedar Narayan1, Julian W. Bess, Jr.2, Chelsea R. Epler2, Krister Wiklund1, Magnus Andersson3. Gregory Q. Del Prete2, Xiongwu Wu3, Amy Moran4, Lisa Hartnell1, 1Physics, Umea˚ University, Umea˚, Sweden, 2Physiology & Biophysics, Lesley A. Earl1, Jeffrey D. Lifson2, Sriram Subramaniam1. Boston Univ School of Medicine, Boston, MA, USA, 3Physics, Umea˚, Umea˚, 1LCB, NIH-NCI, Bethessda, MD, USA, 2AIDS and Cancer Virus Program, Sweden. Leidos Biomedical Research, Inc.,, Frederick, MD, USA, 3Laboratory of Adhesion fimbriae (pili) of uropathogenic and enterotoxigenic Escherichia Computational Biology, NHLBI, Bethessda, MD, USA, 4NIH-NLM, coli (UPEC and ETEC, respectively) facilitate adherence of the bacteria to Bethessda, MD, USA. target cells. Fimbriae are absolutely necessary for colonization and biofilm The proteolytic cleavage of the poly-protein HIV-1 Gag, which is assembled formation in the initiation of disease. The types of fimbriae expressed on on the surface of plasma membranes of infected cells, drives the conversion of the bacterial surface vary with the preferred environmental niche of the bac- the virus from the initial immature, non-infectious form to the functionally terial strain. For example, UPEC that express P-pili are most frequently asso- distinct mature, infectious form. Gag cleavage results in a series of structural ciated pyelonephritis, an infection in the upper urinary tract, whereas changes, ultimately leading to the formation of a mature core. Current models bacteria that express type 1 fimbriae commonly cause cystitis through infec- for assembly of the mature core suggest that the cleaved HIV capsid protein tion of the lower urinary tract. In contrast, ETEC expressing CFA/I and CS2 (CA) nucleates in a concentration-dependent manner, and polymerizes form- pili are associated with diarrheal diseases, initiating disease in the small ing the conical core in a diffusion-controlled process. These models also intestines. postulate that the core begins to grow at its narrow end, and stops growing Although expressed in different enviroments, these fimbriae share basic struc- once it reaches the membrane at the opposite end. Thus, the size of the virus tural and biomechanical features. Structurally, they are all long (1-4 mm), thin itself is expected to be the primary factor that determines core size. Our find- (7-8 nm diameter) helix-like filaments that extend from the bacterial surface. ings challenge this view. Biomechanically, they share the ability to be extended into a thinner filament Cryo-electron microscopic analyses show that along with infectious viruses, (2-3 nm diameter) by unwinding of the helical filament under a constant force. viral isolates also comprise large membranous structures that contain multiple, However, the force required to unwind is specific to each fimbrial type. In freely-floating mature cores. Numerous instances of membrane-attached as- addition, the dependence of the force required to unwind a fimbria on the ve- sembly intermediates with partially formed ‘‘core-rolls’’ that are at different locity of this unwinding, (that is, the kinetics of unwinding), is also type- stages of conversion from a planar Gag lattice to the mature core are also specific and highly variable. These biomechanical parameters are dissimilar observed. These results indicate that the mechanism of core formation in- for UPEC and ETEC expressed fimbriae, separating them into two distinct volves a non-diffusional, cooperative transition triggered by cleavage of the groups. Using force spectroscopy data, helical reconstructions from electron immature Gag lattice, resulting in its rolling away from the plasma membrane microscopy data, and computational simulations, we show in this work how to form sheets that wrap around the viral RNA. Unlike the present models, our these pronounced biomechanical differences may be beneficial for bacterial mechanism predicts that the generation of infectious HIV-1 will be severely survival in a given environment. 528a Wednesday, February 11, 2015

Protein Dynamics and Allostery III a substrate and an allosteric modulator in cell signaling [Lu S, et al. PLOS Comput Biol, 2014, 10: e1003831]. 2669-Pos Board B99 Proposed Thermodynamic Basis for Synaptotagmin Response 2672-Pos Board B102 Anne Hinderliter. Geometric Description of Dynamin Induced Membrane Fission Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, Pablo Va´zquez-Montejo, Zachary McDargh, Markus Deserno. MN, USA. Carnegie Mellon University, Pittsburgh, PA, USA. The synaptic vesicle protein Synaptotagmin I is the calcium ion sensor for The terminal step during the process of clathrin-mediated endocytosis is the neurotransmitter release. are comprised of C2 domains, cal- scission of the connection between the nascent bud and the parent membrane. cium ion dependent, membrane-binding domains. They contain multiple C2 Narrowing of the neck is driven by proteins or their complexes, most promi- domains and all C2 domains are tethered to the membrane. Our recent work nently dynamin, which assembles into rings and spirals that constrict the has redefined Synaptotagmin I has consisting of a series of nearly disordered connection and are believed to generate sufficient force onto the membrane domains that enable the extent of calcium ion and type of phospholipid bound to induce fission. To advance our undertanding of the underlying mechanism, to be cooperatively communicated through the protein. Through applications of in this work we present a geometric framework to study the conformation of thermodynamics to define free energies of stability and of binding, we find C2 a semi-flexible polymer adhering to, or confined by an axially symmetric mem- domains not only from Synaptotagmin I but also in general to have a common brane. The rotational symmetry of the membrane is exploited to obtain a first thermodynamic signature of weak free energies of stabilities in solution and integral of the fourth order Euler-Lagrange equation describing the polymer stabilities that are lipid composition dependent. This plasticity in structure is equilibrium states. In particular, we examine and characterize closed and proposed to underlie the plasticity in function as responsive calcium ion sensors helix-like curves with right-hand chirality, lying on surfaces with the shape whose response is defined by its local membrane environment. of a cylinder and a catenoid. For the cylindrical case, the additional transla- tional symmetry allows to integrate the Euler-Lagrange equation once more, 2670-Pos Board B100 obtaining a quadrature. In this framework the stresses transmitted by the poly- Allosteric Functional Switch in Poliovirus 3C Protease mer onto the membrane are determined entirely in terms of the local geometry Yan M. Chan, David D. Boehr. of the combined system of the helical-dynamin coat wrapping around the mem- Chemistry, Pennsylvania State University, University Park, PA, USA. brane neck,allowing us to analyze the force and torques involved during the Viral genomes are very efficient; they are typically compact but nevertheless constriction process. encode numerous elements that are essential for regulation of both its own replication and packaging, and of the host cell’s machinery. Viruses have 2673-Pos Board B103 developed successful strategies to overcome their biological information stor- Two Pathways Mediate Inter-Domain Allosteric Regulation in Pin1 age problem. For example, the 3Cpro protein from the picornavirus family of Jingjing Guo1,2, Xiaodong Pang1, Huan-Xiang Zhou1. positive-strand RNA viruses is responsible for binding of RNA control se- 1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, quences to regulate translation and replication, interacting with phosphoinosi- USA, 2Department of Chemistry, Lanzhou University, Lanzhou, China. tide lipids (PI) to regulate the maturation of virus replication organelles, and Allostery is an essential means for regulating biomolecular functions and pro- acting as the main protease to cleave host and virus proteins to further regulate vides unique opportunities for drug design, yet our ability to elucidate allosteric host and virus processes. 3Cpro can also be found as a domain in the 3CDpro mechanisms remains limited. Here, based on extensive molecular dynamics polyprotein. 3Cpro by itself and 3CDpro have different protease specificities, simulations, we present an atomistic picture of the pathways mediating the allo- and likely different RNA and PI binding capabilities. The domains in 3CDpro steric regulation of the PPIase domain of Pin1 by its WW domain. Two path- are tethered by a flexible linker and do not make specific 3Cpro-3Dpol interac- ways jointly propagate the action of substrate-WW binding to produce closure tions. Surprisingly, we have found that by extending the C-terminal tail of 3C and rigidification of three PPIase catalytic-site loops. One pathway preexists in with just a few amino acid residues, the RNA and PI binding properties alter the apo protein but remains dormant until substrate-WW binding completes the dramatically. These functional changes are accompanied by changes in the second. The reduction in conformational entropy and preorganization of the structural dynamics of 3C, as measured by NMR relaxation methods. We pro- catalytic-site loops observed here may explain why substrate-WW binding en- pose that these findings have critical bearing on 3C function; proteolytic pro- hances ligand affinity and catalytic activity of the PPIase domain, and suggest a cessing of the C-terminus is the switch from 3CDpro to 3Cpro (by itself) combination drug therapy for Pin1-related diseases. Whereas the traditional activities. Such a simple, but elegant, mechanism does not require any addi- view of allostery has emphasized conformational transition, our study uniquely tional domain-domain interactions in the 3CDpro polyprotein to regulate identifies a distinct role of conformational dynamics in eliciting allostery. 3Cpro function, and can help explain functional differences between 3Cpro and 3CDpro that have confounded virologists and structural biologists. 2674-Pos Board B104 The Impact of Perturbing Dynamic Amino Acid Networks in a (b/a)8 2671-Pos Board B101 Barrel Enzyme The Structural Basis of ATP as an Allosteric Modulator Kathleen F. O’Rourke, Jennifer M. Axe, Nicole E. Kerstetter, Shaoyong Lu1, Wenkang Huang1, Qi Wang1, Qiancheng Shen1, Shuai Li1, David D. Boehr. Ruth Nussinov2, Jian Zhang1. Chemistry, Pennsylvania State University, State College, PA, USA. 1Pathophysiology, Shanghai JiaoTong University, School of Medicine, Amino acid networks describe the web of noncovalent interactions between Shanghai, China, 2Cancer and Inflammation Program, Leidos Biomedical residues spanning an enzyme. These networks may be responsible for the prop- Research, Inc., Frederick National Laboratory, NCI, Frederick, MD, USA. agation of regulatory signals across the protein that influence conformation, Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for en- binding of substrate(s), and catalysis. Tryptophan synthase (TS), the final ergy currency and protein modification. Recent findings uncovered the allo- enzyme in the tryptophan biosynthetic pathway, is a tetramer consisting of a steric function of ATP in cellular signal transduction but little is understood pair of alpha and beta heterodimers arranged in a linear conformation. The about this critical behavior of ATP. Through extensive analysis of ATP in so- alpha and beta subunits are connected by a 25A˚ intramolecular tunnel that lution and proteins, we found that the free ATP can exist in the compact and channels indole, a product from the alpha reaction, to the active site in the extended conformations in solution, and the two different conformational char- beta subunit. In addition to this tunnel, the conformational states of these sub- acteristics may be responsible for ATP to exert distinct biological functions: units are highly coordinated making TS an ideal and heavily studied model for ATP molecules adopt both compact and extended conformations in the allo- substrate channeling and enzyme-enzyme interactions. We used nuclear mag- steric binding sites but conserve extended conformations in the substrate bind- netic resonance chemical shift covariance analysis to delineate amino acid net- ing sites. Nudged elastic band simulations unveiled the distinct dynamic works in the alpha subunit, a (b/a)8 barrel enzyme. We have shown that these processes of ATP binding to the corresponding allosteric and substrate binding observed networks change between the resting state (in the absence of sub- sites of uridine monophosphate kinase, and suggested that in solution ATP pref- strates) and the working state (under active catalytic turnover). Furthermore, erentially binds to the substrate binding sites of proteins. When the ATP mol- the loss of a hydrogen bond between the dynamic b2a2 and b6a6 loops in ecules occupy the allosteric binding sites, the allosteric trigger from ATP to the T183V variant significantly changes these networks and the catalytic rate fuel allosteric communication between allosteric and functional sites is seen in the wild-type. These networks were perturbed by making small Ala stemmed mainly from the triphosphate part of ATP, with a small number to Gly substitutions of surface residues correlated to the catalytic Glu49. These from the adenine part of ATP. The detailed mechanism presented in this study modifications resulted in modest decreases in the catalytic rate, although they may apply to other enzymes in complex with allosteric or substrate ATP mol- are 25 A˚ away from the active site. Amino acid networks are important for the ecules, and provide important insights for the molecular basis of ATP acting as function of an enzyme and may be manipulated to tune its function. Wednesday, February 11, 2015 529a

2675-Pos Board B105 hepatitis C. Various nucleoside analogs (NA) and allosteric non-nucleoside in- Conformational Exchange Coupled to Ionization of Lys Residues Buried in hibitors (NNI) have been shown to bind ns5B polymerase and inhibit the repli- Hydrophobic Environments in Proteins cation of HCV. NNIs have been classified based on the drug binding site as Daniel E. Richman1, Ananya Majumdar2, Bertrand Garcia-Moreno1. observed by crystallography (Thumb I, Thumb II, Palm III, and Palm IV). 1Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA, However, the inherent variability of HCV and the rapid emergence of resis- 2Biomolecular NMR Center, Johns Hopkins University, Baltimore, tance mutations have highlighted the need for a better understanding of the MD, USA. mechanism of inhibition of NNIs at a molecular level. For that purpose, we Ionizable amino acids buried in hydrophobic environments in proteins can have used Hydrogen-Deuterium exchange coupled to mass spectrometry to titrate with anomalous pKa values. Despite the central role of these amino acids characterize the dynamics of ns5B polymerase in the presence and absence in energy transduction and catalysis, the determinants of their unusual proper- of an allosteric thumb II site NNI. As expected, we have determined that inhib- ties are difficult to reproduce with computational approaches. Conformational itor binding to ns5B polymerase causes a significant loss of exchange at the reorganization of the protein backbone is a key determinant of pKa values. This binding site and in regions directly adjacent. Furthermore, we have observed reflects the ability of the protein to sample conformational microstates, which is that large regions of ns5B which are distant from the drug binding site display particularly challenging to reproduce computationally. To characterize struc- a significant loss in protein dynamics as detected by HD exchange. The tural details and timescales of conformational reorganization coupled to ioniza- observed network of allosterically suppressed dynamics encompasses the fin- tion of buried groups, NMR spectroscopy was used to identify locations, gers, fingers extensions, thumb, C-terminal tail and beta-loop regions which measure timescales, and determine the extent of pH dependence of conforma- have all been implicated in one of the multiple steps of the reaction cycle tional exchange in the L25K and L125K variants of staphylococcal nuclease. (de novo initiation, transition to elongation and processive elongation). Lys-25 or Lys-125 titrate with pKa values of 6.3 and 6.2, respectively. Using relaxation dispersion spectroscopy we observed that the b barrel of the L25K 2678-Pos Board B108 protein fluctuates between the native and an alternate state on the 0.4 to 7 ms Dysfunctional Conformational Dynamics of Protein Kinase a from R14 timescale. The HN chemical shifts of the alternate state, assigned using ZZ- Deletion of Phospholamban exchange spectroscopy, were less dispersed than those of the native state, Jonggul Kim1, Larry R. Masterson2,3, Alessandro Cembran4,3, consistent with unfolding of the b barrel. The equilibrium between these states Raffaello Verardi3, Lei Shi1, Jiali Gao1, Gianluigi Veglia1,3. 1Chemistry, University of Minnesota, Minneapolis, MN, USA, 2Chemistry, is strongly pH dependent, with the partially unfolded state dominating at pH 3 values at which the internal Lys residue is charged. In contrast, the L125K pro- Hameline University, St. Paul, MN, USA, Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA, tein exhibited fluctuations near Lys-125 on the ms-ms timescale but in a faster 4 regime than the L25K protein and without pH dependence. These results show Chemistry, University of Minnesota-Duluth, Duluth, MN, USA. the extent of coupling between conformational change and proton binding is Protein Kinase A (PKA) is a ubiquitous signaling enzyme responsible that me- highly dependent on local structural properties. Computational models attempt- diates a myriad of cellular signaling events. A target of PKA during regulation of muscle contraction is Phospholamban (PLN). PLN phosphorylation reverses ing to reproduce the properties and pKa values of buried ionizable groups must its inhibitory effect on the sarcoplasmic reticulum Ca2þ-ATPase (SERCA), be able to represent conformational exchange between native and subglobally 2þ unfolded states on ms timescales. increasing SERCA’s affinity for Ca ions and augmenting diastole. A deletion at the P-2 site of PLN recognition sequence (R14del) found in humans reduces 2676-Pos Board B106 PLN phosphorylation levels, leading to dilated cardiomyopathy. In this study Nmr Investigation of Energy Barriers for Hydrogen-Bond Breakage of we utilize solution state NMR, isothermal titration calorimetry, and MD simu- D Protein Side-Chain NH3 Groups lations to elucidate the molecular mechanism of aberrant substrate recognition. Levani Zandarashvili, Junji Iwahara. Here, we show that this single substrate deletion affects the structural confor- Dept. of Biochemistry & Molecular Biology, University of Texas Medical mation and dynamics of the PKA/PLN complex, causing dysfunctional confor- Branch, Galveston, TX, USA. mational fluctuations throughout the enzyme, disrupting binding and reducing Hydrogen bonds and ion pairs involving charged side chains are important for enzymatic efficiency. These results unveil a crucial role of kinase substrates for protein function. However, their dynamic properties are not well understood. the formation of the catalytically-competent conformation and intramolecular þ Recently we demonstrated that lysine side-chain NH3 groups are extremely allosteric signaling. These findings illuminate a molecular mechanism for the useful probes for NMR investigations of the hydrogen-bond/ion-pair dynamics. pathophysiology of R14del and aberrant Ca2þ signaling, underscoring the þ Previously we found that bond rotations of protein side-chain NH3 groups are importance of a well-tuned structural and dynamic interplay between enzyme nearly as rapid as those of side-chain CH3 groups despite the presence of and substrate to achieve physiological phosphorylation levels for normal car- hydrogen bonds. To understand this phenomenon, we have studied energy bar- diac function. þ riers for NH3 rotations requiring the transient breakage of hydrogen bonds. For the HoxD9 homeodomain-DNA complex, we investigated the temperature 2679-Pos Board B109 þ Dynamical X-Ray Single Molecule Observations of the Molecular Recogni- dependence of the internal motions of lysine side-chain NH3 groups forming þ tion Process in Major Histocompatibility Complex Molecule intermolecular ion pairs with DNA. For these NH3 groups, we determined or- der parameters and correlation times for bond rotations and reorientations at Toshihiro Miyabe1, Yufuku Matsushita1, Kosuke Kasadera2, Yuko Kozono2, four temperatures. The order parameters were virtually independent of temper- Hiroshi Sekiguchi3, Keigo Ikezaki1, Haruo Kozono2, Yuji C. Sasaki1. þ 1Grad. Sch. Front. Sci., The University of Tokyo, Chiba-ken, Japan, 2Res. ature in this range. In contrast, the NH3 bond-rotation correlation times were 3 found to depend strongly on temperature. Based on transition state theory, the Inst. Biomed., Tokyo Univ. of Science, Chiba-ken, Japan, JASRI, JST, þ Hyogo-ken, Japan. energy barriers for NH3 rotations were analyzed and compared to those for þ Major histocompatibility complex (MHC) class II molecule has an important CH3 rotations. Enthalpies of activation for NH3 rotations were found to be role in activities of the immune system. MHC binds peptides from exogenous significantly higher than those for CH3 rotations, which can be attributed to the requirement of hydrogen-bond breakage. However, the transition states in antigens which is engulfed by endocytosis processes of antigen presenting cells which hydrogen bonds with water molecules should be transiently broken are and present it to T cell receptor, then stimulate and activate the immune system. þ In the peptide-loading process of MHC, it is said that DM which is a homolog entropically favorable, and the overall free energies of activation for NH3 ro- of MHC has some activities on that process. The experiment of chromatog- tations are as low as those for CH3 rotations. This entropic reduction in energy barriers can accelerate molecular processes requiring hydrogen bond breakage raphy using peptide labeled with fluorescein on MHC with DM suggests that and may be kinetically important for protein function. This work was supported DM has a relationship between its activity and the length of peptides. Short by Grant CHE-1307344 from the National Science Foundation. peptides tend to be dissociated from MHC, on the other hand, long ones do not. In this research, we used Diffracted X-ray Tracking (DXT) to observe the dy- 2677-Pos Board B107 namics of the peptide bound to MHC’s groove with DM as a single molecule. Thumb II Site Inhibitor Allosterically Suppresses the Dynamics of HCV DXT is the method of detecting the detailed motions of the targets for measure- RNA-Dependent RNA Polymerase ment by labeling a gold nanocrystal. The labeled nanocrystals diffract the inci- Daniel J. Deredge1, Kenneth Johnson2, Patrick Wintrode1. dent x-rays and we obtained the motions of the target by tracking x-ray 1Pharmaceutical Sciences, University of Maryland School of Pharmacy, diffraction spots. We did experiments at SPring-8 (BL40XU, JAPAN). Baltimore, MD, USA, 2Institute of Cellular and Molecular Biology, From the DXT experiments of observing the motions of peptides in MHC’s University of Texas at Austin, Austin, TX, USA. groove with DM, we obtained the time-resolved (100 microsecond) motion Hepatitis C virus RNA-dependent RNA polymerase (ns5B polymerase) is a histograms of peptides. The motions of peptides are obviously different in major target of drug development in the search for an effective treatment of that of being catalyzed by DM. Additionally, the peptide’s motions with DM 530a Wednesday, February 11, 2015 became more complicated than those without DM. We are going to discuss the 2683-Pos Board B113 results and consideration of the experiments. Backbone Flexibility as a Determinant of PKA Values of Buried Ionizable Groups in Proteins 2680-Pos Board B110 Bertrand Garcia-Moreno1, Gabrieal Ortega2, Meredith T. Peck1, Unexpected VASP-EVH1 Interactions with Irak1 and Zyxin: Phosphory- Aaron Robinson1, Jamie L. Schlessman3. lation and Bivalent Binding 1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2Structural Lucila A. Acevedo1, Alex I. Greenwood2, Jeahoo Kwon1, Kun P. Lu3, Biology Unit, CICbioGUNE, Derio, Spain, 3Chemistry, U.S. Naval Linda K. Nicholson1. 1 Academy, Annapolis, MD, USA. Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA, Ionizable groups buried in hydrophobic environments in proteins play essential 2University of Illinois at Urbana-Champaign, Champaign, IL, USA, 3 roles in biological energy transduction. These buried groups usually titrate with Department of Medicine, Harvard University, Boston, MA, USA. anomalous pKa values, shifted in the direction that favors the neutral state VASP is an actin polymerization regulatory protein, and is a founding member of because charges are not compatible with the dehydrated interior of proteins. the Ena/VASP family of proteins. The EVH1 domain, a conserved component of Owing to their importance in catalysis, Hþ transport and other forms of biolog- the Ena/VASP family, localizes these proteins to sites of high cytoskeletal dy- ical energy transduction, the molecular determinants of these pKa values are of namics through its selective binding to the sequence motif (F/W)PxhP, where interest. In particular, the extent to which flexibility of the backbone contributes x is any residue and h is an aliphatic residue. Our NMR studies of VASP- to the apparent pKa values measured experimentally is not well understood. In EVH1 interactions with the innate immunity signaling protein IRAK1 and flexible regions the ionizable groups can sample different microenvironments with the cytoskeletal protein zyxin have revealed an unexpected mechanism with different dielectric properties. The apparent pKa values measured reflect of regulation and an unexpected mode of binding, respectively. These results an ensemble-weighted average of the true pKa values in the different microen- provide important insights into the transient subcellular localization of VASP vironments. To examine the role of backbone flexibility we attempted to modu- in response to innate immunity signaling, and to a novel binding mode that could late the pKa of the internal Lys-66 and Lys-25 in staphylococcal nuclease provide an advantage for specific partners that contain sequential binding motifs. through mutations of selected positions to Gly to increase the flexibility of 2681-Pos Board B111 the backbone. The hypothesis that was tested posits that an increase in flexi- Influence of RNA Binding on the Conformational Dynamics of the Lassa bility would shift the anomalous pKa values of these Lys residues (5.7 for Virus Nucleoprotein Lys-66 and 7.1 for Lys-25) towards more normal values near 10.4 because Jason Pattis, Eric R. May. the removal of conformational restrictions on the backbone would increase Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA. the population of conformational states in which the previously buried ioniz- Lassa virus is an enveloped virus, with a bisegmented ambisense single- able group would become more exposed to water, and this would lead to stranded RNA genome, encoding only four gene products. The most abundant more normal pKa normal values. Besides demonstrating that pKa values are protein is the nucleoprotein (NP) which encapsidates the viral genomic RNA. affected by substitutions to Gly in the manner expected, structural methods Multiple structures of the NP have been determined. The full-length NP was corroborated that an increase in apparent pKa was the result of increased expo- determined in the absence of RNA in a trimeric arrangement, which has sure of the Lys side chains to water. been shown to be the dominant solution form of NP. A second structure of only the N-terminal domain of NP was determined in the presence of a short 2684-Pos Board B114 RNA strand, which revealed an RNA-binding groove. There were significant Regulation of the Molecular Brake Region in FGFR2 Kinase differences between the RNA bound and RNA-free forms in the NP N-terminal Jerome M. Karp, David Cowburn. domain, highlighted by helix 6 adopting a distal (open) orientation, and helix 5 Biochemistry, Albert Einstein College of Medicine of Yeshiva University, undergoing a partial loss of helicity, when RNA is bound. Bronx, NY, USA. The primary focus of this investigation is to understand the mechanism by Unregulated activity of tyrosine kinases (TKS) is responsible for numerous which the Lassa virus NP binds to RNA and how conformational changes in developmental musculoskeletal diseases and cancers. The regulatory mecha- the NP are achieved. We have employed extensive equilibrium molecular dy- nisms of tks and how they are affected by point mutations are still in need of namics (MD) simulations to investigate the influence of RNA on the global mo- elucidation. Fibroblast growth factor receptor (FGFR) kinase is a TK whose tions of the NP, and have identified that RNA-binding induces a closing motion. regulatory element, the ‘‘molecular brake,’’ is thought to prevent constitutive This observation is supported by free energy calculations using both umbrella activation of the receptor, despite not being near the kinase’s active site. sampling and metadynamics methods. The free energy profiles show the closed germline mutations in this ‘‘molecular brake’’ region in FGFR cause musculo- state is more favorable, both in the absence and presence of RNA, and that skeletal disorders such as craniosynostosis and dwarfism, while somatic muta- RNA-binding reduces the energy cost for opening. The observation that the tions can lead to uninhibited cell growth, causing an array of cancers. RNA-bound form prefers a closed configuration appears in disagreement furthermore, it is thought that the majority of pathogenic activating mutations with the open-state crystal structure. However, we believe non-native crystal in protein kinases are in ‘‘molecular brake’’-like regions. However, the mech- contacts are stabilizing the open form of NP, which is supported from energetic anism through which the ‘‘molecular brake’’ of FGFR kinase is disengaged, al- analysis of MD simulations of NP dimers present in the crystal unit cell. lowing for signal transduction, is still unknown. using path-based molecular dynamics (MD) simulations, we have shown that the inward motion of the ki- 2682-Pos Board B112 nase’s activation loop upon tyrosine phosphorylation is correlated with disen- Site-Specific Conformational Diversity in Calmodulin using Cyanylated gagement of the ‘‘molecular brake.’’ results of the string method in collective Cysteine Vibrational Probes along its Binding Interfaces variables, which finds the minimum free energy path connecting the loop-out Kristen L. Kelly, Rebecca Wai, Kanika Ramchandani, Casey Londergan. and loop-in states, indicate found that inward motion of loop occurs within 1 Chemistry, Haverford College, Haverford, PA, USA. frame of ‘‘molecular brake’’ disengagement. Furthermore, umbrella sampling Vibrational spectroscopy was used to probe the modular binding interfaces of shows that in loop-out conformations, the disengaged state of the ‘‘molecular calmodulin. Two hydrophobic amino acid patches allow calmodulin great flex- brake’’ is less stable, while in loop-in conformations, the disengaged state be- ibility in binding to target partners, and these patches contain key methionine res- comes more stable. Our results suggest that the ‘‘molecular brake’’ is allosteri- idues. Site-specific mutagenesis was used to generate numerous single cysteine cally controlled by the activation loop motion. An understanding of the variants of calmodulin, including three mutations at key methionine residues, ‘‘molecular brake’’ disengagement mechanism will provide avenues for tar- which were expressed, purified and cyanylated at cysteine to generate a vibra- geted therapies to counteract pathologic over-activation of fgfr kinase leading tional probe group at each site. Infrared spectroscopy was used to assess the envi- to uncontrolled signal transduction and uninhibited cell growth. ronment surrounding each probe in apo- and calcium saturated conditions, as well as complexed with the binding peptide from skeletal muscle myosin light 2685-Pos Board B115 chain kinase. Circular dichroism (CD) experiments were conducted to ensure PKA Values of Buried Groups in Proteins are Sensitive to the Global the SCN vibrational probe did not perturb calmodulin’s secondary structure, Thermodynamic Stability and isothermal calorimetry was performed for each labeled variant with the Jaime L. Sorenson1, Raidizon Mercedes1, Jamie L. Schlessman2, skMLCK peptide to ensure the probe did not disrupt complex formation. Surpris- Bertrand Garcı´a-Moreno1. ingly, the methionine to cyanylated cysteine mutation at key methionine residues 1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2United States did not lead to a large perturbation in binding thermodynamics. This suggests Naval Academy, Annapolis, MD, USA. that the cyanylated cysteine probe group is relatively innocent even when placed Ionizable groups buried in hydrophobic environments in proteins are essential directly in the binding interface and can report directly on structural dynamics for all forms of biological energy transduction. The molecular determinants of along the binding interface between calmodulin and its many targets. the pKa values of these internal groups are poorly understood. It is increasingly Wednesday, February 11, 2015 531a apparent that conformational reorganization coupled to the ionization of the extends to a cellular environment through cell culture studies. In normal physi- buried group is a major determinant of these pKa values. Specifically, the cre- ology, zinc likely acts as a negative regulator of Hh autoprocessing and inhibits ation of charge in hydrophobic environments can trigger a shift from the fully the generation of Hh ligand and Hh signaling. In many diseases, zinc deficiency folded state to local or partially unfolded states in which the charge can gain and elevated level of Hh ligand co-exist, including prostate cancer, lung cancer, access to water or to an environment where the charge can be solvated. These ovarian cancer and autism. Our data suggest a novel, causal relationship between alternative conformational states are not normally populated owing to the large zinc deficiency and the overproduction of Hh ligand: zinc deficiency likely en- free energy difference between the alternative and fully-folded native states; hances Hh autoprocessing and the production of Hh ligand, thereby activating however, the partially unfolded states can become the new ground state under Hh signaling in diseases. pH conditions where the internal group is charged. If the ionization of an inter- nal group promotes the transition to a new conformational state then its pKa 2688-Pos Board B118 should be sensitive to the global thermodynamic stability (DG) of the protein Probing Factor XIII Substrate Specificity for Fibrinogen aC (233-425) because this determines the energy gap between the ground and the alternative Using Maldi-Tof Mass Spectrometry and 2D 15N-1H HSQC NMR Spec- states. This was tested by measuring the pKa of two internal Lys residues in var- troscopy iants of staphylococcal nuclease with thermodynamic stabilities ranging from Kelly Njine Mouapi1, Jacob Bell1, Kerrie Smith2, Helen Philippou2, 8.4 to 13.8 kcal/mol. The magnitude of the shift in the pK of the internal Robert Ariens2, Muriel Maurer1. a 1 2 Lys residues was found to be sensitive to the DG of the protein confirming Chemistry, University of Louisville, Louisville, KY, USA, Division of that the pKa values of these Lys residues are determined by the probability Cardiovascular and Diabetes Research, University of Leeds, Leeds, United of structural reorganization more than by local dielectric properties of their mi- Kingdom. g croenvironments. These observations imply that structure-based pKa calcula- In blood coagulation, the transglutaminase FXIII introduces covalent -glu- tions for buried groups and other electrostatic processes in hydrophobic tamyl-ε-lysinyl crosslinks into the fibrin clot network. The C-terminal portion environments require accurate treatment of conformational reorganization, of the Fibrinogen Aa chain contains the aC region (221-610) that has a flexible which remains an extremely challenging proposition. aC-connector segment (221-391) and a globular aC domain (392-610). Reactive glutamine (Q) residues from the aC-connector can be covalently 2686-Pos Board B116 cross-linked by activated FXIII (FXIIIa) to reactive lysines (K) located in the Critical Role of a Glycine Residue in an Allosteric Switch C-terminal portion of the aC domain on another fibrin molecule. Studies Dorothy Beckett1, William Cressman2. have shown that aC based crosslinks help generate a clot that has more lateral 1University of Maryland, College Park, MD, USA, 2Chemistry & aggregation, is stronger, and is more resistant to fibrinolysis. Fibrinogen aC Biochemistry, University of Maryland, College Park, MD, USA. (233-425) contains three reactive glutamines (Q237, Q328, Q366) that partic- Thermodynamic coupling in proteins is ubiquitous but mechanistically poorly ipate in cross-linking but in-depth kinetic information is not available on indi- understood, particularly for systems in which the coupling occurs over large vidual residues. A combination of mass spectrometry and NMR-based methods distances. In the E. coli biotin repressor the coupling between homodimeriza- is being used to probe for FXIIIa substrate specificity for fibrinogen aC region 0 tion and allosteric effector, bio-5 -AMP, is 4.0 kcal/mol. The two coupled (233-425). In the MALDI-TOF mass spectrometry assay, FXIIIa-catalyzed processes occur at sites separated by 33A˚ . Structural and thermodynamic cross-linking between reactive glutamines on aC (233-425) and the lysine- studies indicate that the coupling is accompanied by disorder-to-order transi- mimic glycine ethylester (GEE) are monitored following chymotrypsin and tions at the two distant functional sites. Perturbations to the transition at the Glu-C protease proteolytic digests. MALDI-TOF MS runs reveal that FXIIIa ligand binding site via alanine substitutions alter both ligand binding and cross-links GEE to all three reactive glutamines Q237, Q328, and Q366. Q237 coupled dimerization. Alanine substitutions in four loops in the dimerization is cross-linked first followed by Q328 and Q366. Complementary 2D 15N-1H surface yield a range of energetic consequences for BirA dimerization. For HSQC experiments show the incorporation of 15NH4Cl or 15N -GEE into three one of these variants, BirAG142A, the free energies of dimerization and core- separate glutamines on aC (233-425) in the presence of FXIIIa. pressor binding are consistent with complete abolition of coupling. Structural Gel electrophoresis studies further reveal that the lysine mimic dansylcadaver- studies of the variant indicate that the loss of coupling is accompanied by ine and the Q-containing peptide Dansyl-Ahx-a2-antiplasmin(1-15) can each disruption of the disorder-to-order transitions at both functional surfaces. In be crosslinked into aC (233-425). this work the structural basis of coupling between the dimerization and ligand The new knowledge gained about Fibrinogen aC (233-425) and substrate reac- binding surfaces was further investigated by measuring the consequences of tivity may later be used to design therapeutic Aa fibrinogens that could influ- alanine substitutions distributed in three of the dimerization loops on core- ence fibrin clot character. pressor binding. Isothermal titration calorimetry measurements indicate that, in contrast to BirAG142A, the variants, several of which dimerize very weakly, 2689-Pos Board B119 all bind to bio-50-AMP with energetics indistinguishable from wild-type BirA. Ligand Binding to Anion Binding Exosites as Prothrombin is Converted to The results indicate that a single glycine residue serves as a switch for allosteric Active Thrombin regulation of BirA. Ramya Billur, Marina V. Malovichko, Muriel C. Maurer. Chemistry, University of Louisville, Louisville, KY, USA. The blood coagulant thrombin is originally expressed as the zymogen prothrom- Enzymes and Protein Dynamics II bin (ProT). Prothrombin contains the F1.2 (Gla þ Kringle 1 þ Kringle 2) domain and the inactive serine protease prethrombin-2 (PT2). Kringle 2 of F1.2 protects 2687-Pos Board B117 an immature anion binding exosite on ProT called pro-ABE II. An immature, but Zinc Inhibits Hedgehog Autoprocessing: Linking Zinc Deficiency with unprotected, pro-ABE I site also exists on ProT. As part of the zymogen activa- Hedgehog Activation tion process, the prothrombinase complex can cleave ProT after R271 and Jian Xie1, Timothy Owen2, Ke Xia3, Ajay Vikram Singh4, Emiley Tou5, release F1.2 from PT2. Subsequent cleavage of PT2 at R320 helps to generate Lingyun Li3, Leo Q. Wan4, Brian Callahan1, Chunyu Wang1. active thrombin. Both anion binding exosites become fully mature upon produc- 1Biochemistry and Biophysics Program, Rensselaer Polytechnic Institute, tion of thrombin. In the current work, solution NMR studies have been used to Troy, NY, USA, 2Department of Chemistry, Binghamton University, characterize the binding of GpIba (269-286, 15N-labeled L275, 15N-labeled Binghamton, NY, USA, 3Department of Chemistry and Chemical Biology, D277) to (pro)-ABE II and the binding of PAR3 (44-56, 15N-labeled F47, Rensselaer Polytechnic Institute, Troy, NY, USA, 4Department of 15N-labeled D54 ) to (pro)-ABE I. The residues that are 15N-labeled have pre- Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA, viously been shown by X-ray crystallography to make extensive contact with the 5Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, thrombin surface. This observation is further supported by 1D proton NMR line NY, USA. broadening studies on the ligand-thrombin complexes. 15N-1H HSQC NMR ti- Zinc is an essential metal with wide-ranging biological functions while Hedge- trations demonstrate that GpIba (269-286, 15N-labeled L275, 15N-labeled hog (Hh) signaling plays crucial roles in both development and disease. Here we D277) does not bind to the pro-ABE II site on human ProT. Ligand binding af- describe a mechanistic interaction between zinc and Hh signaling. Hh ligand, the finity to ABE II then increases as ProT is converted to PT2 and then thrombin. By upstream activator of Hh signaling, originates from Hh autoprocessing, in which contrast, HSQC NMR titrations reveal that the pro-ABE I site on ProT can Hh precursor protein undergoes self-cleavage and cholesterol modification. In already bind PAR3 (44-56, 15N-labeled F47, 15N-labeled D54). More selective vitro assay showed zinc inhibits Hh autoprocessing with IC50 of 2 mM. Solution therapeutics may take advantage of this immature ABE I site. Binding affinity for NMR revealed that zinc interacts with active site residues of Hh autoprocessing PAR3 (44-56) further increases as active thrombin is produced. After the two domain while ITC indicated that the binding is driven mostly by enthalpy with mature exosites are generated, thrombin is well poised to regulate several coag- 1:1 stoichiometry. We further demonstrated zinc inhibition of Hh autoprocessing ulation and anti-coagulation events. 532a Wednesday, February 11, 2015

2690-Pos Board B120 2693-Pos Board B123 Molecular Dynamics Studies of the Ubiquitin Conjugation Mechanism Use of 2-Photon Fluorescence Correlation Spectroscopy and Electron Serban Zamfir. Microscopy to Ellucidate the Dependence of RLPFK Self Association on Chemistry and Biochemistry, James Madison University, Harrisonburg, Ligand Concentration VA, USA. David Holland, Jeng-Yih Chang, Junjie Zhang, Gregory D. Reinhart. Post-translational modification of proteins can have drastic effect on their struc- Biochemistry and Biophysics, Texas A&M Univ, College Station, TX, USA. ture and function. One such modification involves the attachment of a small Phosphofructokinase (PFK) catalyzes the first committed step of glycolysis. protein, ubiquitin. An important function of ubiquitination is to signal proteins Allosteric regulation of PFK makes its activity, and thus glycolysis, sensitive for cellular degradation. This process occurs in three enzymatic steps. In the to intercellular metabolic conditions. Regulation of PFK from liver is complex second step, ubiquitin transfers to a conjugating enzyme, called E2, which because of the liver’s responsibility to maintain glucose homeostasis, which then transfers ubiquitin to a lysine in the target protein. However, the mecha- means that glycolysis and gluconeogenesis must be regulated reciprocally. nistic details for this final transfer remain obscured. Although it is clear that Allosteric regulation of PFK activity is achieved by modifying the binding af- ubiquitin does bind, there are no studies that show exactly how this happens. finity of its substrate, fructose 6-phosphate (Fru-6-P). However, in vitro studies The two most favored proposals involve a step-wise mechanism with a tetrahe- performed on rat liver PFK (RLPFK) suggest that at physiological concentra- dral oxyanion intermediate and concerted mechanism. This work probes the ac- tions of Fru-6-P RLPFK activity is negligible even in the presence of known curacy of the oxyanion hypothesis. In particular, if the oxygen on the observed allosteric activators. Additional mechanisms must exist to account for the ac- carbonyl carbon can form a stable hydrogen bond with the hydrogen on the ni- tivity required to support flux through glycolysis. The smallest active oligomer trogen of the asparagine side chain, then oxyanion intermediate is plausible. By of RLPFK is a tetramer, however fluorescence polarization studies have previ- using molecular dynamics (MD), combined with umbrella sampling, a free en- ously demonstrated that at a physiological enzyme concentration both Fru-6-P ergy profile of the formation of the breaking and forming of the hydrogen bond and other activators stabilize species much larger than a tetramer. A Weber is constructed to see if its creation is thermodynamically favorable. Further- linkage argument predicts that the highly associated species would demon- more, information about the hydrogen-bonding environment in the active site strate a higher affinity for Fru-6-P resulting in activation. RLPFK exists as a is extracted. tetramer in the dilute enzyme concentrations necessary for in vitro activity as- says whereas, at physiological concentrations, RLPFK can be highly associ- 2691-Pos Board B121 ated. 2-photon fluorescence correlation spectroscopy (FCS) and electron Proteolysis of Abnormal Prion Protein with a Thermostable Protease from microscopy have been used to quantify the oligomeric state of RLPFK at a Hyper-Thermophilic Archaeon Thermococcus Kodakarensis Kod1 high and low enzyme concentrations. FCS performed on alexa-488 labeled Yuichi Koga1, Nami Shimizu1, Akikazu Sakudo2, Shigenori Kanaya1. 1 2 RLPFK suggests a complex self-association behavior in the presence of Fru- Dept. of Engineering, Osaka University, Suita, Japan, University of the 6-P. Electron microscopy indicates that, in the presence of Fru-6-P, rat liver Ryukyus, Nishihara, Japan. PFK can form long fibrils that consist of up to 36 PFK tetramers extending The abnormal prion protein (PrPSc: scrapie-associated prion protein) is over 200 nm in length. These effects are counteracted by MgATP, a known considered to be included in the group of infectious agents of transmissible allosteric inhibitor of the enzyme. Funding: NIH-GM33216, NIH-CBI and spongiform encephalopathies. Since PrPSc is highly resistant to normal ster- Welch-A1543 ilization procedures, the decontamination of PrPSc is a significant public health issue. Tk-subtilisin is a subtilisin-like serine protease identified from 2694-Pos Board B124 a hyperthermophilic archaeon Thermococcus kodakarensis KOD1. Among The Isolated Large Subunit of E. coli Carbamoylphosphate Synthetase De- the subtilisin family of proteases, Tk-subtilisin has significant high heat stabil- viates from Kinetic Mechanism and Allosteric Behavior of the Holoenzyme ity with its highest specific activity at 90C and a half-life of 50 min at 100C. Robert Koenig, Gregory D. Reinhart. In the present study, a hyper-thermostable protease, Tk-subtilisin, was used to Biochemistry and Biophysics, Texas A&M Univ, College Station, TX, USA. degrade PrPSc. Although PrPSc is known to be resistant toward proteolytic Carbamoyl Phosphate Synthetase (CPS) from E. coli is a heterodimeric enzyme enzymes, Tk-subtilisin was able to degrade PrPSc under extreme conditions. translated from the CarA and CarB genes. The polypeptides formed from these The level of PrPSc in brain homogenates was found to decrease significantly transcripts are 42kDa and 118kDa, respectively. CPS catalyzes the synthesis of in vitro following Tk-subtilisin treatment at 100C, whereas some protease carbamoylphosphate through activation of bicarbonate by one equivalent of resistant fractions remain after proteinase K treatment. Rather small amounts MgATP priming bicarbonate for the addition of ammonia; the carbamate is of Tk-subtilisin were required to degrade PrPSc at 100C and pH 8.0. In then phosphorylated by a second equivalent of MgATP. The product of the addition, Tk-subtilisin was observed to degrade PrPSc in the presence of CarB gene contains two active sites, both are functional as partial reactions, sodium dodecyl sulfate or other industrial surfactants. Although several one is responsible for bicarbonate activation by MgATP and the amination proteases degrading PrPSc have been reported, practical decontamination of the activated bicarbonate using an ammonia source (glutamine in vivo), procedures using enzymes are not available. This report aims to provide the second site is responsible for phosphorylation of carbamate and is distal basic information for the practical use of a proteolytic enzyme for PrPSc to the heterodimer interface. The binding site for allosteric regulators is located degradation. on the large subunit proximal to the phosphorylation active site. The smaller subunit contains the active site for deamination of glutamine and is not manda- 2692-Pos Board B122 tory for the activity of the sites on the large subunit. Previous work demon- Kinetic Characterization of Human Liver Phosphofructokinase strated the reverse of the phosphorylation partial reaction of CPS, formation Amanda Tindall, Gregory D. Reinhart. of ATP from MgADP and carbamoylphosphate, is equilibrium ordered and Biochemistry and Biophysics, Texas A&M Univ, College Station, TX, USA. responsive to allosteric ligands. In this work we provide evidence that Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phos- removing the small subunit, thus relieving interfacial constraints 45A˚ from phate (F6P) to fructose 1,6-bisphosphate in an ATP dependent reaction. This the active site, results in reduced allosteric effects. Data shows that the kcat reaction represents the first committed step of the glycolytic pathway and as for the ATP synthesis reaction has decreased by 7-fold with no discernable dif- such plays an important role in metabolism. In liver, this regulation is espe- ference in Kia. kcat ¼ 6.9 sec-1 in holoenzyme and 1 sec-1 for the phosphor- cially interesting, as hepatocytes can alternatively perform gluconeogenesis ylation in isolated large subunit. The Kia determined for MgADP in or glycolysis. While PFKs from the livers of several mammalian species holoenzyme is 510 mM and 700 mM in the isolated large subunit. Supported have been characterized, human liver PFK has never been thoroughly exam- by the grant GM 33216 from the NIH. ined. The gene encoding human liver PFK was synthesized and cloned into an expression vector utilizing the tac promoter. Human liver PFK was ex- 2695-Pos Board B125 pressed in an Escherichia coli cell line, RL257, which contains no native Free-Energy Landscapes of the Translocation of a Substrate in Four PFK. The protein has been purified to a specific activity of 45 U/mg through Proteasome - Activator Complexes Analyzed using Molecular Dynamics a combination of ammonium sulfate precipitations, heat denaturation, and Simulations anion exchange chromatography. Initial characterizations indicate that human Hisashi Ishida. liver PFK is inhibited by ATP more substantially than rat liver PFK, a well Quantum Beam Science Center, Japan Atomic Energy Agency, Kyoto, Japan. characterized mammalian PFK. Additionally, pH changes modify the binding Proteasome is involved in the degradation of proteins. Proteasome activators of F6P to a greater extent than in rat liver PFK. This work seeks to quantify bind to the proteasome core particle (CP) and facilitate opening a gate of the the kinetic and allosteric behaviors of human liver PFK and contrast those be- CP, where Tyr8 and Asp9 in the N-termini tails of the CP form the ordered haviors with those of rat liver PFK. This work is supported by NIH grant open gate. In a double mutant (Tyr8Gly/Asp9Gly), the N-termini tails are GM033216. disordered and the stabilized open-gate conformation cannot be formed. To Wednesday, February 11, 2015 533a understand how proteasome is responsible for the efficient proteolysis of the CheA is a multi-domain histidine kinase which has been demonstrated to play a substrate, four different molecular dynamics (MD) simulations were carried role in the chemosensory array in Escherichia coli, Salmonella typhimurium, out: ordered- and disordered-gate models of the CP complexed with an ATP- Thermotoga maritima and related enterobacteria. The P4 domain of CheA is independent PA26 and ordered- and disordered-gate models of the CP com- of particular interest due to its role in catalyzing ATP/ADP conversion, a pro- plexed with an ATP-dependent PAN-like activator. The free-energies of the cess which involves Mg2þ in the ATP-binding pocket of the enzyme. Recent translocation of a poly-peptide substrate moving through the gate were esti- studies in GHKL superfamily members MutL (from E coli) and BCK (from mated using an MD simulation program called SCUBA developed by the Japan Rattus norvegicus) demonstrate improved activity in the presence of monova- Atomic Energy Agency (JAEA). In the ordered-gate models, the substrate in lent cations Naþ and Kþ. Here we present results from a combined experi- the activator was more stable than that in the CP. The conformational entropy mental and computational analysis to assess the effect of monovalent cations of the N-termini tails of the CP was larger when the substrate was in the acti- on the activity of Salmonella typhimurium CheA and subsequent phosphor- vator than in the CP. In the disordered-gate models, the substrate in the acti- transfer to response regulator CheY. vator was more destabilized than in the ordered-gate models. The mutated N-termini tails became randomized and their increased conformational entropy 2699-Pos Board B129 could no longer increase further even when the substrate was in the activator, Computational and Experimental Characterization of Intramolecular meaning the randomized N-termini tails had lost the ability to stabilize the Regulatory Interactions in Hck substrate in the activator. Thus, it was concluded that the dynamics of the N- Matthew P. Pond, Sunhwan Jo, H. Clark Hyde, Michelle H. Wright, termini tails entropically play a key role in the translocation of the substrate. Lydia Blachowicz, Francisco Bezanilla, Benoit Roux. Department of Biochemistry and Molecular Biology, University of Chicago, 2696-Pos Board B126 Chicago, IL, USA. Development of a Novel Assay for Pink1-Parkin-Miro1 Pathway Src family kinases (SFKs) are a group of nine non-receptor tyrosine kinases that Sungjin Park1, Julian L. Klosowiak1, Alexander V. Statsyuk2, play critical roles in cellular transduction pathways. These highly sought after Sarah E. Rice1. therapeutic targets are prevalent and promiscuous; therefore, they must be 1Department of Cell and Molecular Biology, Northwestern University, tightly regulated to prevent unchecked signaling cascades. Inter-domain inter- Chicago, IL, USA, 2Department of Chemistry, Northwestern University, actions that hinder kinase function are key SFK regulatory mechanisms, and Evanston, IL, USA. detailed information about these interactions is integral to understanding how Mutations in the parkin and PINK1 genes are a major cause of autosomal these enzymes function. recessive early-onset Parkinson disease (PD). Several studies have shown Hck is a SFK primarily found in cells of hematopoietic lineage and is among that PINK1 phosphorylates Parkin, Parkin substrates, and ubiquitin, thereby the most well studied members of the family. Crystal structures of Hck in stimulating ubiquitination of proteins on the mitochondrial surface (i.e. the down-regulated form and solution studies detailing catalytic responses to Miro1) by the Parkin E3 ligase. The ubiquitinated proteins then are degraded, regulatory domain displacement provide a robust framework for characterizing which signals for mitochondrial clearance. Although it is imperative to under- the regulatory interactions. Adding to this knowledge, we provide fundamental stand these events at the molecular level, current biochemical methods are information on Hck regulation by measuring binding free energies of the native cumbersome and lack the resolution to answer these questions thoroughly. domain-peptide interactions. A combined experimental and computational Most importantly, physiological E2 partners for Parkin ligase are contested approach was used to complement and expand the usefulness of the data. in the field. Here, we report a novel assay to study the PINK1-Parkin- The results were compared with bulk FRET measurements made using the Miro1 pathway that fundamentally addresses the aforementioned problems. full-length kinase to observe how these interactions are modified in the context In our assay, the ubiquitin c-terminus is chemically activated as a thioester of the protein. that can undergo transthiolation with the catalytic cysteine of the Parkin E3 Given the high degree of homology among family members, it is unsurpris- ligase, thereby directly charging ubiquitin to Parkin E3s without the need ing that SFKs have some redundant or compensatory functions. However, it for E1, E2 and ATP. We demonstrate that this simplified two-component sys- has been shown that even the most closely related members of the family tem recapitulates two native functions of Parkin, Miro1 ubiquitination and au- cannot always substitute functionally for one another in vivo and the differ- toubiquitination. This simplified E2-bypassing assay will be generally useful ences responsible have yet to be fully elucidated. Our results lay the ground- to study the Parkin mechanism and to screen small molecular Parkin activa- work for comparative analysis between different family members and are tors to treat PD. expected to aid in identifying features that distinguish these enzymes from one another. 2697-Pos Board B127 Modeling the Effect of Direct PKA-PDE Binding in the Formation of 2700-Pos Board B130 Localized Microdomains of Camp in Adult Cardiomyocytes A Nudix Hydrolase Necessary for Mycobacterium Tuberculosis Survival Britton W. Boras. under Oxidative Stress Bioengineering, San Diego, San Diego, CA, USA. Kerstin A. Wolff1, Andres H. de la Pen˜a2, Hoa T. Nguyen3, The b-adrenergic pathway in cardiomyocytes activates protein kinase A (PKA) L. Mario Amzel4, Sandra B. Gabelli5, Liem Nguyen3. 1 to phosphoregulate several Ca2þ handling proteins, including the L-type Ca Molecular Biology and Microbiology, Case Western Reserve University 2 channel, ryanodine receptor, and sarco/endoplasmic reticulum Ca2þ-ATPase School of Medicine, Baltimore, MD, USA, Biomedical Engineering, Johns 3 (SERA) (via phospholamban), resulting in inotropic, lusitropic and chrono- Hopkins University School of Medicine, Baltimore, MD, USA, Molecular tropic responses. Recent studies have postulated a role of direct phosphodies- Biology and Microbiology, Case Western Reserve University School of 4 terases (PDE)/PKA-Regulator (R) subunit interaction to regulate PKA Medicine, Cleveland, OH, USA, Biophysics and Biophysical Chemistry, activation in various compartments of the cardiac cell. This interaction is con- Johns Hopkins University School of Medicine, Baltimore, MD, USA, 5 trol by A kinase anchoring proteins (AKAP)s that select for specific isoforms. Medicine, Johns Hopkins University School of Medicine, Baltimore, In this study we used in vitro kinetic experiments with 3H tagged cAMP, MD, USA. purified R-subunits, and PDEs to quantify this interaction and explore its spec- Despite a constant decrease in tuberculosis (TB) incidence rates, nearly two ificity both for RIa and RIIb with PDE3 and PDE4 isoforms. The experimental billion people worldwide are estimated to have latent TB. Five to ten percent results were combined with a new Markov Model of PKA activation using Vir- of people with latent TB will develop the active form of the disease. Of the tual Cell, a finite volume solver, to suggest the physiological impact of estimated 8.6 million new cases of active TB in 2012, 1.3 million people died including this interaction. This new regulatory pathway and PKA Markov of the disease and 450,000 developed multidrug-resistant TB. Resistance to Model will be included in a whole cell signaling model of cardiomyocyte acti- oxidative stress is essential for Mycobacterium tuberculosis (Mtb) survival vation in response to beta-adrenergic stimulation. in host macrophages and the onset of latent TB infection. We have identified a Nudix (nucleoside diphosphate-linked moiety X) hydrolase, RenU, 2698-Pos Board B128 necessary for Mtb survival in oxidative stress environments. We show that Monovalent Cation Dependence on the Kinase Activity of Salmonella RenU preferentially degrades adenosine derivatives over other nucleoside de- Typhimurium CheA: Experiment and Modeling rivatives. Through a novel fluorescence based assay, we also determined Marie Balboa1, Michael Turvey1, Joseph Falke1, Kene Piasta2, that RenU prefers NADH as a substrate over NAD. Furthermore, we show Margaret Hurley3. that RenU is required for Mtb survival within macrophages. The link be- 1University of Colorado, Boulder, CO, USA, 2Brandeis University, Waltham, tween RenU, NADH, and Mtb survival warrants further investigation as it MA, USA, 3US Army Research Laboratory, Aberdeen Proving Ground, could be the basis for novel therapeutic approaches to prevent and combat MD, USA. latent TB. 534a Wednesday, February 11, 2015

2701-Pos Board B131 matic conformational dynamics. We demonstrated that the enzyme confor- Understanding the Catalytic Mechanism of Laminaripentaose Producing mational flexibility can be regulated by enzyme-substrate interaction, an b-1,3-Glucanase experimental evidence of protein-substrate folding-binding interaction mecha- Xiaochen Zhang, Kevin Osenburg, Shuhua Ma. nism. Our new approach is applicable to a wide range of single-molecule AFM- Chemistry, Towson University, Towson, MD, USA. FRET measurements for protein conformational changes under enzymatic Laminaripentaose-producing b-1,3-glucanase (LPHase) catalyzes the hydroly- reactions, including controls of enzymatic reactivity by mechanical-force sis of a long chain polysaccharide b-1,3-glucan into specific pentasaccharide manipulating protein conformations. oligomers. LPHase is a member of the glycoside hydrolase family 64 (GH- Reference: 64) that play important roles during biomass degradation. Experimentally, 1. Qing Guo, Yufan He, H. Peter Lu, ‘‘Repetitive Force Pulling-Releasing the enzymatic mechanism of LPHase remains to be determined although it Manipulation of Enzymatic Conformational Fluctuations and Enzyme- has been reported that Glu154 and Asp170 may serve as the active site residues Substrate Interactions by Single-Molecule FRET-Magnetic Tweezers Micro- of LPHase. Molecular modelling may offer more insight into the reaction scopy,’’ Phys. Chem. Chem. Phys., 16, 13052 13058 (2014). pathway. 2. H. Peter Lu, ‘‘Sizing up single-molecule enzymatic conformational dy- Several synthetic substrates of LPHase have been reported. We chose laminar- namics,’’ Chem. Soc. Rev., Invited Review Article, 43, 1118-1143 (2014). ihexaose as the substrate and docked one structure of laminarihexaose obtained 3. Yufan He, Maolin Lu, H. Peter Lu, ‘‘Single-Molecule Photon Stamping from the Protein Data bank to the active site of LPHase. Molecular dynamics FRET Spectroscopy Study of Enzymatic Conformational Dynamics,’’ Phys. simulations using a combined quantum mechanical and molecular mechanical Chem. Chem. Phys., 15, 770-775 (2013) (Cover page). (QM/MM) method have been employed to study the hydrolysis of the glyco- 4. Yufan He, Maolin Lu, Jin Cao, H. Peter Lu, ‘‘Manipulating Protein Confor- sidic bond catalyzed by LPHase. Our results on the current LPHase - substrate mations by Single-Molecule AFM-FRET Nanoscopy,’’ ACS Nano, 6, 1221- complex suggest that the proton transfer from the catalytic general acid to the 1229 (2012). glycosidic oxygen be concerted with the nucleophilic attack at the anomeric carbon, and the free energy of activation is about 30 kcal/mol. Additional sim- 2704-Pos Board B134 ulations on LPHase - substrate complexes are underway to characterize the Moonprot: A Database for Proteins that are Known to Moonlight LPHase catalyzed reaction pathway. Constance Jeffery1, Mathew Mani1, Shadi Zabad2, Chang Chen3, Vaishak Amblee1, Haipeng Liu4, Tanu Mathur1, Grant Zwicke1, Bansi Patel1, 2702-Pos Board B132 Jagravi Thakkar1. X-Ray Structure of a Mammalian Stearoyl-Coa Desaturase-1 1Biological Sciences, University of Illinois, Chicago, IL, USA, 2Illinois Yonghong Bai, Jason G. McCoy, Elena J. Levin, Ming Zhou. Institute of Technology, Chicago, IL, USA, 3Bioengineering, University of Baylor College of Medicine, Houston, TX, USA. Illinois, Chicago, IL, USA, 4Pharmaceutical Biotechnology, University of Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces Illinois, Chicago, IL, USA. the first double bond into saturated fatty acyl CoAs. Since the monounsatu- Moonlighting proteins comprise a class of multifunctional proteins in which a rated products of SCD are key precursors of membrane phospholipids, choles- single polypeptide chain performs multiple biochemical functions that are terol esters, and triglycerides, SCD is pivotal in fatty acid metabolism. not due to gene fusions, multiple RNA splice variants, or pleiotropic effects. Humans have two SCD homologs (SCD1 and SCD5), and mice have four The known moonlighting proteins perform a variety of diverse functions in (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when many different cell types and species, and information about their structures fed a high-fat diet because of improved lipid metabolic profiles and insulin and functions is scattered in many publications. We have constructed sensitivity. Thus, SCD1 is a promising pharmacological target in the treatment the manually curated, searchable, internet-based MoonProt Database (http:// of obesity, diabetes, and other metabolic diseases. SCD1 is an integral mem- www.moonlightingproteins.org) with information about the over 200 proteins brane protein located in the endoplasmic reticulum, and catalyzes the forma- that have been experimentally verified to be moonlighting proteins. The avail- tion of a cis-double bond between the 9th and 10th carbons of stearoyl- or ability of this organized information provides a more complete picture of what palmitoyl-CoA. The reaction requires molecular oxygen, which is activated is currently known about moonlighting proteins. The database will also aid by a diiron center, and cytochrome b5, which reduces the diiron center. To researchers in other fields, including determining the functions of genes iden- better understand the structural basis of these characteristics of SCD function, tified in genome sequencing projects, interpreting data from proteomics pro- we crystallized and solved the structure of mouse SCD1 bound to a stearoyl- jects, and annotating protein sequence and structural databases. In addition, CoA molecule at 2.6 A˚ resolution. The structure shows a novel fold information about the structures and functions of moonlighting proteins can comprising four transmembrane helices capped by a cytosolic domain. The be helpful in understanding how novel protein functional sites evolved on acyl chain of the bound stearoyl- CoA is enclosed in a tunnel buried in the an ancient protein scaffold, which can also help in the design of proteins cytosolic domain, and the geometry of the tunnel suggests the structural basis with novel functions. for the regioselectivity and stereospecificity of the desaturation reaction. The structure reveals a dimetal center coordinated by a unique configuration of nine conserved histidine residues that implies a potentially novel mechanism Transcription for oxygen activation. The structure also illustrates a potential pathway for substrate access and product egress, and a possible route for electron transfer 2705-Pos Board B135 from cytochrome b5 to the diiron center. Molecular Origins of Bimodal mRNA Copy Number Distribution Keisuke Fujita, Mitsuhiro Iwaki, Toshio Yanagida. 2703-Pos Board B133 Riken, Suita, Japan. Probing and Manipulating Enzyme Activity and Conformational Dy- Stochastic gene expression contributes to the variability of genetically iden- namics by Single-Molecule Afm-FRET and Magnetic Tweezers-FRET tical cells in identical environments, giving rise to significant consequences Ultramicroscopy in many biological processes. Generally, stochastic gene expression is ex- Qing Guo, Yufan He, H. Peter Lu. plained by a two-state model of gene regulation, where a gene switches be- Department of Chemistry and the Center for Photochemical Sciences, tween on and off states. Transcription kinetics obtained from in vivo Bowling Green State University, Bowling Green, OH, USA. imaging is consistent with the two-state model. A theoretical model predicts Single-molecule conformational manipulation provides unique methods for that the distribution of mRNA copy numbers can be transitioned from a Pois- studying the relationship between function and structure of biomolecules, son distribution to a bimodal distribution by modulating kon and koff. This and for exploring novel properties of biomolecules under complex local envi- transition is considered the origin of cell-to-cell variability. However, despite ronments. Enzymatic reactions are traditionally studied at the ensemble level, the versatility of the two-state model of gene regulation, little is known about despite significant static and dynamic inhomogeneities. We have developed the dynamics of the on-off switching and modulation. Many mechanisms of and applied AFM/Magnetic Tweezers force manipulation combined single- gene regulation have been proposed, including promoter binding of repressors molecule spectroscopy to study the mechanisms and dynamics of enzymatic and activators and, more recently, structural changes of DNA by a DNA bind- reactions involved with kinase and lysozyme proteins. Enzymatic reaction ing protein. turnovers and the associated structure changes of individual protein molecules In this study, we reconstructed prokaryote transcription and transcriptional under pico-Newton force manipulations were observed simultaneously in real- regulation in vitro by using an E coli RNA polymerase (RNAP) and a l phage time by single-molecule FRET detections. By a repetitive pulling-releasing promoter, and visualized mRNA production at the single molecule level by fast manipulation of a Cy3-Cy5 dye labeled kinase molecules under the conditions fluorescence in situ hybridization (fastFISH) to analyze the dynamics of mRNA with and without enzymatic substrates, we observed and analyzed the enzy- production and its modulation. Wednesday, February 11, 2015 535a

2706-Pos Board B136 To determine whether RNAP-CRE interactions are sequence-specific, we con- Uncovering the Dynamics of RNA Polymerase in Live E. coli Cells structed all nucleotide substitutions at each CRE position, and assessed effects Kelsey Bettridge1, Christopher Bohrer1, Xinxing Yang1, Max Klein2, on RNAP-DNA interaction in equilibrium binding experiments and off-rate ex- Jie Xiao1. periments. The results show that RNAP exhibits specificity for T or G at CRE 1Johns Hopkins School of Medicine, Baltimore, MD, USA, 2Johns Hopkins position 4, specificity for T at CRE position þ1, and specificity for G at CRE University, Baltimore, MD, USA. position þ2. Transcription is a highly regulated process in the cell. The kinetics and mech- To identify RNAP residues that mediate specificity at CRE positions þ2, þ1, anism of transcription have been extensively studied in vitro. However, a and 4, we constructed Ala substitutions of RNAP residues and assessed effects living cell is a complex entity; the heterogenous, crowded cellular environ- on RNAP DNA interactions with promoters containing all nucleotide substitu- ment is drastically different from the homogeneous, well-mixed situation tions at CRE positions 4, þ1, and þ2. The results show that Ala substitution in vitro, likely imposing different rules on transcription. In this work we of substitution of bR371 eliminates specificity at CRE position 4, Ala substitu- used a functional, fluorescent protein-tagged RNAP in live E. coli cells to tion of bW183 eliminates specificity at CRE position þ1, and Ala substitution track the dynamics of individual RNAP molecules in time and space. Using of bR151, bD446, or bR451 eliminates specificity at CRE position þ2 quantitative single particle tracking analysis and theoretical modeling, we To define the structural basis of specificity at CRE positions þ1 and þ2, we determined different diffusive states of RNAP and the corresponding transi- determined crystal structures of RPo derivatives containing all nucleotide sub- tion probabilities between states. By further correlating with transcription stitutions at CRE positions þ1 and þ2. The results show that specificity at CRE activities, we provide results illustrating the kinetics of various aspects of positions þ1 and þ2 manifests itself not only in quantitative differences in transcription such as the typical promoter search time and transcription elon- binding thermodynamics and kinetics, but also in qualitative differences in gation time in live cells. structure. In further work, we have used RNAP derivatives containing bR371A, 2707-Pos Board B137 bW183A, and bD446A substitutions as reagents to assess the functional signif- Spatial Organization of Transcription in E. coli Cells icance of RNAP-CRE interactions. The results show that RNAP CRE interac- Xiaoli Weng, Arvin C. Lagda, Jie Xiao. tions affect the sequence specificity of promoter binding and start-site selection Biophysics and Biophysical Chemistry, Johns Hopkins, Baltimore, during transcription initiation and also affect the sequence specificity of trans- MD, USA. location and pausing during transcription elongation. RNA polymerase (RNAP) has been shown to exhibit clustered distribution in bacterial cells. These clusters were hypothesized to be active transcription fac- 2710-Pos Board B140 tories in which concentrated RNAP molecules are engaged in ribosomal RNA Transient-State Kinetic Analysis of the RNA Polymerase I Nucleotide synthesis. To examine this hypothesis, in this study we used photoactivated Addition Cycle localization microscopy (PALM) to map the spatial distribution of RNAP in Francis D. Appling. correlation with active transcription in superresolution. We monitored the co- University of Alabama at Birmingham, Birmingham, AL, USA. localization of RNAP with single mRNA or rRNA gene sites, with nascent tran- RNA polymerases (Pols) are a diverse class of enzymes that fulfill an array of scripts, and with elongation factors NusA and NusB. We further examined the biological requirements. In eukaryotes the task of nuclear RNA synthesis is cellular positioning of rRNA and mRNA genes sharing similar regulatory con- apportioned among three distinct Pols. Unlike all other cellular Pols, Pol I rec- trols under different transcription conditions. Our results provide evidence ognizes a single promoter and synthesizes a single target transcript. Pol I’s rigorously examining the transcription factory model. RNA product is ultimately processed into the three largest RNAs of the ribo- some. It has long been recognized that cellular protein synthetic capacity is 2708-Pos Board B138 linked to cell proliferation and transformation. In summary, Pol I is centrally Bacterial Transcript Elongation Complexes can Retain Sigma Factor located in a process requisite to tumorgenesis. Recently, the Pol I system has throughout RNA Synthesis received attention as a target of chemotherapeutic intervention. Despite Pol Timothy T. Harden1, Larry J. Friedman2, Christopher D. Wells3, I’s involvement in cancer, there are no mechanistic data available to further Ann Hochschild3, Jane Kondev1, Jeff Gelles2. its utility as a drug target. In addition, it is not clear why eukaryotes have 1Physics, Brandeis University, Waltham, MA, USA, 2Biochemistry, Brandeis evolved three distinct Pols to perform the task of nuclear RNA synthesis. A University, Waltham, MA, USA, 3Microbiology and Immunobiology, thorough understanding of the molecular mechanisms governing Pol I tran- Harvard Medical School, Boston, MA, USA. scription will aid the development of Pol I - specific inhibitors as well as further Production of a messenger RNA proceeds through sequential stages of tran- our understanding of the necessity of three nuclear Pols. scription initiation, transcript elongation and termination; all three stages We have developed techniques that enable the reconstitution of Pol I elongation are regulated to control gene expression. As RNA polymerases (RNAPs) tran- complexes from purified protein and nucleic acid components. Combining re- sition to elongation, they lose association with one or more initiation protein constituted elongation complexes with transient-state kinetic approaches we factors required to recognize transcription start sites, and they acquire proteins have characterized the Pol I nucleotide addition cycle and developed a minimal specifically involved in regulating transcript elongation and termination. Es- kinetic framework describing the reaction pathway. Combining these ap- cherichia coli s70 is an initiation factor. Previous studies reached conflicting proaches with structure-based mutagenesis will enable elucidation of the mo- conclusions about the extent to which s70 is retained after elongation begins, lecular mechanisms governing Pol I - catalyzed RNA synthesis. and the rate of its release from RNAP actively undergoing transcript elonga- tion has not been directly measured. We used multiwavelength single- 2711-Pos Board B141 molecule fluorescence co-localization microscopy to observe the association Crowding on DNA as a Source of Bursts in mRNA Production of s70 with RNAP during initiation and throughout the elongation of a Aafke A. van den Berg, S. Martin Depken. 2,400 nucleotide transcript. We observe that a majority of RNAP molecules Bionanoscience, Delft University of Technology, Delft, Netherlands. lose s70 concommitant with promoter escape, while a subpopulation of The time statistics of mRNA production is often non-Poissoinian, characterized elongation complexes retain s70 throughout elongation of the full transcript. by bursts of activity interspersed by periods of inactivity. While a multitude of The fraction of s70 retained on elongation complexes is influenced by the models focuses on initiation dynamics as the source, burstyness is often inde- sequence of the initial transcribed region. The presence of s70 on elongation pendent of promoter sequence and its microscopic origins have remained complexes far downstream of the promoter is expected to substantially alter elusive. We theoretically examine the effects of crowding on the DNA as a uni- their regulation by affecting transcription pausing, termination, and possibly versal and promoter independent mechanism for generating and sustaining re-initiation. bursts. Using a modified version of the totally asymmetric exclusion process, we model RNAp traffic in the presence of DNA binding proteins, higher order 2709-Pos Board B139 genome structures, or other dynamic roadblocks to transcription. Through Sequence-Specific RNAP-DNA Interactions in Transcription Initiation simulations, mean-field solutions, and scaling arguments we provide a state and Elongation: Core Recognition Element (CRE) diagram showing under what conditions and to what extent crowding can be Hanif Vahedian-Movahed, Yu Zhang, Jeremy Bird, Irina Vvedenskaya, expected to induce bursts in mRNA production. We further show how road- Jared Knobloch, Seth Goldman, Bryce Nickels, Richard H. Ebright. block dynamics introduces an effective attraction between polymerases, Waksman Institute, Rutgers University, Piscataway, NJ, USA. that counteracts diffusive spreading and sustains bursts throughout the elonga- The crystal structure of the RNAP-promoter open complex indicates that tion phase. Thus, by accounting for the effect of roadblocks to transcription, RNAP core interacts with the transcription-bubble nontemplate strand segment our model suggests that bursting is a natural side effect of crowding on corresponding to positions 4toþ2 (core recognition element, CRE). the DNA. 536a Wednesday, February 11, 2015

2712-Pos Board B142 combines aspects of tethered particle motion (TPM) microscopy with total in- Live-Cell Single-Molecule Imaging of Sense and Antisense Transcription ternal reflection fluorescence (TIRF) microscopy. This method allows us to of a Yeast Gene watch RNA transcription occurring on dozens of single DNA molecules in par- Antoine Coulon1, Tineke L. Lenstra2, Carson C. Chow1, Daniel R. Larson2. allel and may be scaled for use in higher-throughput investigations of transcrip- 1NIDDK, National Institutes of Health, Bethesda, MD, USA, 2NCI, National tional activity. Institutes of Health, Bethesda, MD, USA. To illustrate the utility of our method, we have been investigating transcrip- The eukaryotic genome is pervasively transcribed, giving rise to various sorts tional interference caused by the histone-like nucleosomal protein (H-NS), of non-coding RNAs whose mechanisms of action are for the most part not un- which is a global transcriptional silencer abundant in many bacteria. Here we derstood. Recent technological advances now allow direct visualization of the use this technique to explore the promoter occupancy and procession of T7 synthesis of nascent transcripts from individual genes over time by decorating polymerase in the presence of H-NS. However, our setup is amenable to the RNAs with fluorescent proteins. Using the orthogonal RNA-binding MS2 and study of a host of different regulatory factors acting on a variety of promoter PP7 bacteriophage coat proteins, we were recently able to tag two regions of the architectures. same RNA in two different colors [Coulon et al. 2014, eLife, in press]. Here, we used this technique to visualize simultaneously sense and antisense transcrip- 2715-Pos Board B145 tion from the GAL10 locus in yeast, during activation of the GAL pathway. DNA Looping both Enhances and Suppreses Transcriptional Noise Fluorescence fluctuations recorded in both channels at the transcription site Jose M.G. Vilar1, Leonor Saiz2. 1 reflect the kinetics of transcription on both strands as the GAL10 gene gets acti- Biophysics Unit (CSIC-UPV/EHU) and Department of Biochemistry and vated in response to galactose. We observe transient antisense transcription Molecular Biology, IKERBASQUE and University of the Basque Country 2 occurring almost exclusively prior to the appearance of sense transcription. UPV/EHU, Bilbao, Spain, Biomedical Engineering, University of Using cross-correlation analysis, we uncovered specific temporal windows California, Davis, Davis, CA, USA. relatively to sense activation where antisense transcription is enriched or DNA looping has been observed to enhance and suppress transcriptional noise depleted - likely reflecting the biochemical mechanisms underlying activation. but it is uncertain which of these two opposite effects is to be expected for given Once transcription of the GAL10 gene starts, transcripts are produced in bursts conditions. Here, we present the derivation of analytical expressions for the separated by periods of inactivity, occasionally leaving the opportunity for anti- main quantifiers of transcriptional noise in terms of the molecular parameters sense transcription to happen. We developed a method for applying fluctuation and elucidate the role of DNA looping [1]. Our results rationalize paradoxical correlation analysis to non-stationary time traces. This allowed us to isolate the experimental observations and provide the first quantitative explanation of bursting kinetics even in the non-steadystate context of a transient response to landmark individual-cell measurements at the single molecule level on the clas- galactose. By modeling the autocorrelation of a bursting gene, we were able to sical lac operon genetic system [2]. infer from our data how the elongation rate, burst size and burst frequency of [1] Vilar J.M.G. and Saiz L., Physical Review E 89, (6) 062703 (2014). the GAL10 gene are modulated by different doses of galactose. This work [2] Choi et al., Science 322, 442-446 (2008). shows how in vivo single-molecule methods and fluctuation analysis can reveal 2716-Pos Board B146 unanticipated mechanisms of transcriptional regulation. Nuclear Actin Dynamics Regulate Nuclear Organization and Transcription 2713-Pos Board B143 Leo Serebryannyy1, Megan Parilla1, Paolo Annibale2, Christina Cruz1, The Mechanism of Transcription Stalling under Torsion Dmitri Kudryashov3, Enrico Gratton2, Cara J. Gottardi4, Primal de Lanerolle1. 1 Chuang Tan1,2, Jie Ma1,2, Jeremy G. Bird3,4, James T. Inman1,2, Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, 2 Jeffrey W. Roberts3, Michelle D. Wang1. USA, Laboratory of Fluorescence Dynamics, University of California at 3 1Laboratory of Atomic and Solid State Physics, Department of Physics, Irvine, Irvine, CA, USA, Chemistry and Biochemistry, Ohio State 4 Cornell University, Ithaca, NY, USA, 2Howard Hughes Medical Institute at University, Columbus, OH, USA, Medicine, Northwestern University, Cornell University, Ithaca, NY, USA, 3Department of Molecular Biology and Chicago, IL, USA. Genetics, Cornell University, Ithaca, NY, USA, 4Waksman Institute, Rutgers Actin is an important and elegant mechanotransducer that transfers messages University, Piscataway, NJ, USA. from the extracellular environment into the nucleus, thus conferring changes During transcription, RNA polymerase (RNAP) translocates along DNA and in both the physical properties and the genetic program of the cell. Although introduces torsional stress, and excessive torque accumulation can lead to tran- actin in the nucleus has been established in a growing number of functions, scription stalling. We previously measured this stall torque for E. coli RNAP the form of nuclear actin remains poorly understood as there are no apparent during transcription1. However the mechanism and regulation of RNAP stalling actin filaments in the nucleus. Therefore, we tested how disrupting the form of under torsion has remained elusive. Here, we investigate the transcriptional dy- nuclear actin impacts function and what effects this may have on the nucleus. namics of RNAP in the presence and absence of GreB, a transcription elonga- To do so, we formed stable nuclear actin filaments using a variety of methods, tion factor known to rescue backtracked RNAP. Using an angular optical trap including the nuclear enrichment of actin binding proteins supervillin and assay, we found that the presence of GreB can significantly increase the stall a-E-catenin and the expression of a mutant form of skeletal a-actin. Using torque of RNAP. In addition, RNAP often exhibited distinct reverse motion fluorescence recovery after photobleaching (FRAP), we found that stabiliza- upon stalling in the absence of GreB, while this was rarely observed in the pres- tion of nuclear actin filaments significantly impairs actin dynamics within ence of GreB. These results suggest that backtracking is the primary mecha- the nucleus. The formation of nuclear actin filaments coincides with striking nism of RNAP stalling on DNA under torsion and demonstrate a potential changes in nuclear structure and overall nuclear topography as determined by regulatory role for GreB in assisting an elongating RNAP in overcoming the confocal microscopy and raster image correlation spectroscopy. Using a com- torsional barrier of DNA. bination of FRAP analysis, structured illumination microscopy and immuno- 1J. Ma, L Bai, and M.D. Wang. Transcription under torsion. Science 340:1580- logical assays, we found that nuclear actin filaments reduce the association of 3 (2013). actin with RNA polymerase II and this correlates with impaired RNA poly- merase II dynamics, localization and gene recruitment. Moreover, we were 2714-Pos Board B144 able to recapitulate our findings in purified nuclear extract by using in vitro A High-Throughput Single-Molecule Assay for Screening Transcriptional transcription assays with the covalent actin crosslinking domain (ACD) of Interference V. cholerae MARTX toxin. Based on our data, which help explain the absence Amir Mazouchi1, Tai-Wei Su1, Joshua Milstein2. of nuclear actin filaments in the interphase mammalian nucleus, we propose a 1Chemical and Physical Sciences, University of Toronto, Mississauga, ON, model where nuclear actin dynamics are critical for maintaining proper nu- Canada, 2Physics, Chemical and Physical Sciences, University of Toronto, clear function. Mississauga, ON, Canada. Transcription of genetic information from DNA to RNA for protein synthesis and regulatory control is a fundamental biological process. Gene expression in the Chromatin and the Nucleoid cell is regulated, and eventually silenced, for various purposes such as to trigger a developmental pathway, to protect the genome from infectious DNA elements 2717-Pos Board B147 or to respond to environmental stimuli. Gene silencing techniques are used in Epigenetics: How Much Physics Do We Need to Understand It? biomedical research and in the development of novel therapeutics for treating Christophe Lavelle. various cancers, infectious diseases and neurodegenerative disorders. CNRS UMR7196 / INSERM U1154, CNRS, Paris, France. We present a simple single-molecule assay for studying transcription and as- Eukaryotic genomes are divided into chromosomes, each consisting of a single sessing the effects of small molecules on gene transcription. Our approach molecule of several centimeters of DNA compacted into a nucleoprotein Wednesday, February 11, 2015 537a substance known as ‘‘chromatin’’. In the recent years, more and more evidence interaction probabilities, and the Rabl arrangement. In addition, has accumulated pointing out chromatin polymorphism and dynamics as a pri- Chr-NE interactions affect loci specific and averaged chromosomal diffusion. mary mean of control of genome accessibility in time and space, driving the Based on these results we conclude that the presence of Chr-NE interactions focus on this complex polymer as a critical player in gene regulation. A thor- may delay the decay of the biologically relevant fractal globule state in vivo. ough characterization of chromatin properties would then be a prerequisite step in our understanding of differential gene expression, e.g. ‘‘epigenetics’’ 2720-Pos Board B150 in its original definition by Waddington as ‘‘the study of the causal mechanisms Biophysical Models of Nucleosome Positioning by which the genes of the genotypes bring about phenotypic effects’’. Razvan V. Chereji, David D. Clark. We wish here to emphasize some physical characteristics of genome organiza- NICHD, National Institutes of Health, Bethesda, MD, USA. tion in order to provide a more complete framework in which to interpret the con- A human body contains enough DNA to circle the Earth’s Equator more than trol of gene expression. Indeed, as various molecular motors push, pull and twist 2.5 million times. The basic units of DNA packaging are called nucleosomes. DNA, transient forces and torques develop within chromatin, with expected con- Their locations along the chromosomes play an essential role in gene regulation. sequences on transcription and other DNA metabolism events such as repair or We study nucleosome positioning in yeast, fly and mouse, and build biophysical recombination. In addition to discussing some basic mechanical and topological models in order to explain the genome-wide nucleosome organization. We issues, we will also present some recent quantitative and qualitative insights from show that DNA sequence is not the major cause of the regular arrays of nucle- our lab into chromatin organization and dynamics, including the still controver- osomes observed in vivo near the transcription start sites (TSS). We construct a sial role of ions in DNA compaction and the mechanical action of recombinases. minimal model in which nucleosomes are positioned by potential barriers Boule´ JB, Mozziconacci J and Lavelle C. (2014). The polymorphism of the located in the gene promoters, and which accurately reproduces the genome- chromatin fiber. J Phys Cond Mat (in press). wide nucleosome occupancy patterns observed over the transcribed regions in Lavelle C. (2014). Pack, unpack, bend, twist, pull, push: the physical side of living cells. Our statistical mechanics model allows us to study nucleosome gene expression. Curr Opin Genet Dev 25:74-84. phasing against potential barriers and wells [1, 2], sequence-dependent nucleo- Huet S, Lavelle C & al. (2014). Relevance and limitations of crowding, fractal, some affinity [2], nucleosome unwrapping [3], competition between different and polymer models to describe nuclear architecture: is a unified picture out of DNA-binding proteins, and accessibility of transcription factors [4, 5] to target reach? Int Rev Cell Mol Biol 307:443-479. sites which are found in nucleosomal DNA, among others. We also discuss alternative nucleosome positioning mechanisms: nucleosome anchoring [6] 2718-Pos Board B148 and active nucleosome positioning by ATP-dependent remodelers [7]. Nucleosome Kinetics and Accesibility of DNA [1] RV Chereji, D Tolkunov, G Locke, AV Morozov, Phys. Rev. E 83, 050903 Jyotsana J. Parmar1, Dibyendu Das2, Ranjith Padinhateeri1. (2011) 1Biosciences and Bioengineering, Indian Institute of Technology Bombay, [2] RV Chereji and AV Morozov, J. Stat. Phys. 144, 379 (2011) Mumbai, India, 2Physics Department, Indian Institute of Technology [3] RV Chereji and AV Morozov, Proc. Natl. Acad. Sci. U.S.A. 111, 5236 Bombay, Mumbai, India. (2014) Crucial cellular processes like gene regulation, transcription, and replication [4] N Petrenko, RV Chereji, MN McClean, AV Morozov, JR Broach Mol. Biol. require access to DNA that is covered with nucleosomes. Many experiments Cell 24, 2045 (2013) suggest that nucleosome organization and dynamics can significantly influence [5] N Elfving*, RV Chereji*, V Bharatula, S Bjo¨rklund, AV Morozov, JR exposure and accessibility of various locations on the genome. In this work we Broach, Nucleic Acids Res. 42, 5468 (2014) (* contributed equally) investigate the kinetics of DNA exposure as a result of nucleosome dynamics. [6] RV Chereji, AV Morozov, YM Moshkin, in preparation We consider binding and dissociation of nucleosomes taking into account both [7] D Ganguli*, RV Chereji*, J Iben, HA Cole, DJ Clark, Genome Res. (2014) sequence specificity and ATP-dependent activity, and study accessibility of (* contributed equally) DNA near different kinds of barriers (e.g. a well-positioned protein or a nucle- 2721-Pos Board B151 osome free region near transcription start site). Using analytical calculations Prediction of Chromosome Conformations with Maximum Entropy and numerical simulations, we find the following results. We show that the Principle timescale of exposure of a DNA site near a barrier can be very diverse and Bin Zhang, Peter G. Wolynes. crucially depends on the DNA sequence and the initial nucleosome organiza- Rice University, Houston, TX, USA. tion. We show how nucleosome-mediated cooperativity can emerge when mul- The genomes’ three-dimensional (3D) organization is crucial in regulating tiple transcription factors are binding at nearby locations and we investigate many biological processes, including gene regulation, DNA replication, and how multi-nucleosome correlations influence the time scale of accessibility cell differentiation. A high-resolution chromosome structure thus will signifi- as a function of the distance from the barrier. We discuss ramifications of our cantly advance our understanding of these important processes. A major step findings in understanding gene regulation and stochasticity in gene expression. toward building a structural model of the chromosome is the inventions of chro- mosome conformation capture methods, 5C and Hi-C, that aim at detecting 2719-Pos Board B149 physical contact frequencies between pairs of genomic loci. However, compu- Chromosome-Nuclear Envelope Interactions Have Multiple Effects on tational approaches to construct 3D structures that are consistent with these Chromosome Folding Dynamics in Simulation experimental contact frequency measurements remain lacking. Nicholas A. Kinney1, Igor V. Sharakhov2, Alexey V. Onufriev3. 1 We develop a statistically rigorous approach based on maximum entropy prin- Genomics, Bioinformatics, and Computational Biology, Virginia Tech, ciple to determine a least-biased potential energy landscape that reproduces Blacksburg, VA, USA, 2Entomology, Virginia Tech, Blacksburg, VA, USA, 3 experimentally determined Hi-C contact frequency between genome pairs. Computer Science, Virginia Tech, Blacksburg, VA, USA. The resulting energy landscape supports a knotless chromosome conformation, It is well recognized that the chromosomes of eukaryotes fold into non-random which has been highly anticipated since complex knotted conformations configurations within the nucleus. In humans and fruit flies, chromosomes likely prohibit the access of gene information for transcription and hinder DNA repli- adopt a particular 3D configuration called the fractal globule (FG) which has mul- cation. We further show that the topologically associating domain signal alone tiple biologically significant properties. However, the fractal globule is a meta- also enforces a chromosome structure free of knots. Our results highlight stable state which, over time, transitions to a less biologically favorable state the importance of local interactions in determining the global topology of the called the equilibrium globule. One of the key questions is how the FG state is chromosome structure. Finally, the derived landscapes for multiple chromo- stabilized in-vivo? We use simulations to study the effects of chromosome- somes support the formation of territories that have long been observed in mi- nuclear envelope (Chr-NE) interactions on the dynamics of the fractal globule croscopy experiments. Together with Hi-C experiments, our approach provides within a model of Drosophila melanogaster (fruit fly) interphase chromosomes. a coherent picture of the 3D architecture of the genomes that is consistent with The computational model represents chromosomes as self-avoiding walks many the available experimental data. (SAW) bounded by the nuclear envelope (NE). Model parameters such as nucleus size, chromosome persistence length, and chromosome-nuclear envelope inter- 2722-Pos Board B152 actions are taken directly from experiment. Several key characteristics of the Modeling the Binding of H-NS to DNA non-equilibrium FG state are monitored during each simulation’s progress: chro- Eva C. van Mastbergen, Jocelyne Vreede. mosome territories, intra-chromosomal interaction probabilities, loci specific van ‘t Hoff Institute for Molecular Sciences, University of Amsterdam, diffusion constants, and presence of the Rabl (polarized) chromosome arrange- Amsterdam, Netherlands. ment. Next, we compare the outcomes of simulations which include or exclude Bacterial chromosomal DNA is organized within a structure called the nucleoid, Chr-NE interactions. We find that Chr-NE interactions reinforce the non- which is distinctly different from the rest of the cytoplasm. Bacteria have a equilibrium properties such as chromosome territories, high intra-chromosome number of nucleoid-associated proteins that influence the organization of the 538a Wednesday, February 11, 2015 nucleoid by bending, wrapping or bridging DNA. The Histone-like Nucleoid coiling in the fibers with L ¼ 10n and 10nþ5 bp, different stiffness of the two Structuring protein H-NS can bridge DNA by binding to two separate DNA du- types of fibers, and a correlation between the local NRL and the level of tran- plexes, or shield the DNA by binding to distant sites on the same duplex, depend- scription in different parts of the yeast genome. ing on external conditions. H-NS occurs in Gram-negative enterobacteria and silences genes involved in bacterial virulence and antibiotic resistance. The cur- 2725-Pos Board B155 rent view reflects that the formation of an H-NS - DNA assembly starts with the Multivalent Targeting of Nucleosomes by the BRG1 At-Hook and Bro- initial binding of an H-NS dimer to a specific nucleotide sequence, followed by modmain additional H-NS dimers interacting with bound H-NS and binding to adjacent Daniel P. Farrell, Katayoun Varzavand, Catherine A. Musselman. sites on DNA. Several nucleotide sequences have been identified to which H- Biochemistry, University of Iowa, Iowa City, IA, USA. NS binds strongly. Despite enormous progress in methods aimed at resolving Beyond providing an avenue for packaging the genome into the nucleus, chro- molecular structures, which resulted in resolving the structures of the dimeriza- matin provides elegant mechanisms for the dynamic regulation of the eukaryotic tion domain and the DNA binding domain, it is still impossible to experimentally genome. Chromatin structure undergoes dramatic spatial and temporal reorgani- obtain detailed structural information of the entire complex, whereas dynamic zations throughout the life cycle of the cell, which require extensive remodeling properties are even harder to investigate in experiments. Molecular simulation of the nucleosome, the basic subunit of chromatin. A major mechanism for can complement experiments by modeling the dynamical time evolution of bio- modulating chromatin structure is through ATP driven nucleosome remodeling. molecular systems in atomistic detail. Employing molecular dynamics simula- The switching/sucrose non-fermenting (SWI/SNF) chromatin remodeling com- tions, we studied the binding mechanism of H-NS to DNA. Our results show plex facilitates ATP dependent remodeling of nucleosomes critical in gene regu- that H-NS binds strongly to AT-rich dsDNA in the minor groove. Furthermore, lation. SWI/SNF activity is mediated by one of two possible ATPases, Brahma we found that H-NS binds transiently to dsDNA with high GC content, in the ma- (BRM) or Brahma related gene 1 (BRG1). The chromatin targeting, occupancy jor groove. These observations are in excellent agreement with experimental and activity of the complex are positively affected by histone acetylation, and it data. By using transition path sampling, we were able to further probe the mech- has been found that this is mediated through the C-terminal Bromodomains anism of H-NS binding to AT-rich DNA, resulting in the identification of two (BDs) of BRG1 and BRM. One possible mechanism for this is that BD recogni- different modes of interaction. tion of acetylated histones targets and/or retains SWI/SNF at chromatin leading to increased activity. This is supported by previous reports of acetylation and BD 2723-Pos Board B153 dependent recruitment and retention of SWI/SNF at gene promoters. However, The Binding Landscapes of the H3/H4 and CENP-A/H4 Dimers the BRG1 BD has been shown to have very poor affinity and conflicting speci- Haiqing Zhao1, Yamini Dalal2, Garegin A. Papoian1. ficity for acetylated histone peptides in vitro, calling into question the importance 1Institute for Physical Science and Technology, University Of Maryland, of this interaction. We propose that the nucleosome context is critical for proper College Park, MD, USA, 2Center for Cancer Research, National Cancer activity of the BD in binding acetylated histones, and suggest that an adjacent Institute, Bethesda, MD, USA. AT-hook DNA binding domain contributes to BD activity. Here we present Centromere protein A (CENP-A) is a centromere-specific variant of histone H3 our recent results using NMR spectroscopy and TIRFM to investigate the multi- and shares almost 50% amino acid identity to canonical histone H3. CENP-A is valent interaction of the BRG1 AT-hook and BD with nucleosomes, including required for packaging the centromere, to facilitate separation of sister chroma- details of the structural basis by which the AT-hook-BD motif interacts with tids during mitosis. Indeed, significant structural similarities have been reported the 601-mononucleosome, the kinetic and thermodynamic basis of complex for- to exist between CENP-A/H4 dimers and H3/H4 dimers in co-crystals. In this mation, as well as the effect of histone acetylation. work, we used molecular dynamics simulations to map the binding free energy 2726-Pos Board B156 landscape for the CENP-A/H4 and H3/H4 dimers. The Associated memory, Quantification of Interphase Chromatin Dynamics in Fission Yeast Water mediated, Structure and Energy Model (AWSEM) and umbrella sam- Takeshi Sugawara1, Kenta Masuda2, Jun-ichi Uewaki1, Akinori Awazu1,3, pling constraints were applied for each simulation system towards obtaining Hiraku Nishimori1,3, Masaru Ueno1,2. two-dimensional free energy profiles of monomeric protein association and 1Research Center for the Mathematics on Chromatin Live folding. Surprisingly, our calculations revealed significant thermodynamic dis- Dynamics(RcMcD), Hiroshima University, Hiroshima, Japan, 2Department tinctions between dimerization profiles of CENP-A/H4 and of H3/H4 pairs. of Molecular Biotechnology, Graduate School of Advanced Sciences of Furthermore, we also investigated the actions of various histones chaperones, Matter, Hiroshima University, Hiroshima, Japan, 3Department of finding that free energy landscapes of the CENP-A/H4 dimer is significantly Mathematical and Life Sciences, Faculty of Science, Hiroshima University, remodeled in the presence of its cognate chaperone HJURP. The obtained re- Hiroshima, Japan. sults are in general agreement with the available experimental data and provide In mammals and other higher organisms, interphase chromosomes remain sepa- new thermodynamic insights into the mechanisms that form the basis of canon- rated from each other and compartmentalized into chromosome territories. In ical and histone variant CENP-A nucleosomes assembly in vivo. yeast, chromosomes adopt a Rabl configuration, with arms extending from cen- tromeres tethered at the spindle pole body(SPB), functionally equivalent to the 2724-Pos Board B154 centrosome, to telomeres at the opposite nuclear envelope. These organizations Topological Polymorphism of Two-Start Nucleosome Fibers: A Stereo- generally constrain chromatin motion, and contribute to gene positioning inside chemical Analysis the nucleus. On the other hand, active genes escape from such physical con- Davood Norouzi, Victor B. Zhurkin. straints and are located at active regions such as transcription factories and nu- NCI/NIH, Bethesda, MD, USA. clear pore complexes(NPCs). In spite of extensive studies for these phenomena, The spatial organization of nucleosomes in 30-nm fibers remains unknown in how gene loci sustain and change their positioning during the cell cycle still re- detail. To address this issue, we analyzed all stereochemically possible config- mains unclear. In order to elucidate chromatin dynamics during interphase, we ¼ urations of two-start nucleosome fibers with short DNA linkers L 13 - 37 bp visualized several gene loci and the centromeres (and the SPB) on the fission ¼ (nucleosome repeat length NRL 160 - 184 bp). Four superhelical parameters yeast S. pombe chromosomes that show a Rabl configuration, and then tracked – inclination of nucleosomes, twist, rise and diameter – uniquely describe a uni- the positions of the loci on a longer time scale than conventional one. The sta- form symmetric fiber. The energy of a fiber is defined as the sum of four terms: tistical analysis of the physical distance from the SPB to each locus suggested elastic energy of the linker DNA, steric repulsion, electrostatics and a phenom- that the gene loci show not only restricted diffusion due to the physical enological (H4 tail - acidic patch) interaction between two stacked nucleo- constraint of the Rabl configuration, but also a novel dynamic property, that somes. By optimizing the fiber energy with respect to the superhelical is quite different from simple diffusive behaviors reported so far. In this presen- parameters, we found two types of topological transition in fibers (associated tation, we will discuss functional roles that such chromatin dynamics possibly with the change in inclination angle): one caused by an abrupt 360 change play within the interphase nucleus. in the linker DNA twisting, and another caused by over-crossing of the linkers. (The first transition is characterized by change in the DNA linking number, 2727-Pos Board B157 DLk ¼ 1, and the second one by DLk ¼ 2.) To the best of our knowledge, Effect of Architecture of Cell Nucleus on the Folding Principles of 3D this topological polymorphism of the two-start fibers was not reported in the Genome of Budding Yeast computations published earlier. Importantly, the optimal configurations of Gamze Gu¨rsoy, Yun Xu, Jie Liang. the fibers with linkers L ¼ 10n and 10nþ5 bp are topologically different. Bioengineering, University of Illinois at Chicago, Chicago, IL, USA. Our results are consistent with experimental observations, such as the inclina- Architecture of the cell nucleus and the spatial organization of genome are crit- tion 60-70 (the angle between the nucleosomal disks and the fiber axis), helical ical for nuclear functions. Single-cell imaging techniques and chromosome rise, diameter and left-handedness of the fibers. In addition, we make several conformation capture (3C) based methods provide a wealth of information testable predictions, among them existence of different degree of DNA super- on the spatial organization of chromosomes. Computational tools for modeling Wednesday, February 11, 2015 539a chromosome structure have broad implications in studying effects of the geom- nism remains unknown. We have developed the first theoretical model on etry of nucleus on higher-order genome organization and nuclear functions. ParA/ParB-mediated motility. We establish that the coupling between the Here we describe a multi-chromosome constrained self-avoiding chromatin ParA/ParB biochemistry and its mechanical action works as a robust engine. model for studying ensembles of structural genome models to understand the It powers the directed movement of plasmids, buffering against the diffusive folding principles of budding yeast genome. We successfully generated a large motion. Our work thus sheds light on a new emergent phenomenon, in which number of model genomes of yeast under different geometrical constraints and elaborate mechanochemical couplings of non-motor proteins can work collec- found that spatial confinement of cell nucleus and molecular crowding in the tively to propel cargos to designated locations, an ingenious way shaped by nucleus are key determinants of the folding behavior of yeast chromosomes. evolution to with the lack of a processive motor protein in bacteria. Furthermore, the relative positioning of chromosomes and the interactions be- tween them are found to be due to presence of nuclear landmarks such as 2731-Pos Board B161 centromere tethering to spindle pole body. Nucleosome Repeat Length Relates to the Gene Expression Level in Yeast Ataur R. Katebi1, Davood Norouzi1, Feng Cui2, Victor Zhurkin1. 2728-Pos Board B158 1National Cancer Institute, National Institute of Health, Bethesda, MD, USA, Torque Mediated Kinetics of CENP-A Nucleosomes Reveals Resilience 2College of Science, Rochester Institute of Technology, Rochester, to Tension NY, USA. Rifka Vlijm1, Yamini Dalal2, Cees Dekker1. We are investigating correlation between the DNA folding in 30-nm chromatin 1Departmnent of Bionanoscience, Kavli Institute of Nanoscience, Delft fiber and the level of gene expression. The 30-nm fiber is generally character- University of Technology, Delft, Netherlands, 2Center for Cancer Research, ized by the nucleosome repeat length (NRL) - that is, the length of the core National Cancer Institute, Bethesda, MD, USA. DNA, 147 bp, plus the linker DNA length, L. We found previously that there In eukaryotic cells, DNA is wrapped around histone octamers, forming nucle- are two families of the two-start chromatin fiber structures characterized by osomes. In the centromere, the region of the chromosome that links sister chro- different DNA topology and flexibility. (Depending on the NRL value, the matids, histone H3 is replaced by CENP-A (Centromere protein A). Since energetically optimal fiber structure belongs to one of the two families.) CENP-A chromatin is the point of contact between the microtubules/kineto- Here we analyze the high resolution nucleosome positioning data to find chore complex and the rest of the chromosome, these regions endure very whether there is any correlation between the NRL and the gene expression level high forces. Whereas canonical nucleosomes unwrap at 3pN and disassemble in yeast. We calculate the NRL values for the two groups of genes - 25% highly at 15-20pN of force, the estimated forces applied by microtubili are much expressed and 25% lowly expressed genes (out of ~3,500 yeast genes that are at higher. To investigate how CENP-A nucleosomes respond to externally applied least 1,000 bp long). Our results show that the average NRL¼161-162 bp for forces and torque, we studied how CENP-A chromatin responds to defined the highly active genes (i.e., linker L¼14-15 bp), whereas NRL¼167-168 bp stretching forces and torque generated by magnetic tweezers. With this tech- (i.e., linker L¼ 20-21 bp) for the lowly transcribed genes. Based on these find- nique, a single DNA molecule containing a few (up to 10) nucleosomes is teth- ings, we conclude that the highly and lowly active gene sets have distinct nucle- ered between a glass surface and a magnetic bead. By applying stretching osome fiber organization with the linker Lz10nþ5 and 10n, respectively. We forces at constant negative and positive supercoiling, the forces needed for hypothesize that organization of the most active genes in fibers with Lz10nþ5 disassembly of canonical H3 and CENP-A nucleosomes can be obtained and (which are more flexible than the fibers with Lz10n) facilitates formation of compared. By measuring the DNA end-to-end length as a function of applied gene loops, thereby inducing transcription of these genes. rotations before and after CENP-A nucleosomes are removed from the DNA, the linking number of the nucleosomes can be determined. Interestingly, while 2732-Pos Board B162 inducing supercoiling at constant low (<0.5pN) forces, unlike canonical nucle- Using FRET to Monitor Nucleosome Movement by the Chd1 Chromatin osomes, CENP-A nucleosomes appear to respond to the applied torque. By Remodeler 1 1 2 further analyzing the structural stability of CENP-A nucleosomes under Robert Levendosky , Kyle Horvath , Srinivas Chakravarthy , Matt Hobson3, Gregory Bowman3. stretching force and torque, we hope to unravel the mechanism by which 1 2 CENP-A nucleosomes resists disassembly during mitosis in vivo. Biology, Johns Hopkins University, Baltimore, MD, USA, Argon National Laboratory, Chicago, IL, USA, 3Biophysics, Johns Hopkins University, 2729-Pos Board B159 Baltimore, MD, USA. The Chd1 Chromatin Remodeler can Sense a Protein Bound at the Edge of The chromatin remodeler, Chd1, creates regularly spaced arrays of nucleosomes. the Nucleosome and is Sensitive to DNA Unwrapping This spacing behavior correlates with a preference for shifting mononucleo- Ilana Nodelman1, Kyle Horvath1, Jessica Winger1, Robert Levendosky1, somes away from DNA ends. However, the mechanism by which Chd1 senses Ashok Patel2, Ming Li3, Michelle Wang3, Gregory Bowman1. extranucleosomal DNA is not understood. To investigate how sliding activity 1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2Kusuma may be regulated, we are adopting a FRET-based nucleosome sliding assay to School of Biological Sciences, Indian Institute of Technology, Delhi, India, measure the kinetics of Chd1 directed nucleosome sliding. By labeling the end 3Physics, Cornell University, Ithica, NY, USA. of the DNA with Cy3 and the nucleosome with Cy5, we should be able to monitor Chromatin remodelers are essential for establishing and maintaining the place- nucleosome sliding as the loss of quenching of Cy3 by Cy5 as the dye pair sep- ment of nucleosomes along genomic DNA. However, how remodelers respond arates. Using a similar system, others have observed single or double exponential to transcription factors and other bound factors that might influence chromatin increases in Cy3 fluorescence corresponding to nucleosome sliding. Curiously, organization is poorly understood. Here we use the Lac repressor to investigate with Chd1 we observe an initial increasing phase followed by a pause and then a how the Chd1 remodeler responds to a protein bound to the edge of a nucleo- second increasing phase. In addition, after an initial drop in Cy5 fluorescence ex- some. We found that Lac repressor effectively provided a barrier for nucleo- pected from FRET, a later phase shows an increase in Cy5 fluorescence above its some sliding by Chd1. This barrier did not require an absolute block in initial level. These results suggest that not all of the fluorescence signals are re- sliding, but instead was achieved through the bidirectional movement of nucle- porting on DNA movement alone, and are likely being influenced by Chd1 bind- osomes, with a higher preference for sliding nucleosomes away from occupied ing to the labeled nucleosome. We are investigating other dye pairs and Lac repressor sites. The presence of Lac repressor did not markedly diminish nucleosome constructs to isolate nucleosome sliding activity from binding or the affinity of Chd1 for nucleosomes, suggesting that Lac repressor is sensed other molecular interactions that may occur during the reaction. after nucleosome sliding has been initiated. Nucleosome sliding rates were also reduced by DNA unwrapping, suggesting a mechanism by which Chd1 2733-Pos Board B163 may indirectly sense factors bound at the edge of the nucleosome. Development of a ‘‘Realistic’’ Model of the Dynamic Chromatin Fiber using an Integrated Computational Approach 2730-Pos Board B160 Stefjord Todolli1, Nil Rawal2, Nicolas Clauvelin1, Willy Wriggers3, Mechanochemistry of Persistent Plasmid Movement Wilma K. Olson1. Longhua Hu, Jian Liu. 1BioMaPS Institute, Rutgers University, Piscataway, NJ, USA, 2Rutgers National Institutes of Health, Bethesda, MD, USA. University, Piscataway, NJ, USA, 3Mechanical and Aerospace Engineering, The segregation of DNA prior to cell division is essential to the faithful inher- Old Dominion University, Norforlk, VA, USA. itance of the genetic materials. In many bacteria, the segregation of the low- DNA in eukaryotic cells is highly compacted into a hierarchical chromatin copy-number plasmids involves an active partition system composed of ParA structure to fit inside the nucleus. Linker histones play an important role in ATPase and DNA-binding ParB protein, which stimulates the hydrolysis activ- this packing. Their interaction with the nucleosomes and intervening DNA ity of ParA. Both in vivo and in vitro experiments have shown that the ParA/ linkers, in combination with linker length, are believed to affect chromatin ParB system can drive the persistent movement of the plasmids in a directed folding and long-range interactions. The details of chromatin structure at this fashion, just like a processive motor protein. However, the underlying mecha- level of compaction are still an open question but have profound implications 540a Wednesday, February 11, 2015 on biological processes, such as gene expression. Two major challenges are that work we continue in our efforts to elucidate the mechanism and function of linker histones contain a long intrinsically disordered C-terminal domain the chromatin dynamics in interphase by investigating the dynamic contribution (CTD) and determination of high-resolution structures of chromatin has not of major nuclear motors such as DNA polymerase, RNA polymerase II, and been possible due to the large size of the system and its dynamic nature. topoisomerase II combining DCS and molecular perturbations. We are taking advantage of the recently published 11-A˚ resolution electron mi- [1] Zidovska A, Weitz DA, Mitchison TJ, Micron-scale coherence in interphase croscopy maps of two chromatin fibers (Song et al., 2014), to construct a com- chromatin dynamics, PNAS, 110 (39), 15555-15560, 2013 plete low-resolution model of chromatin. High-resolution crystal structures of [2] Bruinsma R, Grosberg AY, Rabin Y, Zidovska A, Chromatin Hydrody- nucleosome core particles are fitted to the electron density maps and the inter- namics, Biophys. J., 106 (9), 1871-1881, 2014 vening segments of linker DNA are modeled based on a sampling protocol that utilizes new DNA optimization methods. Situs is used to score all models 2736-Pos Board B166 against the density maps. Linker histones are modeled on the densities ex- Unique Mechanical Properties of Cell Nuclei Regulated by Chromatin 1,2 2 2 2 tracted after accounting for the nucleosomes and linker DNA. The modeled Chii J. Chan , Wenhong Li , Jana Scholze , Mirjam Schu¨rmann , 2 paths of the DNA linkers and the sites of the nucleosomes provide spatial con- Jochen Guck . 1Cavendish Laboratory, University of Cambridge, Cambridge, United straints for positioning the globular core and modeling the folding of the disor- 2 dered CTD of the linker histone. The pathways of the CTD are selected from Kingdom, Biotechnology Center, TU Dresden, Dresden, Germany. ensembles of conformations obtained using ab initio structure prediction tools Nuclear mechanics and structure could affect gene regulation and gene expres- like Rosetta. We are using the model in coarse-grained Monte Carlo simula- sion. Chromatin, a major component of cell nuclei, could play an important role tions of long-range interactions along arrays of precisely positioned nucleo- in maintaining nuclear integrity and their mechanical properties. Previous somes in the presence of linker histones. studies on nuclear mechanical properties have focused largely on the role of the nuclear lamina, using techniques such as AFM and micropipette aspiration. 2734-Pos Board B164 In this work, we explicitly address the contributions of chromatin to nuclear Matrix Constraints Regulate Transcription Dependent 3D Organization of rheology after isolation from the cell using a microfluidic optical stretcher. Chromosomes We find that isolated nuclei swell in volume under uni-axial stress and exhibit Yejun wang1, Mallika Nagarajan1, G.V. Shivashankar1,2. significant softening with increased nuclear size, which can be described by a 1Mechanobiology Institute, Singapore, Singapore, 2FIRC Institute for filtration model for the nuclear membrane and a cortical chromatin model, Molecular Oncology (IFOM), Milan, Italy. respectively. In addition, changes to the state of chromatin condensation via his- Recent studies have revealed that cell geometric constraints regulate cytoskel- tone modifications or chromatin remodeling processes (ATP, topoisomerase II) eton as well as nuclear architecture, and gene expression. In addition, the spatial can strongly impact nuclear morphology and compliance. Moreover, isolated and temporal organization of chromosomes has been shown to modulate gene nuclear mechanics is also sensitive to ionic conditions: nuclei stiffen with expression. However, the role of 3D organization of the nuclear architecture increasing ionic strength of the buffer and exhibit a transition from stretch to and chromosome assembly in facilitating this geometric-constraints regulated contraction in the presence of multivalent ions (only). Finally, we find that in genome regulation is unclear. To address this, we used NIH 3T3 fibroblast cells contrast to other studies suggesting a high refractive index of cell nuclei cultured on fibronectin coated mircofabricated patterns, and combined fluores- compared to the cytoplasm, the refractive index of isolated cell nuclei of a variety cence in situ hybridization (FISH) with confocal imaging. We found that alter- of cell types can be lower than the refractive index of the cells. The presented ation of cell geometry changed the radial position of individual chromosomes, as work establishes a quantitative link between nuclear mechanical properties well as the relative position of specific chromosome pairs. Interestingly, such and the compaction state of chromatin, which can be modulated by a change chromosome reorganization was accompanied with the enrichment of active in nuclear volume, chromatin remodeling or electrochemical environment. RNA polymerase II (phospho S5CTD) in intermingling regions. Consistent 2737-Pos Board B167 with microarray results, we observed nuclear localization of serum response co- Tracking Chromosome Conformation in Live Cells with CRISPR Imaging factors (MKL) in flattened nuclei, while that of inflammatory response transcrip- Juan Guan1, Jeffrey Hu1, Baohui Chen1, Sakaya Sekine1, tion factors (p65) in spherical nuclei. Supporting this, super-resolution imaging Sanjeev Balakrishnan2, Bo Huang1. of these factors showed spatial colocalization of SRF, MKL and pol2 in rectan- 1Pharmaceutical Chemistry, UCSF, san francisco, CA, USA, 2Cell and Tissue gular patterns, while colocalization of p65 and pol2 in circular patterns. More Biology, UCSF, san francisco, CA, USA. interestingly, binding-activatable localization microscopy (BALM) on open In each cell cycle, chromosomes go through dramatic large-scale structural chromatin spreads revealed specific transcription dependent clustering of chro- changes, oscillating between being relatively open at interphase and highly mosomal contacts depending on nuclear morphology. In summary, our results compact at metaphase. However, little is known about how they change be- reveal highly modular changes in 3D chromosome organization to facilitate tween the two extremes. Therefore it is desirable to monitor the long-term dy- co-clustering of genes and its co-regulation, depending on cell geometry. namics of chromosome structure in live cells. Here we use a modified CRISPR system to directly image specific genomic loci in hundreds of live cells with 2735-Pos Board B165 high temporal and spatial resolution. By following dozens of diffraction- Mechanism and Function of Chromatin Positional Fluctuations in Inter- limited fluorescence spots sparsely decorating a single chromosome, specific phase genomic elements can be localized with a precision of ~30 nm. We study Alexandra Zidovska. how topological domains and long range interactions between chromosome Center of Soft Matter Research, Department of Physics, New York loci are maintained or re-established through cell cycle. As CRISPR imaging University, New York, NY, USA. allows us flexibility and specificity in imaging any genomic loci, the method Chromatin structure and dynamics control all aspects of DNA biology yet are developed here could be easily adapted to explore other systems where long- poorly understood. In interphase, time between two cell divisions, chromatin fills term live cell imaging is required. the cell nucleus in its minimally condensed polymeric state. Chromatin serves as substrate to a number of biological processes, e.g. gene expression and DNA 2738-Pos Board B168 replication, which require it to become locally restructured. These are energy- Conformational Sampling of Unmodified and Acetylated H3 Histone Tails consuming processes giving rise to non-equilibrium dynamics. Chromatin dy- on a Nucleosome by All-Atom Model Molecular Dynamics Simulations namics has been traditionally studied by imaging of fluorescently labeled nuclear Jinzen Ikebe, Shun Sakuraba, Hidetoshi Kono. proteins and single DNA-sites, thus focusing only on a small number of tracer Japan Atomic Energy Agency, Kyoto, Japan. particles. Recently, we developed an approach, displacement correlation spec- A nucleosome, 146 or 147 base-pair DNA wrapped around a histone octamer troscopy (DCS) based on time-resolved image correlation analysis, to map chro- composed of two copies of each H3, H4, H2A, and H2B histone proteins, is matin dynamics simultaneously across the whole nucleus in cultured human a compact unit structure to store eukaryotic DNA into the cell nucleus. cells[1]. DCS revealed that chromatin movement was coherent across large re- Although the X-ray conformations of the core region have been already deter- gions (4-5mm) for several seconds. Regions of coherent motion extended beyond mined, the conformations of the disordered histone terminal regions (histone the boundaries of single-chromosome territories, suggesting elastic coupling of tails) remain poorly understood. Recent experimental evidences suggest that motion over length scales much larger than those of genes[1]. These large-scale, chemical modifications on the histone tails regulate DNA functions, such as coupled motions were ATP-dependent and unidirectional for several seconds. transcription, duplication, and splicing. To understand the regulation mecha- Following these observations, we developed a hydrodynamic theory of active nism, it is necessary to elucidate difference between conformational states of chromatin dynamics, using the two-fluid model and describing the content of unmodified and modified histone tails. Molecular dynamics (MD) simulations cell nucleus as a chromatin solution, which is subject to both passive thermal by generalized ensemble methods such as replica exchange MD and multica- fluctuations and active (ATP-consuming) scalar and vector events[2]. In this nonical MD are effective means to investigate the conformational ensemble Wednesday, February 11, 2015 541a of biological systems and have been applied for protein systems with small with almost one linker histone per nucleosome, and regulate gene expression dur- charge polarization. However, these means are not suitable to obtain the ing developmentand withinsomatic cells. We usedfluorescence resonance energy ensemble of histone tails in a form of a nucleosome, because strong electro- transfer (FRET) to detect changes to nucleosome wrapping, which is sensitive to static interactions between positively charged histone tails and negatively both H1 binding and transcription factor binding within the nucleosome. H1 bind- charged DNA compared with those in regular protein systems makes difficult ing induces an increase in nucleosome wrapping and FRET, while transcription to realize a sufficient sampling. Thus, we applied adaptive lambda square dy- factor binding within the nucleosome decreases nucleosome wrapping and namics (ALSD) simulation we developed recently to investigate the conforma- FRET. We find that H1 suppresses but does not abolish TF binding suggesting tions of H3 histone tails. ALSD dynamically scales the simulation parameters that H1 dynamically regulates nucleosomal DNA accessibility to TF binding. (charge, van der Walls and torsion energies) only for the histone tails during the We then prepared nucleosomes with histone H3 acetylated at lysine 56 simulations. This successfully sampled various histone tail conformations. In (H3K56ac). This modification is involved transcriptional regulation and increases this poster, we introduce the ALSD simulation results and the differences in DNA unwrapping. We find that this modification abolishes H1 suppression of TF conformational ensembles between unmodified and acetylated H3 histone tails. binding within the nucleosome. This result suggests that H3K56ac can encode, within a nucleosome, H1 regulation of DNA accessibility and provides a mecha- 2739-Pos Board B169 nism by which H1 regulation of DNA accessibility is targeted through the genome. Dynamics of the Nucleosome Core Particle Revealed from a New Database of High-Resolution X-Ray Crystallographic and Simulated Structures 2742-Pos Board B172 Gautam Singh, Andrew V. Colasanti, Nicolas Clauvelin, Wilma K. Olson. Mechanism of Nucleoid Collapse in E. coli BioMaPS Institute for Quantitative Biology, Rutgers University, Piscataway, Elio A. Abbondanzieri. NJ, USA. TU Delft, Delft, Netherlands. The nucleosome core particle is a highly conserved structure which can play All living cells must organize their DNA into dynamic three-dimensional archi- diverse roles depending on the organism, cell, or part of chromatin in which tectures that are compatible with essential cellular processes such as transcrip- it resides. The Protein Data Bank currently contains approximately 90 nucleo- tion, translation, and DNA repair. Recently it has been shown that DNA is able some core particle structures, most of which were determined in the last five to condense into a crystalline lattice in bacterial cells exposed to stressful con- years. The recent emergence of the field of epigenetics, and the increase in ditions due to the action of a single gene. The protein responsible for creating data available from experiments, warrants a need to develop new approaches these DNA ‘biocrystals’ is Dps (DNA-binding protein from starved cells) and it to compare features of interest across multiple structures. is essential in helping cells survive conditions ranging from starvation to anti- The growing ensemble of structural data garnered from in vitro and in silico ex- biotic exposure. When present at sufficiently high concentrations, Dps drives periments provides a unique platform to study the mechanochemical properties the condensation of DNA into a biocrystal both in vitro and in vivo. We inves- of the nucleosome. We have developed a database and new computational tools tigate the cooperative transition that allows Dps to rearrange the genome using to allow researchers to analyze and compare the nucleosome core particle struc- a variety of techniques. We have developed a novel single molecule assay to tures deposited in the Protein Data Bank. The features of the DNA-protein assem- probe the physical interactions between fluorescently tagged Dps and DNA. bly can be examined in novel coordinate frames placed on the structure, allowing We find that Dps induced collapse is rate limited by a slow nucleation event researchers to obtain a better understanding of the organization and subtleties of but then proceeds extremely rapidly (< 100 ms). We also used magnetic twee- the macromolecular complexes. This comparison allows one to examine the ‘mo- zers to apply loads to DNA in the presence of Dps and determined collapse is tion’ of specific residues of interest, including specific sites of post-translational much more concerted than previously assumed. We find that Dps binding to histone modification. The database also includes DNA-histone contact points, DNA displays the qualitative features of an Ising system and we have devel- DNA conformational parameters, and information about protein features such oped a mean field model that captures important features of the collapse. We as the secondary structure in the globular histone core and the ‘motion’ of the his- look at nucleoid dynamics in single cells to test this model. tone tails. Along with these features, we also characterize the dynamics of the global structure of the nucleosome core particle, including changes in the super- 2743-Pos Board B173 helical path of the DNA, rearrangements of the histone tetramers, and nucleo- The Bacterial Nucleoid Drives Cytoplasmic Dynamics some stacking inside crystals. This information allows us to understand and Stella Stylianidou, Nathan J. Kuwada, Paul A. Wiggins. model the critical role of mono-nucleosome structural propensity in processes Physics, University of Washington, Seattle, WA, USA. such as carcinogenic modifications of the DNA, and nucleosome remodeling. Bacterial cells exhibit complex, cell-cycle-dependent subcellular organization despite the lack of membrane-bound organelles. One of the most popular pro- 2740-Pos Board B170 posed mechanisms for this cellular ultrastructure is physical exclusion from The Effect of Nucleosome Unwrapping on Chd1-Induced Chromatin the dense bacterial nucleoid. To quantitatively investigate this hypothesis, we Remodeling visualized and mapped the motion of fluorescently-tagged ectopic MS2- Jessica Winger, Gregory Bowman. mRNA complexes in thousands of growing E. coli cells. We find that the the mo- Johns Hopkins University, Baltimore, MD, USA. lecular complexes’ motion strongly depends on their spatial position along the The tight wrapping of DNA into nucleosomes reduces access and can provide a long-axis of the cell and that their dynamics are well characterized by a quanti- barrier for gene expression. Chromatin remodelers are responsible for sliding tative model that requires only two physical contributions: nucleoid exclusion the nucleosome into a position where the DNA can be readily processed but and membrane confinement. Strikingly, we also find that the mobility of the mo- the precise mechanism is unclear. The DNA in the nucleosome dynamically un- lecular complexes is highest in regions of high nucleoid density, and that pertur- wraps and rewraps around the histone core, which may affect how the chro- bations to nucleoid structure tend to increase cytoplasmic mobility. These results matin remodeler binds to the nucleosome and facilitates sliding. Unwrapping provide strong quantitative support for two modes of nucleoid action: (1) orga- is a natural response to proteins binding to DNA at the edge of the nucleosome, nizing the cell through physical exclusion forces and (2) as a facilitator of rapid and can also be affected by DNA sequence. Chromodomain Helicase DNA motion throughout the cytoplasm. These results have potentially important bio- Binding Protein 1 (Chd1) is an ATP-dependent chromatin remodeler that is logical implications and suggest that the nucleoid may play a much more direct responsible for transcription-related chromatin remodeling, generating evenly role, than previously thought, in the organization and transport of subcellular spaced nucleosomal arrays across the genome. We made unwrapping variants components, including large protein complexes and plasmids. by perturbing DNA-histone interactions to see what effect this might have on Chd1’s ability to slide nucleosomes. We find that unwrapping DNA from the 2744-Pos Board B174 histone slows Chd1-induced sliding. We are investigating the effects of pro- Acto-Myosin Contractility Regulates Nuclear and Chromatin Plasticity Ekta Makhija1, D.S. Jokhun1, G.V. Shivashankar1,2. teins binding to the edge of the nucleosomes as well as sequence effects to un- 1 2 derstand how these influence Chd1 sliding and positional preference. Mechanobiology Institute, Singapore, Singapore, FIRC Institute for Molecular Oncology (IFOM), Milan, Italy. 2741-Pos Board B171 The cytoskeleton can exert forces on the nucleus and chromatin via direct phys- Acetylation of Histone H3 Lysine 56 Abolishes Linker Histone Regulation ical links on the nuclear envelope. Such forces provide prestress on the nucleus of Transcription Factor Binding thereby governing its morphology. However, the role of these forces in modu- Morgan Bernier1, Yi Luo1, kingsley Nwole1, Jennifer J. Ottesen2, lating nucleus and chromatin plasticity has not yet been explored. In this proj- Poirier G. Poirier1. ect, we used micro-contact printing of fibronectin to create different substrate 1Physics, Ohio State Universtiy, Columbus, OH, USA, 2Chemistry and geometries for culturing NIH3T3 cells so as to obtain specific cytoskeletal or- Biochemistry, Ohio State Universtiy, Columbus, OH, USA. ganization and then measured the nuclear and chromatin dynamics in these The linker histone H1 is an ubiquitous regulator of chromatin structure and dy- cells. We observed that nuclei of cells on constrained isotropic geometry (small namics. These linker histones are highly abundant in metazoan somatic cells circle) were much more plastic than those on elongated anisotropic geometry 542a Wednesday, February 11, 2015

(big rectangle). This nuclear plasticity, measured as projected nuclear area fluc- 2747-Pos Board B177 tuations, showed a non-monotonous relation to actin polymerization state. Polydopamine as an Efficient Polymer to Prepare Biologically Relevant Also, myosin contractility was determined to be necessary for such nucleus Supported Lipid Bilayers plasticity. The effect of cytoskeletal organization and their active forces on Souryvanh Nirasay1, Antonella Badia2, Jerome P. Claverie1, chromatin plasticity was further quantified by tracking the dynamics of Isabelle Marcotte1. condensed chromatin regions, which showed increased dynamics correspond- 1Chemistry, Universite´ du Que´bec A` Montre´al, Montreal, QC, Canada, ing to enhanced nuclear plasticity. In summary, using cells of defined geome- 2Chemistry, Universite´ de Montre´al, Montreal, QC, Canada. tries to specify cytoskeletal organization, our work demonstrates the role of Solid-supported lipid bilayers (s-SLBs) are widely used as versatile biological active cytoskeletal forces in regulating nuclear and chromatin plasticity. membrane mimics. However, lipid-solid surface interactions and ensuing fric- tions lead to a decrease in lipid mobility. To address this issue, we prepared polymer-supported lipid membranes using a soft polymer cushion as a lubri- Membrane Physical Chemistry III cating layer. A bioinspired polymer, polydopamine was chosen due to its ability to easily form thin coatings onto a wide range of surfaces, with a control of the 2745-Pos Board B175 thickness depending on the immersion time of the substrate. Amino Acids and Peptides Stabilize Fatty Acid Membranes against Salt- Polydopamine coated-mica was used to support zwitterionic phospholipid bila- Induced Flocculation yers of dimyristoylphosphatidylcholine (DMPC) or dioleoylphosphatidylcho- Moshe Gordon1, Roy A. Black2, Matthew C. Blosser3, Sarah L. Keller3. line (DOPC). Atomic Force Microscopy (AFM) was performed to verify the 1Chemistry, University of Washington, Seattle, WA, USA, 2Bioengineering, deposition of DMPC and DOPC bilayers, revealing the presence of lipid patches University of Washington, Seattle, WA, USA, 3Chemistry & Physics, on the polymer surface. The addition of positively charged cholesterol (DC- University of Washington, Seattle, WA, USA. Chol) in the membrane composition greatly improved the lipid surface coverage The prebiotic formation of biopolymers (specifically DNA, RNA, and proteins) (up to 90%). Best results were obtained for 20 and 30% DC-Chol containing has long been a mystery and is important for understanding the origin of life on membranes.They were also further characterized with other surface-sensitive earth. These bio-molecules are composed of building blocks that would have techniques such as fluorescence microscopy to assess the phospholipid mobility. been dispersed in early oceans. Our previous work has shown that RNA bases Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance with Dissi- and ribose bind to and stabilize fatty acid vesicles [Black et al. PNAS 110, pation (QCM-D) results showed the irreversible deposition of the lipid bilayers 13272 (2013)]. Our results implied that the building blocks of a biological poly- on the polymer surface. The polydopamine polymer film proved to be efficient to mer could have spontaneously associated with components of the first mem- maintain fluidity of the phospholipid bilayers, thus enhancing this class of mem- branes to form stable structures. We have now shown that protein building brane model. We will present practical application of our s-SLBs onto porous fil- blocks, too, stabilize fatty acid vesicles against salt-induced flocculation. Using ters as an improved model of Parallel Artificial Membrane Permeability Assay spectrophotometry, we measured the presence of flocs (and other structures) in (PAMPA) to better predict passive permeation of orally administered drugs. fatty acid solutions, with and without amino acids and over a range of tempera- tures. Using fluorescence microscopy, we identified the structures that caused 2748-Pos Board B178 changes in absorbance in our spectrophotometric assays. We found that the Polymer and Silica Supported Bilayer Formation Studied through Time two most hydrophobic prebiotic amino acids, leucine and isoleucine, prevent Resolved Spatial Point Pattern Analysis of Vesicle Deposition salt-induced flocculation. Moreover, although alanine and glycine, which are Stephen Cross1, Oliver Birkholz2, Jacob Piehler2, Matthew Peel1, less hydrophobic, had little effect on flocculation, dipeptides composed of these Suman Peel1. 1 2 amino acids preserved vesicles in the presence of salt even at 60 degrees C. These University of Bristol, Bristol, United Kingdom, University of Osnabru¨ck, vesicles appeared to be primarily multilamellar structures, which may promote Osnabru¨ck, Germany. reactions between components of biopolymers more effectively than unilamellar Due to their optimal separation from the sensor-substrate, polymer supported vesicles. Thus prebiotic membranes could have facilitated the formation of pep- bilayers (PSBs) provide biologically-relevant mimics of cellular membranes tides by bringing amino acids together, and peptides could have increased the whilst staying amenable to numerous analytical, imaging and fluidic tools - a formation of stable membranes. Such an auto-amplifying system, combined dual precondition for drug discovery through function-interaction analysis of with selection for more effective peptides, could have led to the first cells. cell-surface receptors and other membrane-interacting proteins. Although silica supported bilayers (SSBs) spontaneously form from silica-adsorbed vesicles, 2746-Pos Board B176 successful PSB formation via a similar method has thus far been limited by Measurement of the Viscosity of E. coli Membranes using Molecular insufficient understanding of the underlying processes. Here, we generated a Rotors and Flim polymer support through incubation of poly-L-Lysine conjugated to alkyl chain Jacek T. Mika1, Alexander J. Thompson2, Johan Hofkens1, terminated poly(ethylene)glycol on silica. This substrate yielded efficient Marina K. Kuimova2. vesicle deposition and spontaneous bilayer formation thereby providing a 1 2 Chemistry, KU Leuven, Leuven, Belgium, Chemistry, Imperial College rare opportunity to address the mechanism of PSB formation. London, London, United Kingdom. Currently there is a lack of consensus about the mechanism of SSB formation, We have employed molecular rotors, small organic molecules whose fluores- with putative mechanisms invoking (i) preferential vesicle adsorption at the cence lifetime is sensitive to the viscosity of the environment, to assess the vis- edges of bilayer patches, (ii) vesicle dispersion through sequestration of sub- cosity of the E.coli plasma membrane. We used Fluorescence Lifetime Imaging strate binding sites and (iii) complete spatial randomness, where each scenario Microscopy (FLIM) which allowed us to measure the fluorescence lifetimes is expected to show a unique point pattern for vesicle adsorption. In order to (and thus viscosities) on the level of single cells. We probed the viscosity of differentiate between these scenarios, we measured deposition of SUVs encap- membranes both in livee cells and in spheroplasts, where the outer membrane sulating sulforhodamine B (SRB) over silica and polymer substrates, with these was removed by lysozyme treatment. Viscosity values obtained for both envi- surface bound SUVs imaged via TIRF microscopy. As the SUVs bind to the ronments were similar implying that the molecular rotor used indeed localized solid substrate, they yield a diffraction limited point spread function (PSF) to the plasma membrane as shown previously for fluorophores of similar struc- that is fit with a two-dimensional ellipsoidal Gaussian distribution. Rapid dye tures. Measurements on life cells show a rather broad spread of viscosities be- photo-bleaching facilitates the observation of only the freshly deposited tween individual cells in population; such heterogeneity of physical parameters SUVs. The obtained pattern of vesicle locations is compared to a complete of the cell has been reported previously for the diffusion of protein in the cyto- spatial random distribution using Ripley’s K-function analysis. Preliminary re- plasm of bacteria. The viscosity of membranes was temperature dependent as sults indicate random deposition during the early stages of bilayer formation we have observed a change in viscosity when cells grown at 37 degrees Celsius (<4% lipid surface coverage), but the observed deviations at higher loading were measured at lower temperatures than the growth. indicate exclusion from vesicle/bilayer regions. Subjecting the cells to a hyperosmotic shock by increasing the medium osmo- larity by adding NaCl also elicited a change in viscosity and yet a larger spread 2749-Pos Board B179 of viscosity values between individual cells, which is consistent with previous Controlled Modulation of Lipid Bilayer State by a Photosensitive Mem- observations that a fraction of cell within a population seems to respond to the brane Effector osmotic shock more strongly than the others. Chen Shen1, Lars Jørgensen1,2, Dordaneh Zargarani3,4, Benjamin Runge5, The values of viscosity measured for the plasma membrane of E.coli in this Bridget Murphy5, Olaf Magnussen5, Beate Klo¨sgen1. study are higher than those measured previously in E.coli lipid extracts or in 1Phys. Dept., Univ. Southern Denmark, Odense M, Denmark, 2National Food the plasma membrane of life eukaryotic cells but slightly lower than what Institute, Technical University of Denmark, Lyngby, Denmark, 3Otto-Diels- was reported previously for the Gram-positive bacterium Bacillus subtilis. Insitute of Organic Chemistry, Christian-Albrechts-University of Kiel, Kiel, Wednesday, February 11, 2015 543a

Germany, 4CAT Catalytic Center, RWTH-Aachen University, Aachen, 2752-Pos Board B182 Germany, 5IEAP, Christian-Albrechts-University of Kiel, Kiel, Germany. Ion-Mobility Mass Spectrometry Assay for Incorporation of Phytanic Acid The lipid membrane matrix represents a 2-D liquid-crystal, the properties of into Muscle Phospholipids which, at fixed other conditions, are locally modulated by the presence of effec- Glen Humphrey1, Peter S. Backlund2, Paul S. Blank1, Joshua Zimmerberg1. tors as e.g. cholesterol (passive) or proteins (passive and active). Not only does 1PPB NICHD NIH, Bethesda, MD, USA, 2NICHD NIH, Bethesda, the incorporation of effectors into the host matrix locally or even globally MD, USA. modify the initial state of the host per se, most probably the state of the matrix Human dysferlinopathies (Limb-Girdle Muscular Dystrophy 2b, Myoshi Myop- in turn serves as a control tool for regulating the work of functional units (e.g. athy) are muscle-wasting syndromes caused by mutations in the dysferlin pro- proteins). tein. Dysferlin loss impairs sarcolemmal repair that may contribute to disease Results are presented on a membrane model system that was inoculated with a progression. One therapeutic approach is to treat subjects with compounds photosensitive and thus active variety of cholesterol. Azobenzene-cholesterol that fortify the natural membrane tendency to reseal after damage, and minimize (azo-chol) exhibits a reversible trans-cis transition (365nm: trans- to cis-; the inflammation that impedes regeneration and amplifies tissue destruction. 455nm: cis- to trans-). In a membrane, the azobenzene group, covalently con- Phytanic acid is a saturated branched chain fatty acid comprised of a 16 carbon nected to the cholesterol by an ester bond, is confined into the headgroup re- aliphatic chain with 4 methyl groups (4ME 16:0); it is incorporated into phospho- gion. The system was explored by a combination of spectroscopic (UV-vis, lipids and triglycerides. Model phospholipid bilayers containing phytanic acid NMR, mass spectroscopy), thermodynamic (Langmuir compression, calorim- exhibit greater electrical stability than bilayers composed of straight-chain phos- etry) and structural studies (X-ray/neutron reflectometry, grazing incidence pholipids. Phytanic acid-containing phospholipids may improve the resistance X-ray diffraction). The conformational change of the guest upon illumination of the muscle fiber sarcolemma to stretch-induced damage. To measure phytanic is coupled into the host system, inducing a transition of the whole membrane. acid incorporation into muscle phospholipids, dysferlin-deficient A/J mice were The increased demand of headgroup area for the cis-azo-group pushes the maintained on a defined diet supplemented with 2% phytol for three weeks; con- membrane into a more compressed state, and vice versa for trans-azo-chol. trol animals received the defined diet without phytol. Muscle tissue lipids were The switching process between the two final states exhibits first-order kinetics. analyzed by mass spectrometry. The muscle phosphatidylcholine species pro- The state of the host bilayer is modulated as a response to the conformational files were similar between phytol and control diet, except that some additional switching of the guest effector via external light illumination. In a more general species were detected in the phytol diet muscle. The two most abundant novel context, similar behavior may be found upon the conformational changes of species (m/z 790.7 and 862.7) are tentatively identified as PC 20:0-16:0 and membrane proteins during work. PC 20:0-22:6. To confirm the presence of phytanic acid, the ion mobility of these species was compared with diphytanoyl PC standard and endogenous straight 2750-Pos Board B180 chain PC phospholipids. The ion mobility is intermediate between PC species Creating Fluid Supported Lipid Bilayers with High Amounts of Phospha- containing either zero or two phytanic acid chains, consistent with having one tidylethanolamine phytanic acid chain. We estimate that these species represent 5% of PC. Using Anne Sendecki, Matthew F. Poyton, Tinglu Yang, Paul S. Cremer. this methodology, we will identify phytanic acid containing species in the other Chemistry, Pennsylvania State University, University Park, PA, USA. classes, in order to determine the total amount of phytanic acid-containing phos- Phosphatidylethanolamine (PE) comprises 20-50% of overall phospholipid pholipid incorporated in the muscle. content in human cell membranes and constitutes 70-80% of the membranes in gram-negative bacteria. Its presence is specifically required for the proper 2753-Pos Board B183 folding of numerous membrane proteins, the function of active transport Fluctuation-Induced Interactions between Membrane-Bound Proteins systems, cell division, fusion, blood coagulation, and may play a role in Kayla Sapp, Lutz Maibaum. neurodegenerative diseases. Unfortunately, it is hard to work with this lipid Department of Chemistry, University of Washington, Seattle, WA, USA. in model systems like supported lipid bilayers and there is correspondingly The spatial organization of membrane-bound proteins is in part determined by less information known about its basic physical properties in bilayers. Specif- interactions that originate from long-range correlations due to the membrane’s ically, the role of PE in lipid raft formation, vesicle fusion, cholesterol inter- elastic behavior. Even basic geometric mechanisms, such as the suppression of actions, ion binding, and lipid flip-flop needs to be elucidated to understand membrane height fluctuations near protein binding sites, can lead to nontrivial its part in cell membrane function and disease pathways. To this end, interactions between proteins that might result in their aggregation. To study my research has two goals. First, I have developed supported lipid bilayer sys- the effect of such membrane-induced interactions, we devise a simple model tems that can operate with high mole percentages of PE. Second, I have begun that captures (a) a nonspecific repulsion between proteins, (b) elastic properties to exploit such systems to explore the properties and functions of PE in of the membrane, and (c) a local harmonic coupling between proteins and membranes. membrane shape. The model’s dynamics is governed by Langevin equations to faithfully capture entropic effects and the importance of rare fluctuations. 2751-Pos Board B181 We find that the membrane induces an attractive interaction between the pro- Biophysical Analysis of a Successful Protocol to Reconstitute Tetramers of teins, which aggregate to mitigate the entropic cost of suppressing membrane the M2 Muscarinic Receptor fluctuations. This generic mechanism might help explain the spatial patterns Helen Y. Fan, Dar’ya S. Redka, Heiko Heerklotz. induced by membrane sculpting proteins. Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada. 2754-Pos Board B184 The human G protein-coupled receptor M2 muscarinic receptor has been Effect of Phosphatidylinositol-Bisphosphate (PIP2) Lipids on Membrane functionally reconstituted in its tetrameric state into mixed lipid bilayers Structure and Forces (Redka et al. 2013). This is achieved by first solubilizing the receptor in mixed Sourav Haldar, Paul S. Blank, Joshua Zimmerberg, Donald C. Rau. detergent micelles composed of digitonin and sodium cholate, then reconsti- Program on Physical Biology, National Institute of Child Health and Human tuting it into vesicles composed of phosphatidylcholine, phosphatidylserine Development, National Institutes of Health, Bethesda, MD, USA. and cholesterol, followed by detergent removal. To understand how the indi- It is widely accepted that the interaction between lipid bilayers at closest separa- vidual detergent and lipid components used in this empirical protocol tion is dominated by a repulsive hydration force arising from the structuring of contribute to the stability and activity of the receptor, we used isothermal water molecules on the lipid bilayer surface. This force has been recognized as titration calorimetry (ITC) to study the self-assembly of the mixed surfactant a major activation energy barrier preventing fusion of bilayers. Our working hy- system; differential scanning calorimetry and pressure perturbation calorim- pothesis is that the orientation of the hydrogen bond network on the bilayer sur- etry to probe the phase behavior of the membrane; ITC, fluorescence (time- face determines the amplitude of the hydration force. We are testing this resolved) leakage assays and dynamic light scattering to characterize hypothesis using the 1,2-dioleoyl-sn-glycero-3-phospho-[10-myo-inositol- 0 detergent-lipid interactions. The results suggest ideal mixing between digi- 4’,5 -bisphosphate](PI(4,5)P2) lipid and its isomers which differ in the position tonin and sodium cholate in the formation of mixed micelles that stabilize of phosphate groups on the inositol ring. Phosphatidylinositol-bisphosphate the receptor. Differences in membrane-partitioning behavior between the (PIP2) lipids are pivotal in signaling and play an important role in exocytosis. two detergents and the presence of a significant fraction of gel phase at the Specifically, we have utilized small angle X-ray diffraction (SAXS) and moni- temperature used in the protocol contribute to non-equilibration of the deter- tored the structural consequences of osmotic pressure in multilamellar suspen- gents in the bilayer. The insights gained from this biophysical approach will sions of dioleoylphosphatidylcholine (DOPC) in the presence of PI(4,5)P2. aid in the mechanistic selection of detergents and conditions that influence Our preliminary data (powder X-ray diffraction and reconstructed electron den- function, oligomeric state, orientation, and accessibility of membrane proteins sity profiles) show that there are notable changes in bilayer structure, particularly in future studies. the lamellar repeat distance, thickness, and forces in the presence of PI(4,5)P2. 544a Wednesday, February 11, 2015

We are currently investigating whether the effects of PIP2 lipids are dependent on at a certain chain length although the partition coefficient still increases. This is the phosphate group position, ionic strength, and temperature. Since the relative known as cut-off effect. Although the cut-off effect is seemingly contradicting position of the hydrogen bond acceptor in phosphates are determined by their the Meyer-Overton correlation, we would like to show that the universal valid- exact molecular structure, our results will be relevant in the context of molecular ity of the Meyer-Overton correlation still applies. recognition involving PIP2 lipids. Instead of anaesthetic potency, the cut-off effect can also be described by the melting transition temperature of lipids. Recently, we have provided a thermo- 2755-Pos Board B185 dynamic theory to describe the principle of the freezing point depression, i.e. Phospholipid Giant Unilamellar Vesicles (GUVS) Melt Like Large the anaesthetic solves better in the fluid phase of the membrane than the gel (LUVS), Not Mulitilamellar (MLVS), Vesicles phase. Similarly, it has been shown that the longer n-alcanols cause an increase Mark A. Kreutzberger, Paulo F. Almeida. in the transition temperature. This indicates that these non-anaesthetic n-alcanols Chemistry and Biochemistry, UNCW, Wilmington, NC, USA. solve better in the gel phase of the membrane than the fluid phase, and we, there- The disparity between the excess heat capacity curves for the melting of large fore, suggest to extend the Meyer-Overton correlation to differentiate between unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) has given rise solubility in the membrane and solubility in the fluid phase of the membrane. to two hypotheses. The first is that the size of the vesicles results in a difference for how the two types of vesicles undergo their phase transitions. The second is 2758-Pos Board B188 that there is communication between the bilayers in an MLV when it transitions Association of Model Neurotransmitters with Lipid Bilayer Membranes from its ordered gel phase to its liquid disordered phase, resulting in increased Brian Josey1, Frank Heinrich1,2, Mathias Lo¨sche1,2, Robert Cantor3. cooperativity. To test these hypotheses, differential scanning calorimetry 1Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2Center for (DSC) was performed on giant unilamellar vesicles (GUVs) of pure DPPC. Neutron Research, National Institute of Standards and Technology, The GUVs were prepared using electroformation, and visualized with confocal Gaithersburg, MD, USA, 3Chemistry, Dartmouth College, Hanover, NH, USA. fluorescence microscopy. The average size of the GUVs determined from the The conventional model of synaptic transmission between neurons is based images was about 7 micrometers. For comparison, the size of LUVs is about on the specific binding of neurotransmitters to ligand-gated ion channels. 100 nm. DSC was then performed of the GUVs, and their excess heat capacity Fast perfusion electrophysiological studies of receptor responses to neuro- curve was recorded. The excess heat capacity curves for the GUVs closely transmitters have revealed complex kinetic behavior that cannot be repro- resemble the curves for LUVs. Both GUV and LUV curves are much broader o o duced unless the standard kinetic model is expanded to include additional (halfwidth ~ 1 C) than those of MLV curves (halfwidth ~ 0.1 C). The similarity conformational states. However, if one invokes neurotransmitter adsorption of the GUV and LUV excess heat capacity curves indicate that the size of the to the lipid membrane, the electrophysiological data can be reproduced vesicles does not impact how they undergo their phase transitions. In addition, with a simpler kinetic model that includes only the standard set of three the results provide evidence that there is cooperativity between the bilayers of conformational states [1]. This indirect mechanism of influence of neurotrans- an MLV when it undergoes its melting phase transition. mitters on receptor conformational transitions is assumed to be nonspecific. 2756-Pos Board B186 Unlike anesthetics, experimental verification has been difficult because of Biomolecules Altering the Lipid Molecular Shape in Model Non-Lamellar the low binding affinities of neurotransmitters to lipid bilayers [2]. We quan- Membranes tify this interaction by measuring the equilibrium dissociation constant of Chandrashekhar V. Kulkarni1, Yogita Patil-Sen1, Mukta Kulkarni2, neurotransmitters on membranes with surface plasmon resonance (SPR) spec- Ales Iglic2. troscopy and characterize neurotransmitter association with bilayers through 1Centre for Materials Science, University of Central Lancashire, Preston, neutron reflectometry (NR) on artificial membranes. Sparsely-tethered bilayer United Kingdom, 2Faculty of Electrical Engineering, University of Ljubljana, lipid membranes (stBLMs) composed of zwitterionic (PC) and anionic (PS Ljubljana, Slovenia. and PG) lipids were assembled and their interactions with serotonin and g- Intracellular membranes exhibit complex, highly dynamic and non-lamellar aminobutyric acid (GABA) were studied as model systems. SPR shows a morphologies.1 Structural resemblance of such membranes with bicontinuous range of binding affinities for different neurotransmitters. Consistent with cubic and hexagonal lipid phases has been recognised in past two decades.2 How- these results, NR shows that the ligand with the largest affinity (serotonin) ever, principles behind their dynamic organization and structural roles in cellular penetrates the membrane deeply whereas GABA, for which the affinity is a processes still remain elusive. Model membrane systems like liposomes, planar tenth of serotonin, associates with the bilayer peripherally. Overall, we estab- lipid bilayers and micelles have been largely used to interact various biomole- lish that some neurotransmitters interact non-specifically with the lipidic cules to determine structures and functions of biomembrane systems.3 However, membrane matrix at physiologically relevant concentrations and that this these models are too simple to mimic complex intracellular membranes. interaction differs vastly for different neurotransmitters. These results could Non-lamellar lipid self-assemblies exhibit some fundamental features that can have a significant impact on our understanding of the molecular mechanism be crucial while mimicking convoluted biomembrane architectures like, for of synaptic transmission. example, continuous bilayer and aqueous networks of bicontinuous phases. 1. Sonner and Cantor, Annu. Rev. Biophys. 42, 143 (2013). Here we employed a model lipid-monoolein (MO) to form non-lamellar phases.4 2. Wang, et al., J. Phys. Chem. B. 155, 196 (2011). It is an unsaturated monoglyceride with C-18 chain. The average molecular shape is inverse conical (typical for majority of lipids), which is usually described by a 2759-Pos Board B189 ‘shape factor’, also called ‘critical packing parameter’ (>1 for inverse phases). Effects of Archaeal Tetraether Lipids on Membrane Partitioning of the By synchrotron small angle x-ray scattering we investigated changes in MO Antifungal Drug Nystatin Melvin Hudson1, Parkson Chong2, Umme Ayesa1. (Pn3m cubic) phase due to addition of a range of biomolecules (four different 1 2 lipids, a protein, a sugar, a surfactant and three different oils). We further calcu- Temple University, philadelphia, PA, USA, Temple University, lated molecular shapes and found that despite of variable sizes of lattice param- Philadelphia, PA, USA. eters the shape factor values stay within particular ranges. Each additive Nystatin is a polyene antibiotic that is frequently used in the treatment of cuta- molecules studied here affects the molecular shape differently which helped us neous, vaginal, and oral fungal infections. It interacts with ergosterol in the to understand their structural roles in non-lamellar biomembrane architectures. fungal cell plasma membrane, causing membrane disruption and leakage, 1. Lavoie, C. et al. Progress in Histochemistry and Cytochemistry 46, 1-48 and leading to cell death. Liposomal nystatin has good antifungal activity but (2011). with significantly less systemic toxicity compared to free nystatin. In this study, 2. Tomas, L. FEBS Letters 369, 13-17 (1995). we attempt to use archaeal tetraether lipids to increase the stability and drug 3. Chan, Y.-H. M. & Boxer, S. G. Current Opinion in Chemical Biology 11, loading of liposomal nystatin. Using a fluorometric method, we have deter- 581-587 (2007). mined the partition coefficient of nystatin into liposomes composed of choles- 4. Kulkarni, C. V., et al. Phys Chem Chem Phys 13, 3004-3021 (2011). terol and POPC (80 mol%) in the absence and presence of archaeal tetraether lipids. We found that nystatin partition into liposomes increases significantly 2757-Pos Board B187 (by a factor of ~4-10) with the addition of 0.5 mol% archaeal tetraether lipids Does the Meyer-Overton Correlation Need to be Modified PLFE to replace 0.5 mol% cholesterol. PLFE is the polar lipid fraction E Henrike Sasse-Middelhoff, Thomas Heimburg. isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius. This Niels Bohr Institute, Copenhagen, Denmark. dramatic change in nystatin partitioning engendered by PLFE was surprising The linear relationship between the partition coefficient and anaesthetic po- and may result from the presence of interfacial regions between diester and tet- tency is known as Meyer-Overton correlation. The homologous series of n-al- raether lipid domains. This result plus our previous findings in PLFE-enhanced canols, upon elongation, shows increasing anaesthetic potency accompanied by membrane stability suggest that tetraether lipids can be a useful additional an increase of the partition coefficient. However, the anaesthetic potency drops component in liposomal nystatin formulations. Wednesday, February 11, 2015 545a

2760-Pos Board B190 plexes were collected by using FTIR spectrometer BRUKER Tensor 27 in Polystyrene Nanoparticles Alter the Stability of Model Cell Membranes the temperature range 2-40C. The phase transitions in examined systems David Van Doren, Luke Cuculis, Shelli L. Frey. were studied by using the differential scanning calorimetry (DSC). Ability of Chemistry Department, Gettysburg College, Gettysburg, PA, USA. creating of stable complexes in DNA-surfactant systems studied was confirmed Due to their small size, nanoparticles have the ability to penetrate pulmonary using electrophoresis on agarose gel. For all formed stable lipoplexes the com- and vascular tissue, and as a result, are classified as potential human carcino- plete reduction of electrophoretic mobility was observed. Finally the transfec- gens. On the other hand, nanoparticle insertion into targeted cells can play a tion efficiency of selected systems were also tested on HeLa cells. key role in drug delivery and gene therapy applications, prompting a need to more thoroughly characterize nanoparticle/membrane interactions. Because 2763-Pos Board B193 nanoparticle interactions with biological membranes exist, but have not been Photothermal Manipulation of Membranes fully characterized, the stability of model cell membranes was observed in Scott K. Shaw, Bradley D. Smith. the presence of particles. Giant unilamellar vesicles (GUVs) composed of ca- Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, nonical ternary mixture of lipids of dipalmitoylphosphatidylcholine (DPPC)/ USA. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol (1:1:1) with There is emergingevidence that highlycontrolled nanoscale heating processes can 0.8% mol fluorescent lipid were made in the presence of functionalized poly- be used to manipulate biological membrane events such as pore formation, bilayer styrene nanoparticles at varying concentrations. A change in the vesicle size translocation, and fusion. These processes may have diverse applications in trig- distribution in the presence of NP indicates that inclusion of particles affects gered drug release, gene transfection, and hybridoma formation. The goal of this the stability of bilayer curvature. Aminated polystyrene particles were shown work is to devise new methods of producing nanoscale heating and to exploit this to limit the size of stable GUVs even at low particle concentrations of 0.025 phenomenon as a way to manipulate biomembrane structure and function. Photo- wt%. Association of the positively charged particles at the lipid/water interface thermal gold nanoparticles have been explored for these purposes but suffer from resulted in extruded lipid tubules from the vesicle surface. Carboxylated parti- size-restricted diffusion limitations and poor clearance profiles, while many pho- cles produced a less dramatic effect. This may be attributed to the greater tothermal organic dyes are susceptible to photobleaching. Our lab has developed charge density of the carboxylated particles, compared to those with amine high performance near infrared (NIR) dyes and dye loaded nanoparticles which generate heat upon absorption of NIR laser light, making them ideal for in vivo functionalization, such that a higher inter-particle repulsion could prevent 1,2 nanoparticle arrangement on the surface. In both cases, high nanoparticle con- applications. These photothermal agents were incorporated into cells and artifi- centration completely prevented the formation of GUVs, indicating a concen- cial membranes, and heat was produced with precise spatiotemporal control. tration dependent effect. The effect of the nanoparticles on the membrane Membranes sensitive to temperature were shown to release encapsulated contents material properties of the vesicles will also be discussed. and to have increased bilayer translocation rates. Recent work with artificial tem- perature insensitive membranes and cell membranes is also discussed. Chem. Sci. 4 2761-Pos Board B191 (1) Spence, G. T.; Hartland, G. V.; Smith, B. D. 2013, , 4240 How Nsaids Affect Lipid Monolayer and Bilayer Properties (2) Spence, G. T.; Lo, S. S.; Ke, C.; Destecroix, H.; Davis, A. P.; Hartland, G. Chem. Eur. J. 20 Jaime Larsen, Melanie Grooms, Brendan G. Ashton, David D. Busath. V.; Smith, B. D. 2014, , 12628 Brigham Young University, Provo, UT, USA. 2764-Pos Board B194 Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin synthe- Theoretical and Experimental Insights into Lipopolysaccharides- sis by blocking cyclooxygenase and influence gramicidin channel lifetimes in Polymyxin B Interactions Using Genetically Modified Enterobacterial planar lipid bilayers. In the past we studied how the NSAIDs salicylic acid, ace- Strains tylsalicylic acid, acetaminophen, ibuprofen and diclofenac influenced the Daniel R. Aguayo, Javier Salazar, Jaime Huerta, Mackarenna Alarco´n, pressure-area curve in the aqueous subphase on dipalmitoyl-PC pressure-area Bele´n Navarro, Nicola´s Pacheco, Hegaly Mendoza. curves at modestly supratheraputic dosages (1 mM concentrations in the sub- Center for Bioinformatics and Integrative Biology, Universidad Andres phase). We observed consistent changes that imply these NSAIDs interact Bello, Santiago, Chile. strongly with the lipid head groups of monolayers. Here we report subsequent The Gram-negative Outer Membrane Lipopolysaccharides (LPS) is the first findings using umbrella sampling molecular dynamics. Each titratable NSAID molecular barrier of the antimicrobial peptide (AMP). Each LPS monomer is was simulated, both protonated and unprotonated, with DPPC bilayers. The composed of a lipid-A and a polysaccharide chain of multiple modular- neutral NSAIDs have free energy wells as deep as 10 kcal/mol in the head- subunits with defined chemical structure and properties. There is great interest group region, whereas charged molecules were uniformly repulsed from the in understanding how AMP, like Polymyxin-B, associate and permeabilizate bilayer. These findings call for further experiments on the pH dependency of bacterial membranes; nevertheless, this process is poorly understood and drug-bilayer interactions and suggest that neutral NSAIDs, including proton- further studies are needed. To understand the Polymyxin-B interaction with ated aspirin in the low pH chyme of the stomach, interact with cell membranes the LPS, we have combined molecular dynamics simulations of LPS and could cause adverse side effects. containing-bilayers and in vivo and in vitro experimental assays using geneti- cally modified Escherichia coli and Salmonella Typhimurium strains to display 2762-Pos Board B192 LPS of controlled chemotypes. The dynamical characterization of the Studies of Zwitterionic Lipoplexes - Nanosystem Based on Phospholipids Polymyxn-B interactions with LPS-subunits gives a new insight of their contri- and Surfactants as Innovative Delivery Systems for Gene Therapy bution to the outer membrane destabilization process, which is essential to Joanna Wolak, Michalina Skupin, Zuzanna Pietralik, develop new antibiotics strategies. Granted by FONDECYT N 11130576. Weronika Andrzejewska, Maciej Kozak. Department of Macromolecular Physics, Adam Mickiewicz University, 2765-Pos Board B195 Poznan, Poland. Interaction of Thymol with Cell Membranes Models Studied with Tensi- The gene therapy is the one of the most promising method of treatment in ometry, Vibrational Spectroscopy and Molecular Simulation contemporary medicine. This way of therapy is very useful in the treatment a Joa˜o Victor N. Ferreira, Tabata M. Capello, Leonardo J. Amaral de Siqueira, dozen of incurable or fatal diseases. This method of treatment is leaned on imple- Joa˜o Henrique G. Lago, Luciano Caseli. ment a gene into patient’s cells with the use of dedicated delivery systems (vec- Ciencias Exatas e da Terra, Universidade Federal de Sao Paulo, Diadema-SP, tors). The main problem of gene therapy is to find the best vector which will be Brazil. effective and will be not toxic for human cells. A good approach seems to be use Thymol (2-isopropyl-5-methylphenol) is a natural compound that acts as a mi- of non-viral vectors like delivery system based on lipid-surfactant mixtures. crobicide, with a probable effect on cell membrane surfaces. However, the mech- The aim of this study was to examine the possible application of selected anism of action when it interacts with lipid surfaces is not sufficiently known. For amphoteric surfactants (zwitterionic alkyl derivatives of sulfobetaine) as com- this reason, it is important to understand at the molecular level interactions be- plexing agents (with and without helper lipid) for nucleic acids (siRNA, low tween the drug and biointerfaces, and using models for cell membranes can be and high-molecular weight DNA). an appropriate strategy for this purpose. In this study, we employed Langmuir The studies of DNA conformation in selected DNA - zwitterionic surfactant monolayers of lipids as cell membrane models, with the drug incorporated in lipoplexes were performed using the circular dichroism (CD) spectroscopy. monolayers of zwitterionic lipids, namely DPPC (dipalmitoyl phosphatidyl CD spectra were recorded in the spectral range 350 - 200 nm by using J-815 choline), and negative lipids, namely DPPS (dipalmitoyl phosphatidyl serine), spectrometer (Jasco). The results obtained indicate that the DNA maintains and compared to data obtained with Molecular Simulation. Combining data on the B-form for wide range of surfactant concentrations in the solution. The Surface Pressure-Area Isotherms with Polarization Modulation Infrared structure and organization of lipoplexes was also independently analyzed by Reflection-Absorption Spectroscopy (PM-IRRAS), the effect of the thymol on the Fourier transform infrared spectroscopy. The absorption spectra for lipo- lipid monolayers was compared by in view of the chemical and molecular 546a Wednesday, February 11, 2015 structure of the lipids and the drug. The adsorption of the drug at the monolayers to which divalent cations (Ca2þ and Mg2þ) were added. X-ray fluorescence decreases the order of the lipid film, in a molecular mechanism that involves both of the PIP2 monolayer on the buffer shows an eight fold surface enhancement polar head groups and alkyl tails from the lipids. Also the adsorption of thymol is (within the x-ray penetration depth below the critical angle, ~5 nm) of mono- modulated by the lipid monolayer composition since it adsorbed in a higher valent Kþ compared to its bulk concentration. When physiological levels of extend in negative charged lipid monolayers. Data obtained from Molecular calcium are added (1-100 mM), the Ca2þ gradually replaces bound Kþ ions, Simulation corroborate with Langmuir monolayer experiments suggesting spe- leading to a significant change in the organization of the PIP2 model membrane. cific sites of interaction between lipid and drug. A model is then proposed in At higher concentrations (100-1000 mM), which might be achieved during cal- which thymol interacts with lipids at the air-water in such a way that the interac- cium signaling, we observe a 1000 fold surface enhancement of Ca2þ. Similar 2þ tions are maximized owing to geometrical adaptations on behalf of the contact experiments with Mg ions also show strong ion binding to PIP2 at physiolog- between specific groups between the lipid and the drug. ical levels (1 mM) with a lesser structural effect on the monolayer compared to that induced by Ca2þ. For mixed solutions of Mg2þ and Ca2þ we find that Ca2þ 2766-Pos Board B196 occupies the majority of binding sites, and at mM concentrations completely Effects of Cations on the Material Properties of Model Cell Membranes removes the Mg2þ ions from the interface. Surprisingly, with both 1 mM Michael J. Counihan, Carly R. Strelez, Shelli L. Frey. Mg2þ and 1 mM Ca2þ in the subphase there is still a fourfold surface enrich- Chemistry Department, Gettysburg College, Gettysburg, PA, USA. ment of Kþ ions at the headgroup region. The outer leaflet of a cell membrane can be modeled with a lipid monolayer, of which structure and material properties can be measured as a function of lipid 2769-Pos Board B199 composition, insertion of macromolecules (peptides or nanoparticles), and ion Charge Dependence of POPG:POPC-Liposome Repulsions in Deionized presence, among other variables. Cation effects on these monolayers are not Water fully characterized, especially at higher ion concentrations, and due to the Joel A. Cohen1, Ming-Tzo Wei2, H. Daniel Ou-Yang2. greater variety of multivalent cations promise to be more complex than well- 1Biomedical Sciences, University of the Pacific, San Francisco, CA, USA, understood anion effects. Since biological processes take place in buffered en- 2Physics, Lehigh University, Bethlehem, PA, USA. vironments, an understanding of ion effects on lipid macromolecular structures Shear moduli of deionized suspensions of electrostatically interacting 100 nm is necessary to study fundamental cell membrane interactions. POPG:POPC liposomes were measured by microrheological methods. Lipo- These experiments investigated effects that cations of different charge (NaCl, some compositions spanned the entire range of POPG:POPC ratios. The lipo- MgCl2, and CaCl2) and concentration (0.1 M to 1.0 M) have on the structure somes were sized by extrusion and diluted in deionized water at volume and material properties of dipalmitoylphosphatidylcholine (DPPC) lipid mono- fractions between 0.094 and 0.167. Liposome mean size, polydispersity, and layers. To determine trends due to ion type and concentration, isotherm features electrophoretic mobility were also measured. Polydispersity indices ranged were quantified and compared, namely: area per molecule at liftoff, surface pres- from 0.08 to 0.15, indicating quite monodisperse suspensions. When illumi- sure at the phase transition plateau, layer compressibility prior to collapse, and nated by white light, POPG:POPC ¼20:80 suspensions displayed optical Bragg area per molecule and surface pressure upon collapse into the subphase. Small scattering, indicative of particle ordering. At volume fraction 0.167 the depen- ion concentrations (0.1 M) allowed lipids in the monolayer to pack more closely dence of shear modulus on composition was non-monotonic, first increasing compared to a model membrane on a pure water subphase due to electrostatic to 6000 d/cm2 at POPG:POPC ~20:80, then decreasing to 1000 d/cm2 at screening. At higher concentrations, the surface pressure at defined areas per POPG:POPC ¼100:0. Other concentrations behaved similarly. This effect molecule was greater compared to pure water (indicating expansion of the struc- may be attributed to the screening behavior of fully deionized suspensions, ture), and the monolayer underwent an additional structural transition, likely a where counterion screening due to dissociated protons depends exponentially rearrangement of the lipid tails with respect to the air-water interface before on particle charge. A 100 nm POPG liposome has ~50,000 phosphate groups collapse. The divalent salts caused a decrease in monolayer compressibility, indi- on its outer surface, each bearing 1 negative charge when dissociated. Since cating fluidization, and faster increase of liftoff area at higher salt concentrations. shear moduli reflect the mean particle charge of an interacting suspension, These trends were noticeably different compared to the monovalent cations. they provide information on interaction-mediated surface-charge regulation These effects can be explained by considering local electrostatic interactions. (protonation). Shear moduli were analyzed by the DLVO theory of screened 2767-Pos Board B197 Coulomb repulsions between charged spheres. Liposomes are well-suited to A Study of How Chelating Agents Interact with Neutral Lipid Membranes such studies since their titratable charge can be accurately controlled. James L. Wallis, Merrell A. Johnson, Bruce D. Ray, Horia I. Petrache. Physics, IUPUI, Indianapolis, IN, USA. 2770-Pos Board B200 Chelating agents are used in a range of areas, from treating metal poisoning in The Effect of Compartmentalization on the Kinetics of Transition Metal medicine to removing unwanted heavy metal ions from a given solution in Ion-Induced LDL Peroxidation biochemistry. Specifically, ethylenediaminetetraacetic acid (EDTA) is widely Dov A. Lichtenberg, Ilya Pinchuk. used in biochemistry and other areas of science to sequester polyvalent cations Tel Aviv University, Tel Aviv, Israel. such as Ca2þ and Fe3þ from solutions. Small angle x-ray scattering [1] has The typically observed kinetic profiles of transition metal ion-induced lipid per- shown that low concentrations (mM range) of EDTA in solution introduce a oxidation can be described in terms of a limited number of characteristic time- phase coexistence in homogeneous 1-palmitoyl-2-oleoyl-sn-glycero-3-phos- points derived from experimental time-dependencies and presented in terms of phocholine (POPC) multilamellar vesicles. This suggests that there are interac- rate constants and concentrations, as calculated based on mechanistic consider- tions taking place between EDTA and the lipid head group. We investigate the ations. The critical part of such analysis is that it is valid only if the experi- interaction of EDTA with lipids in the presence of various cations, using 1H mental system behaves as if it is homogeneous, i.e. as if the reaction occurs NMR. A discussion of the interactions present between EDTA and lipids will in a solution. In spite of the uncertainties due to the latter assumptions, we ob- be formulated based on observed proton chemical shifts and relaxation rates. tained a reasonable agreement between the experimental data and the theoret- [1] Johnson et al, Langmuir 2014. ically predicted dependencies, which supports our previous theoretical treatment. Yet, several previous findings could not have been explained in 2768-Pos Board B198 terms of our (‘‘quasi-homogeneous’’) model, indicating that the model is valid Specificity and Competitive Cation Association to Phosphatidylinositol- not under all conditions. One example is that under certain conditions, rapid 4,5-Bisphosphate Model Membranes peroxidation of lipids occurs prior to complete consumption of LDL- Edgar E. Kooijman1, Zachary T. Graber2, Wenji Wang3, Ivan Kuzmenko4, associated Tocopherol. We think that uninhibited (‘‘rapid’’) peroxidation be- David Vaknin3. comes apparent when considerable fractions of the particles lose all their 1Biological Sciences, Kent State University, Kent, OH, USA, 2Chemistry and Tocopherol (i.e. before the time predicted for ‘‘homogeneous’’ system). The Biochemistry, Kent State University, Kent, OH, USA, 3Ames Laboratory and lack of Tocopherol is an essential but insufficient demand for rapid peroxida- Department of Physics, Iowa State University, Ames, IA, USA, 4x-ray tion. Another demand is that the particle should contain at least a critical num- science division, advanced photon source, Argonne National Laboratory, ber of hydroperoxides molecules. In the present investigation, we show that the Argonne, IL, USA. results of all our kinetic studies can be understood if we consider compartmen- Phosphatidylinositol-4,5-bisphosphate (PIP2) is an active signaling lipid impli- talization. Specifically, for any given composition of the particles (LDL and/or cated, among other functions, in the regulation of cell growth by activating the HDL), the kinetic results are probably governed by the distribution and rate of tumor suppressor PTEN. Using synchrotron surface-sensitive x-ray diffraction exchange of antioxidants and hydroperoxides between particles. Our analysis is and fluorescence techniques we determined the preferential cation binding to of special importance for systems containing more than one population of lipo- PIP2 monolayers. The natural, highly unsaturated PIP2 was spread as a Lang- protein particles. The possible effects of compartmentalization should be muir monolayer on a physiological buffer containing 100 mM KCl at pH 7.2 considered in other reactions that occur in inhomogeneous systems. Wednesday, February 11, 2015 547a

Membrane Active Peptides and Toxins II 2774-Pos Board B204 Membranes can Finely Tune Peptide-Induced Lipid Extraction by 2771-Pos Board B201 Modulating their Lipid Composition The Presence of Antiparallel Beta-Sheets in Toxic Fibrils Formed by Alexandre Therrien, Michel Lafleur. ABeta on GM1 Clusters Dept. of chemistry, Universite´ de Montre´al, Montre´al, QC, Canada. Yuki Okada1, Hiroshi Ueno1, Yoshiaki Yano1, Masaru Hoshino1, Membrane binding of lytic peptides, and the resulting peptide-induced lipid Hikari Itoh-Watanabe2, Akira Naito2, Katsumi Matsuzaki1. extraction (membrane solubilization) are influenced by the nature of lipids 1Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, forming the membranes. However, the impact of various lipid species on these Japan, 2Yokohama Naitional University, Yokohama, Japan. two phenomena cannot be directly inferred one from the other; distinct peptide/ The abnormal aggregation of amyloid b-protein (Ab) is considered to be central lipid interactions are pivotal in these distinct events. in the pathogenesis of Alzheimer’s disease. We have focused on ‘membrane- Melittin is a small cationic peptide with a secondary amphipathic helical mediated’ amyloidogenesis because Yanagisawa et al. identified a specific character. It interacts spontaneously with lipid bilayers, and induces lipid form of Ab that was bound to monosialoganglioside GM1 in brains exhibiting extraction. We investigated how phosphatidylserine (PS), cholesterol, and the early pathological changes associated with the disease. We have found that phosphatidylethanolamine (PE) modulate melittin binding to phosphatidylcho- amyloid fibrils formed on GM1 clusters were more toxic than those formed in line (PC) membranes as well as their impact on lipid extraction. solution [1, 2]. The less toxic fibrils formed in solution are considered to be First, PS, a negatively charged phospholipids, increases melittin affinity for composed of in-resister parallel b-sheets, whereas the structure of the toxic membranes but inhibits melittin-induced lipid efflux. It is proposed that the fibrils is unknown, although FTIR spectra suggested the presence of antiparallel attractive electrostatic interactions between melittin and PS, which are respon- b-sheets [1, 2]. sible for the increased affinity, anchor melittin at the bilayer interface and prevent In this study, we investigated the structure of the toxic fibrils in detail. Solid- its relocation required for lipid extraction. Second, cholesterol reduces melittin state NMR measurements using site-specifically [15N, 1-13C]-labeled Abs membrane affinity. In parallel, it inhibits the extent of lipid extraction induced suggested that the fibrils contained both parallel and antiparallel b-sheet struc- by melittin and LC-MS analysis indicates that lipid/melittin particles resulting tures. Chemical cross-linking experiments using Cys-substituted Abs also sup- from the extraction are cholesterol- depleted relative to the cholesterol content ported this conclusion. Thus, the toxic fibrils were found to possess a novel of the original membranes. It is proposed that the phospholipid ordering caused unique structure. by cholesterol is unfavorable to peptide binding and inhibits the overall melittin [1] Fukunaga, S. et al., Biochemistry 51, 8125 (2012). action. Third, PE decreases the affinity of melittin for PC-based bilayers, an in- [2] Matsuzaki, K., Acc. Chem. Res. 47, 2397 (2014). hibition more significant in the gel than in the fluid phase. The limited amount of melittin that can be accommodated in PE-containing gel-phase bilayers appears 2772-Pos Board B202 to make the bilayers more susceptible to lipid extraction. As a consequence, PE Structural Transformation of Amyloid Peptides Interacting with Lipid can act as a promotor or an inhibitor of melittin-induced lipid extraction, depend- Membranes ing on its proportion. These findings demonstrate that membranes can tune Yen Sun, Huey W. Huang. peptide-induced lipid extraction by altering their lipid composition. Physics and Astronomy, Rice University, Houston, TX, USA. 2775-Pos Board B205 The inherent cytotoxicity of protein aggregates implies a common mechanism Mechanism of Action of b-Hairpin Antimicrobial Peptides for amyloid diseases (Bucciantini et al). However, accumulated evidence sug- Richard B. Lipkin, Themis Lazaridis. gests that the insoluble fibrils or aggregates are not the culprit. On the other Chemistry, City College, New York, NY, New York, NY, USA. hand, amyloid peptides in soluble form do interact with lipid bilayers, sug- Previous work showed that the b-hairpin antimicrobial peptide (AMP) prote- gesting that the cell plasma membranes are a target of amyloid pathogeneses. grin forms stable octameric b-barrels and tetrameric arcs (half barrels) in In particular, Keyed et al. have demonstrated that amyloid peptides all in- both implicit and explicit membranes. Here, we extend this investigation to crease membrane ion conductance without any evidence of discrete channel several AMPs of similar structure: tachyplesin, androctonin, polyphemusin, go- or pore formation. In this study we try to find the common molecular process mesin, and the retrocyclin q-defensin. We also examine synthetic b-sheet pep- of soluble amyloid peptides interacting with lipid membranes that might tides selected from a combinatorial library for their ability or inability to form induce membrane conductivity. It is difficult to study this molecular process pores in lipid membranes. When heptameric, octameric, and decameric b-bar- for most amyloid peptides because of its propensity to fibrillize at relatively rels and tetrameric arcs of these peptides were initially embedded in preformed low solution concentration. PrP 106-126 is a random coil in its soluble neutral and anionic lipid pores, a variety of behaviors and membrane binding form. We study its kinetics of structural transformation in the presence of lipid energies were observed. The synthetic peptides bound very strongly and vesicles. The time dependence of the structural changes was analyzed as a formed stable barrels and arcs in both neutral and anionic pores. The natural function of the lipid concentration. We demonstrate that the soluble peptides AMPs exhibited unfavorable or marginally favorable binding energy and ki- transform from random coils to alpha-helices upon binding to lipid bilayers. netic stability in neutral pores, consistent with the lower hemolytic activity The helical state is stable, as long as the bound peptide to lipid ratio P/L of some of them compared with protegrin. Binding to anionic pores was on the lipid vesicle is below a critical value P/L*. But as P/L exceeds more favorable, but significant distortions of the barrel or arc structures were P/L*, the peptides transform from the helical state to beta-aggregates. This sometimes noted. These results are discussed in light of the available experi- is consistent with previous studies of penetration and hIAPP in multiple lipid mental data. The diversity of behaviors obtained makes it unlikely that the bar- bilayers. Thus we found the common fibrillization process of amyloid rel and arc mechanisms are valid for the entire family of b-hairpin AMPs. peptides interacting with lipid bilayers. Our proposal is that the process of peptides’ transformation from random coils to helices and then to beta- 2776-Pos Board B206 aggregation creates defects in the membranes that allow ion permeation to Attack on Single Escherichia Coli Spheroplasts by Antimicrobial Peptides occur as observed by Keyed et al. Tzu-Lin Sun, Yen Sun, Huey W. Huang. Physics, Rice University, Houston, TX, USA. 2773-Pos Board B203 Studies of the molecular mechanisms of AMPs are mostly performed with lipid Alpha-Synuclein Stabilizes Small Unilamellar Vesicles by Reducing Both bilayers.Thus there is a persistent question as to whether the action of AMPs on Membrane Surface Tension and Rigidity bacterial membranes can be reproduced on lipid bilayers. Recently Weisshaar Anthony R. Braun, Jonathan N. Sachs. and coworkers have studied the actions of AMPs on E. coli and Bacillus subtilis Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA. by time-lapse fluorescence microscopy. The direct observation of the action of Alpha-Synuclein (aSyn) stabilizes small unilamellar vesicles (SUV) by AMPs on bacteria revealed two key steps. The first is growth halting due to reducing the vesicle’s surface tension and membrane rigidity. Using coarse- direct interference of AMP with cell wall synthesis and is recoverable. The sec- grained molecular dynamics simulations we explored the membrane remodel- ond is permeabilization of the cytoplasmic membrane which is not recoverable. ing characteristics of aSyn bound to DPPC lipid vesicles. By combining our Here we study the direct action of AMPs on the cytoplasmic membranes by us- recently developed Spherical Harmonics fluctuation analysis with 3-dimen- ing E. coli spheroplasts, the cell form from which the outer membranes have sional pressure tensor calculations we were able to characterize aSyn’s effect been removed. The purpose is to compare the action on bacterial membranes on both structure and mechanical properties of DPPC vesicles. Our findings with that on lipid bilayers. The key question is how to reveal the mechanisms highlight a dramatic reduction in membrane surface tension and increased of AMPs on bacterial membranes. First we observe the action of AMPs on giant membrane fluctuations, suggesting a less rigid bilayer, for the aSyn vesicle sys- unilamellar vesicles (GUVs) made of E. coli total lipid extract.We used the tem relative to pure DPPC. aspiration method to hold the GUV in a solution containing a soluble dye 548a Wednesday, February 11, 2015 molecule and a fluorescence labeled melittin to obtain the lipid bilayer response and positively charged at slightly acidic pH, can thus exhibit pH dependent to an AMP. The same method was applied to spheroplasts to observe the action activity. 2H solid state NMR spectroscopy was used to study the effect of of AMPs on bacterial membranes. In both cases, we found first the binding of Gad-1 and Gad-2 on model membranes at different pH values. The addition peptides expanded the membrane area. As the membrane area increased to of peptides to the model membrane system POPE/POPG-d31 (3:1) results in a ~2-3%, the dye molecules began to leak in. After a time, we found the sphero- change in the splitting of spectrum both at pH 7 and pH 5. Further investigation plasts lost their phase contrast indicating the lose of the cell content. The same of peptide effects on lipid acyl chain motion were determined through calcula- experiments were repeated with LL37 and magainin. We found that sytox green tions of orientational order parameter values (SCD). The order parameter profile is not a good indicator of membrane permeability. demonstrated that Gad-1 causes more lipid acyl chain disordering than Gad-2, at both pH 7 and pH 5. These results are consistent with Gad-1’s greater activity in 2777-Pos Board B207 assays for bacterial growth inhibition. On the other hand, while Gad-2 is more Insights into the Effects of Antimicrobial Peptides on Live E. coli Cells active at inhibiting bacterial growth at low pH than at neutral pH, its effect on using Time-Lapse Fluorescence Microscopy lipid acyl chain disorder is the same at pH 7 and pH 5. Ranga Rajan, James C. Weisshaar. Chemistry, University of Wisconsin Madison, Madison, WI, USA. 2780-Pos Board B210 Time-lapse fluorescence microscopy enables detailed observations of the ef- The Transmembrane State of Antimicrobial Piscidin in Bacterial Cell fects of natural and synthetic antimicrobial peptides (AMPs) on live E. coli Membrane Mimics Dramatically Alters the Polar-Nonpolar Segregation cells. Apart from tracking membrane permeabilization caused by AMPs, we of the Bilayer also observe the effect of AMPs on intracellular components such as the Myriam Cotten1, Jorge I. Hernandez2, Kimberly Bogardus1, nucleoid and the ribosome. Mihaela Mihailescu3. 1 2 Using our assays, we have uncovered differences in the mechanisms of action Chemistry, Hamilton College, Clinton, NY, USA, Chemistry, Clemson 3 of synthetic peptides known to permeabilize LUVs. Our single-cell, real-time University, Clemson, SC, USA, Institute for Bioscience and Biotechnology data is rich in spatial and temporal resolution, and is biologically relevant. Research, University of Maryland, Rockville, MD, USA. Piscidin 1 (P1) and piscidin (P3) 3 are twenty-two-residue-long antimicrobial 2778-Pos Board B208 peptides that are membrane-active. While their amphipathic structures lend Histidine-Rich Designer Peptides with pH-Dependent Membrane Topol- themselves to forming pores by the carpet/toroidal pore mechanism, direct evi- ogy, Antimicrobial, Nucleic Acid Transfection and Viral Transduction dence for their mode of action is lacking. Previously, we determined the high res- Capabilities olution structures of both peptides bound parallel to the surface of bacterial cell Christopher Aisenbrey1, Philippe Bertani1, Andrea Farrotti2, David Fenard3, mimics constituted of 3:1 phosphocholine (PC)/phosphoglycerol (PG). In a Anne Galy3, Elise Glattard1, Arnaud Marquette1, Jesus Raya1, comprehensive effort to decipher their mechanism of action, we combined Evgeniy S. Salnikov1, Joachim Seelig4, Lorenzo Stella2, Louic Vermeer1, dye leakage assays, oriented CD (OCD), X-ray diffraction (XRD), and neutron Nathaliia Voievoda1, Burkhard Bechinger1. diffraction (ND) in 3:1 PC/PG to simultaneously investigate at various peptide- 1Institute of Chemistry UMR7177, University of Strasbourg/CNRS, to-lipid (P/L) ratios the permeabilization capability of the peptides, the physical Strasbourg, France, 2Dipartimento di Scienze e Tecnologie Chimiche, state of the bilayer, and the bilayer orientations and locations of the peptides. University Tor Vergata, Roma, Italy, 3UMR_S951, Genethon, INSERM, The OCD results show that P1 adopted an inserted state at a lower P/L than P3 University of Evry, Evry, France, 4Biocenter, University of Basel, Basel, (1:25 versus 1:16) in agreement with its stronger permeabilization capability. Switzerland. XRD data showed that the bilayer gradually thinned from 52.5 A˚ in the The synthetic LAH4 peptides were designed to investigate the interactions that peptide-free state to 48.5 A˚ in the presence of piscidin at 1:16 P/L. ND exper- determine the membrane topology of helical peptides (1). Their core consist of iments at 1:25 P/L confirmed the OCD and XRD results. Because we calculated alanines, leucine and four histidines arranged to form an amphipathic helix, as the ND profiles for samples containing peptides that were 2H-labeled at either well as two lysines at each terminus. Through protonation of its histidines (pKs the C-end or at both the N- and C-ends, we could determine the bilayer orien- between 5.4 and 6.0) the alignment of the helices is transmembrane at neutral tation and location of each peptide. Importantly, the combined OCD and ND pH and in-plane at pH <5.5 (1). data revealed that P1 has a transmembrane orientation almost parallel to the The LAH4 peptides exhibit membrane pore-formation and antimicrobial action bilayer normal. Further investigation of this state at a 1:16 P/L demonstrated at both neutral and at acidic pH including against clinical isolates where the low that it induces a dramatic broadening of the lipid headgroup distribution indi- pH configuration is more active (2). The LAH4 peptides have been found to cating that it abolishes the strict nonpolar-polar segregation of the bilayer. also exhibit potent DNA and siRNA transfection activities (3). LAH4 can The implications for piscidin’s mode of action will be discussed. therefore act as a non-viral vector and has indeed been used for the delivery of quantum dots or protein-based vaccines. Furthermore, transduction by 2781-Pos Board B211 adeno-associated viruses or lentiviruses is enhanced by LAH4 peptides (4). Antimicrobial Peptide Piscidin Permeabilizes Bacterial Membranes and Ongoing biophysical and structural investigations will be reported which aim Binds Intracellular Targets at Sub-Lethal Concentrations to understand these activities at atomic resolution (5-8). Mason Schoeneck, Robert Hayden, Gina Goldberg, Bryan Ferguson, 1. B. Bechinger, J.Mol.Biol. 263, 768 (1996). Michael McCormick, Herman Lehman, Myriam Cotten. 2. A. J. Mason, et al., J. Biol. Chem. 284, 119 (2009). Chemistry, Hamilton College, Clinton, NY, USA. 3. B. Bechinger, V. Vidovic, P. Bertani, A. Kichler, J Pept Sci 17, 88 (2011). Piscidins 1 (P1) and 3 (P3) are cationic a-helical membrane active antimicro- 4. D. Fenard, D. Ingrao, A. Seye, J. Buisset, S. Genries, S. Martin, A. Kichler, A. bial peptides originally isolated from the hybrid striped seabass. The peptides, Galy, Molecular Therapy-Nucleic Acids 2, doi:10.1038/mtna.2013.17 (2013). which possess broad-spectrum activity against bacteria, fungi, and viruses, bind 5. C. Aisenbrey, B. Bechinger, Langmuir 30, 10374 (2014). to anionic membranes and lyse them once a sufficient concentration has been 6. R. Macha´n, P. Jurkiewicz, T. Steinberger, B. Bechinger, M. Hof, Langmuir reached. In this study, we used confocal microscopy and FITC-labeled pisci- 30, 6171 (2014). dins to show that at sub-inhibitory concentrations (0.75 mM), P1 and P3 are 7. B. Perrone, A. J. Miles, E. Salnikov, B. Wallace, B. Bechinger, Eur. Biophys. able to translocate across the membrane of Gram-positive and -negative bacte- J. epub. Sep. 3, (2014). ria. The FITC signal of the peptides was co-localized with the DAPI signal of 8. A. Farrotti, G. Bocchinfuso, A. Palleschi, N. Rosato, E. Salnikov, N. Voie- the nucleoid region of the bacteria. CD using a 15 piece of DNA and a voda, B. Bechinger, L. Stella, (2014, submitted). gel retardation assay using a 1782 base pair piece of DNA confirmed that P1 and P3 could bind to DNA in vitro. It appears that P3 binding was more disrup- 2779-Pos Board B209 tive to the structure of DNA, and this may be due to the extra arginine residue in Effect of Histidine Rich Antimicrobial Peptides, Gad-1 and Gad-2, On P3’s sequence. However, these results did not indicate whether the peptides Model Membranes at Different pH Values translocated through the membrane or entered the cells via pores formed in Gagandeep K. Sandhu, Michael R. Morrow, Valerie Booth. the membrane. To probe this question, we used the bacterial strain E. coli Physics and Physical Oceanography, Memorial University of Newfoundland, ML35 in a permeabilization assay to determine at which peptide concentration St. John,s, NL, Canada. the membrane became permeabilized. Our data indicate that at the lowest con- Antimicrobial polypeptides (AMPs) are an important component of the innate centration tested (0.1 mM) the membrane of ML35 cells became permeabilized immune system of many different organisms and are generally amphipathic by P1 and P3. Our results agree with prior findings that permeabilization of bac- and cationic in nature. Electrostatic interactions between positively charged res- terial cell membranes can occur prior to lysis and cell death. The implications idues of the AMP and negatively charged lipids in pathogen membranes are an of piscidin’s ability to translocate into bacterial cells and bind to intracellular important component of AMP mechanism and specificity. AMPs such as Gad-1 targets at sub-lethal concentrations will be discussed in terms of its mechanism and Gad-2 that are rich in histidine, an amino acid that is uncharged at neutral pH of action, which was previously believed to be based on its membrane activity. Wednesday, February 11, 2015 549a

2782-Pos Board B212 with the lipid extracting effect of daptomycin. Here we make use of the CD Molecular Dynamics Simulations Reveal Mechanistic Details of Polymyxin spectral change with Ca2þ ion concentration and with PG concentration to Penetration into both Membranes of E. coli analyze the nature of the daptomycin-Caþþ-PG aggregates. The data shows Syma Khalid1, Nils A. Berglund1, Peter J. Bond2, Thomas J. Piggot3. a sharpe threshold (or micellization) effect on the Ca2þ ion concentration 1School of Chemistry, University of Southampton, UK, Southampton, United dependence, indicating that each aggregate contains 20 or more calcium Kingdom, 24 Bioinformatics Institute (A*STAR), Singapore, Singapore, ions. This result shows that the aggregates are large, inconsistent with the 3Detection Department, Defence Science and Technology Laboratory, idea of daptomycin aggregates forming oligomeric pores, since such a pore Salisbury, United Kingdom. must be made of a relatively small number of daptomycin. Once we realize Gram-negative bacteria such as E.coli are protected by a surprisingly complex the micellization effect, the stoichiometry of the daptomycin-Caþþ-PG aggre- cell envelope. The cell envelope is composed of two membranes that form a gates can be analyzed from the calcium and PG concentration dependence. protective barrier around the cells, and control the influx and efflux of solutes via various routes. Lysing the cell by disrupting the membranes, or permeating 2785-Pos Board B215 across them to gain access to the cell interior are key properties for antimicro- Lipid Selectivity of Fungicidal Lipopeptides bial agents. Polymyxins are a class of antibiotics that have been shown to be Sebastian Fiedler, Quang Huynh, Hiren Patel, Heiko Heerklotz. highly active against Gram-negative bacteria. It is thought they enter bacterial Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, cells through a self-uptake mechanism, although the molecular details of the Canada. mechanism are still unclear. Cyclic lipopeptides act against a variety of pathogens and, thus, constitute highly We present, to our knowledge, the first molecular dynamics simulation study of efficient crop-protection agents. For example, commercially available fungicides an antimicrobial peptide, with both membranes of E.coli. Our model of the contain mixtures of different Bacillus subtilis lipopeptide families, such as sur- outer membrane contains lipopolysaccharide molecules in the inner leaflet factins (SF), iturins (IT), and fengycins (FE). Besides other effects, the fungicidal and a mixture of ohosphilipids in the inner leaflet. In contrast, the inner mem- activity of these peptides is mainly mediated by permeabilizing the membrane. brane is comprised only of phospholipids. Our simulations reveal the effects of Accordingly, surfactins behave like extremely powerful surfactants and induce Polymyxin B1 (PMB1) binding on the physical properties of each membrane. graded membrane leakage, iturins synergize with surfactins, and fengycins Thus they are able to identify potentially different mechanisms for membrane follow an all-or-none leakage mechanism. However, little is known about how disruption by PMB1. Peptide aggregation and insertion of one peptide tail was the lipid composition of the membrane affects the capability of these lipopeptides observed in the outer membrane. In contrast, PMB1 peptides insert readily as to induce membrane leakage. To shed more light on their lipid selectivity, we per- monomers, accompanied by water penetration into the inner membrane. Our formed fluorescence-lifetime-based leakage assays on various types of lipid- simulations demonstrate the importance of capturing relevant details of biolog- bilayer compositions. To this end, we compare leakage efficiencies and mode ical complexity, in molecular models of biological membrane systems. of actions of SF, IT, FE, and mixtures of them in lipid vesicles composed of phos- phatidylcholine/phosphatidylethanolamine (PC/PE), PE/phosphatidylglycerol 2783-Pos Board B213 (PE/PG), and analyze the effect of phosphatidylserine and cardiolipin. Further- Thermodynamics Govern the Mechanism of Antimicrobial Lipopeptides: more, we assessed the influence of ergosterol as a main component of fungal Insights from Coarse-Grained Molecular Dynamics Simulations cell membranes. Our results aid in the understanding of the mechanism of Dejun Lin, Alan Grossfield. lipopeptide-induced fungicidal activity and demonstrate that B. subtilis tailors University of Rochester, Rochester, NY, USA. these lipopeptide mixtures to specifically attack fungal membranes. Antimicrobial lipopeptides (AMLPs) are a series of acylated cationic peptides 2786-Pos Board B216 with broad-spectrum antimicrobial activity and low hemolytic activity. We Structure and Membrane Topology of the Pore-Forming Peptide used microsecond-scale coarse-grained molecular dynamics simulations with Maculatin 1.1 the MARTINI force field to understand AMLPs’ modes of action. Using Marc-Antoine Sani1, Terry P. Lybrand2, Frances Separovic1. rigorous free energy calculations with a novel reaction coordinate, we quanti- 1School of Chemistry Bio21 institute, Univeristy of Melbourne, parkville, fied formation of lipopeptide micelles in solution, as well as the the affinity of Australia, 2Center for Structural Biology, Vanderbilt University, Nashville, those micelles for different membrane compositions. The results yield both ki- TN, USA. netic and thermodynamics arguments for the lipopeptides selectivity for bacte- Antimicrobial peptides (AMP) that target membranes are an attractive alterna- rial over mammalian membranes. Our results indicate that the acyl chain of tive to classic antibiotics, since they do not require internalization nor target a C16-KGGK, one of the AMLPs, is mainly responsible for its affinity to mem- specific stereo-structure, thus limiting development of bacterial resistance. Their brane while the peptide portion determines the selectivity towards different mode of action involves the disruption of lipid membranes; however, the rela- membrane lipid compositions. The micelle results suggest both kinetic and tionship between lipid membrane structure and peptide potency remains unclear. thermodynamics arguments for the lipopeptides selectivity for bacterial over We present the structural investigation of the AMP maculatin 1.1 (Mac1) in DPC mammalian membranes. Our results provide biophysical insights into the micelles and DHPC/DMPC isotropic (q¼ 0.5) bicelles. Using solution and solid- mechanism of lipopeptides’ antimicrobial action. state NMR with paramagnetic relaxation enhancement agents (PRE) and molec- ular dynamics (MD), we demonstrate the important role of the membrane 2784-Pos Board B214 structure in modulating the structure and location of Mac1. HSQC of specifically The Nature of Daptomycin Aggregates 15N labeled Mac1 in buffer displayed a narrow chemical shift dispersion that is 1,2 3 4 4 Ming-Tao Lee , Wei-Chin Hung , Yen-Fei Chen , Huey W. Huang . typical of random coil structures. Introduction of micelles and bicelles produced 1Life Science Group, National Synchrotron Radiation Reasearch Center, 2 chemical shift dispersions characteristic of helical structures, with differences Hsinchu, Taiwan, Department of Physics, National Central University, suggesting that Mac1 adopts a different degree of helicity dependent on the cur- Jhongli, Taiwan, 3Department of Physics, R. O. C. Military Academy, 4 vature. 3D TROSY-NOESY allowed assignment of the sequential 15N labeled Fengshan, Taiwan, Department of Physics & Astronomy, Rice University, residues, and determination of a 3D helical structure in phospholipid micelles Houston, TX, USA. and bicelles, the latter producing the greatest helical stretch. Titration of the Daptomycin is the first FDA-approved member of a new structural class of anti- PRE agent Gd3þ-(DTPA) showed that the central core of Mac1 is protected biotics_the cyclic lipopeptides. It is an important drug against multidrug- in bicelles while in micelles only the N-term is exposed to the PRE effect. resistant gram-positive pathogens. The peptide interacts with the lipid matrix MD simulations in DPC micelles revealed N-term exposure to the solvent, of cell membranes, inducing membrane permeability to ions, but its molecular and they also suggested that Mac1 bent to adapt the curved micelle structure. mechanism has been a puzzle. Unlike the ubiquitous membrane-acting host-de- Experiments will be repeated with 4 and 8 peptides per micelles. fense antimicrobial peptides, daptomycin does not induce molecular-leaking pores in the cell membranes–no calcein leakage was detected from lipid vesi- 2787-Pos Board B217 cles. Thus how it affects the membrane permeability to ions is not clear. The Structure of Transmembrane Pores Stabilized by Antimicrobial Peptides antibacterial activity and induced ion leakage by daptomycin correlate with Magainin and PGLa the target membrane’s content of phosphatidylglycerol (PG) and occur only Almudena Pino Angeles, John M. Leveritt III,, Themis Lazaridis. þ in the presence of Ca2 ions. Fluorescence resonance energy transfer (FRET) Chemistry, City College of New York, New York, NY, USA. experiments and the recently discovered lipid extracting effect have shown Antimicrobial peptides are found in many organisms as part of their defense sys- daptomycin aggregation in membranes, but the chemical structure of daptomy- tem against bacterial infections. They have similar structural and functional fea- cin gives no clue to the nature of daptomycin aggregates. Jung et al discovered tures: most consist of amphiphilic helices that bind to a membrane and disrupt it an inversion of the CD spectrum of daptomycin that occurs only in the simul- by diverse methods. Among these peptides, magainin 2 and PGLa are found in taneous presence of PG and Caþþ. We have correlated the inversion of the CD the frog skin and exhibit synergistic effects in lipid bilayer disruption by the 550a Wednesday, February 11, 2015 formation of transmembrane pores [1]. Experimental methods, such as solid- spectra and NMR measurements of amide temperature coefficients for aqueous state NMR, have shed light on the relative orientation of magainin 2 and KK36 are not consistent with a canonical 310-helical conformation. We present PGLa on membranes with different lipid composition [2,3], but there is a general here the complete titration of amide proton chemical shifts for KK36 and KK45 lack of information on the structure of the pore and the specific interactions that from aqueous (90:10 H2O/D2O) solution to DMSO-d6 in order to identify the lead to its stabilization. In the present work, we studied the structure and dy- residues that are undergoing local environment changes as the bulk environ- namics of transmembrane pores formed by magainin 2 and magainin 2/PGLa ment is changed. Measurements of the CD spectra of both peptides aqueous (2:2) tetramers by all-atom molecular dynamics simulations performed at the solution as a function of temperature will be correlated with the amide temper- Anton supercomputer (Pittsburgh Supercomputing Center). For both systems ature coefficient data from NMR to obtain a more complete picture of solvent- we observed stabilization of a pore. The 9-ms simulation time allows a detailed induced conformational changes. Ultimately the correlation of structure and analysis of its structure and properties, the role of the lipids surrounding it, and sequence will inform future antibiotic peptide design. the relative orientation of magainin 2 and PGLa in the membrane. References 2790-Pos Board B220 [1] Matsuzaki, Mikani, Akada et al. Biochemistry (1998) 37:15144-15153 Anisotropic Membrane Curvature Sensing by Antibacterial Peptides 1 2 [2] Tremouilhac P, Strandberg E, Wadhwani P and Ulrich AS. J. Biol. Chem. Jordi Go´mez-Llobregat , Martin Linde´n . 1Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, (2006) 281:32089-32094 2 [3] Strandberg E, Zerweck J, Wadhwani P and Ulrich AS. Biophys. J. (2013) Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. 104: L09-L011 Some proteins and peptides have an intrinsic capacity to remodel lipid bilayers and sense membrane curvature via a curvature-dependent membrane binding en- 2788-Pos Board B218 ergy. This is crucial for many biological processes. For example, antimicrobial Modulation of the Interaction Between Detergent Micelles and Model peptides are believed to disrupt bacterial membranes by producing pores, which Peptide Antibiotics by Varying the Peptide Charge Distribution are highly curved structures. In this work, we explore a new computational John Weirich1, Brianna Haight1, Olivier Lequin2, Lucie Khemte´mourian2, method to investigate curvature sensing by simulating the interaction of single Ludovic Carlier2, Adrienne Loh1. peptides with a buckled lipid bilayer, using the coarse-grained Martini model. 1Chemistry & Biochemistry, U. Wisconsin - La Crosse, La Crosse, WI, USA, We analyze three canonical antimicrobial peptides, magainin, melittin, and LL- 2Laboratoire des Biomole´cules, UMR 7203 UPMC-ENS-CNRS, Universite´ 37, and find qualitatively different sensing characteristics. In particular, melittin Pierre et Marie Curie, Paris, France. and LL-37 show anisotropic curvature sensitivity, but with different preferred ori- With rising disease rates and decreasing effectiveness of conventional antibi- entations relative to the direction of greatest curvature. These findings provides otics, there is an immediate need for new antibiotics. One promising solution new insights into the microscopic mechanisms of curvature sensing and its role is through cationic antimicrobial peptides, which act by perturbing bacterial in membrane remodeling, and should motivate experimental development to membranes. We are investigating model peptide antibiotics composed primarily simultaneously measure position and orientation of membrane-bound proteins. of the hydrophobic dialkylated amino acid Aib (a-aminoisobutyric acid), which 2791-Pos Board B221 imparts a strong 310-helical bias due to steric hindrance at the a-carbon. Cationic Membrane Interaction of an Anti-Bacterial AApeptide Defined by EPR lysine residues were placed in adjacent locations in the center of the helix Spectroscopy (KK45) or one full turn apart (KK36). Micelles of dodecylphosphocholine Pavanjeet Kaur1, Yaqiong Li2, Jianfeng Cai2, Likai Song1. (DPC) or sodium dodecyl sulfate (SDS) were used as zwitterionic or anionic 1National High Magnetic Field Laboratory and Florida State University, membrane models, respectively. The interaction of model peptides with micelles Tallahassee, FL, USA, 2University of South FLorida, Tampa, FL, USA. can provide valuable information about the role of helical structure and peptide Antibiotic resistance is one of the major threats to public health. AApeptides are charge distribution on peptide-membrane interactions. Here we present thermo- a new class of synthetic anti-bacterial peptidomimetics that are not prone to anti- dynamic and spectroscopic data characterizing the peptide-micelle interactions. biotic resistance, and are highly resistant to protease degradation. The broad- Binding enthalpies for the interactions of KK36 and KK45 with DPC and SDS spectrum anti-bacterial activities of AApeptides are believed to be related to micelles were measured using isothermal titration calorimetry (ITC). Prelimi- their unique structural features, which are capable of disrupting bacterial mem- nary data suggests that binding to SDS micelles is exothermic, while binding branes selectively over human eukaryotic cells. How AApeptides selectively to DPC micelles is endothermic. In both cases, KK45 has a more favorable bind- interact with bacterial membranes and alter lipid assembly and properties is un- ing enthalpy than KK36. Measurements of longitudinal relaxation times (T1) in clear, but such information is essential in order to understand their antimicrobial the absence and presence of a gadolinium line broadening reagent indicate that activities. Here, by using electron paramagnetic resonance (EPR) techniques at 9 KK45 is more buried than KK36 in SDS micelles, and that both peptides are more and 95 GHz, we have characterized the membrane interaction and destabilizing buried in SDS micelles than in DPC micelles. These results suggest that the activities of an AApeptide, cyclic-g-AApeptide, on liposomes mimicking bacte- enthalpy of binding is dominated by hydrophobic interactions between the Aib rial and eukaryotic cell membranes. The analysis revealed specific interactions sidechains and the detergent molecules. These interactions are enhanced in between cyclic-g-AApeptides and negatively-charged lipid molecules. Subse- KK45, possibly because the charges are more localized to the center of the helix. quently, the AApeptide interacts strongly with the bacteria-mimic liposomes containing negatively-charged lipids, and thereby inhibits membrane fluidity. 2789-Pos Board B219 Furthermore, AApeptide binding induces significant lipid-lateral-ordering of Isomeric Model Antibiotic Peptides Differing Only in Charge Placement the bacteria-mimic liposomes, detected by EPR at 95 GHz. In addition, Adopt Different Helical Conformations AApeptide binding increases the membrane permeability of the bacteria- 1 1 2 2 Jayna Sharma , Riley Larson , Olivier Lequin , Lucie Khemte´mourian , mimic liposomes. By contrast, minimal membrane fluidity and permeability 2 3 4 1 Ludovic Carlier , Kevin Larsen , Theodore Savage , Adrienne Loh . changes were observed for liposomes mimicking eukaryotic cell membranes, 1Chemistry & Biochemistry, U. Wisconsin - La Crosse, La Crosse, WI, USA, 2 consisting of neutral lipids and cholesterol, upon AApeptide binding. The results Laboratoire des Biomole´cules, UMR 7203 UPMC-ENS-CNRS, Universite´ revealed that the intrinsic features of AApeptides are important for their ability Pierre et Marie Curie, Paris, France, 3Biophysics Program, Stanford 4 to selectively disrupt bacterial membranes, the implications of which extend to University School of Medicine, Stanford, CA, USA, Wisconsin State Lab of developing new antibacterial biomaterials. Hygiene, Madison, WI, USA. The efficacy of existing antibiotics is slowly declining as species of bacteria are 2792-Pos Board B222 evolving, increasing the need to find new antibiotics. One promising opportu- Activity of Antimicrobial Peptide Protegrin-1 is Tuned by Membrane nity lies in peptide antibiotics, which are commonly helical and cationic. Our Cholesterol Content model antibiotic peptides are composed primarily of the hydrophobic dialky- J. Michael Henderson1, Kathleen D. Cao1, Zhiliang L. Gong1, 2 1 3 1 lated amino acid Aib (a -aminoisobutyric acid), which imparts a strong 310-he- Gregory T. Tietjen , Charles T.R. Heffern , Daniel Kerr , Nishanth Iyengar , lical bias due to steric hindrance at the a-carbon. Cationic lysine residues are Indroneil Roy4, Alan J. Waring5,6, Mati Meron7, Binhua Lin7, Sushil Satija8, substituted into strategic locations in the sequence. Previous studies have re- Jaroslaw Majewski9, Ka Yee C. Lee1. ported that substitution of monoalkylated amino acids into an Aib-rich 1Chemistry, University of Chicago, Chicago, IL, USA, 2School of sequence can impact helical shape. We report here the effect of charge place- Engineering & Applied Science, Yale University, New Haven, CT, USA, ment on peptide helical structure in two solvents: DMSO (dimethyl sulfoxide), 3Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, and water. We are focusing on two octameric peptides, in which two lysine res- USA, 4Chemical Engineering, City College of New York, New York, NY, idues are placed in adjacent locations in the sequence (KK45), or one turn apart USA, 5University of California Los Angeles, Los Angeles, CA, USA, 6 7 (KK36). NMR data indicates that in DMSO, KK36 adopts a canonical 310-he- University of California Irvine, Irvine, CA, USA, Center for Advanced lical structure, while the KK45 helix is kinked. However, circular dichroism Radiation Sources, University of Chicago, Chicago, IL, USA, 8NIST Center Wednesday, February 11, 2015 551a for Neutron Research, National Institute for Standards and Technology, biotics remodel host membranes to achieve desirable activity and selectivity. Gaithersburg, MD, USA, 9Los Alamos Neutron Science Center, Los Alamos Here we study the structure-activity relationship of a series of polymer molec- National Laboratory, Los Alamos, NM, USA. ular brushes (PMBs) with well-defined nanoscale architectures that mimic The ability of antimicrobial peptides (AMPs) to target and lyse the harmful mi- spherical and rod-shaped viruses. Our preliminary data based on PMBs with crobial membrane over that of a host’s is a unique characteristic, making these hydrophilic polymer brushes reveal that: (1) amphiphilicity is not a required innate immune effectors promising candidates to fill a growing therapeutic trait - hydrophilic PMBs can be designed to have potent antibiotic performance void resulting from antibiotic drug resistance. This selectivity is believed to as well with no hemolytic side effect; (2) the nanoscale architecture of PMBs depend on the chemical and structural properties of the lipids that comprise the defines their double selectivity, not molecular weight per se; (3) PMBs are cell membrane. The selectivity of AMPs can be based on the electrostatic attrac- far more powerful antibiotics than individual linear-chain polymers that tion of these predominately cationic peptides for the bacterial membrane surface make up the PMBs; and (4) nanostructured PMBs induce topological changes heavily populated with negatively charged lipid components. We have previ- of membranes by forming membrane pores that unlikely fit in with any known ously shown with atomic force microscopy that zwitterionic dimyristoylphos- models of AMP action. These findings challenge existing wisdom and suggest phatidylcholine (DMPC) bilayers display concentration-dependent structural that the spatially-defined, multivalent interactions inherent to PMBs is of great transformations induced by protegrin-1 (PG-1) that progress from finger-like in- significance for the development of polymer-based antibiotics. stabilities at bilayer edges, to the formation of pores, and finally to a network of worm-like micelles. The increasing degree of membrane disruption in charge- 2795-Pos Board B225 neutral membranes demonstrates that a more complex interaction than that sug- Analysis of Piscidin and Lipopolysaccharide Interactions: A Step Towards gested by a simple electrostatic argument is needed to explain AMP selectivity. Characterizing Immunomodulation We propose that in addition to an electrostatic element, specific membrane Laura McCormick, Myriam Cotten. compositional differences between host and pathogen tunes AMP activity to Chemistry, Hamilton College, Clinton, NY, USA. selectively disrupt microbial membranes. We have tailored our investigations Lipopolysaccharide (LPS) is one of the most extensively studied pathogen- to utilize membrane components which eukaryotes and prokaryotes contain in associated molecular patterns (PAMPs), as it composes 90% of the membrane drastically different proportions, specifically the presence and absence of choles- in Gram-negative bacteria. Recognition of micellar aggregates of LPS by the im- terol. In these results we have employed a variety of biophysical techniques to mune system subsequently mounts an inflammatory response against infection. elucidate how increasing cholesterol content in both phospholipid monolayers However, if this process is over exaggerated, high levels of cytokines may become and bilayers attenuates the ability of PG-1 to induce membrane disruption. detrimental, leading to organ shutdown and septic shock. Various cationic antimi- Atomic force microscopy and isothermal titration calorimetry were used to assess crobial peptides (AMPs) have been shown to decrease the transcription and the propensity for peptide insertion and pore formation. X-ray and neutron reflec- release of pro-inflammatory cytokines associated with LPS recognition. tivity measurements were advantageous in providing molecular level detail on Piscidin is a cationic antimicrobial peptide first isolated in the mast cells of fish. In the location and orientation of PG-1 with respect to the membrane. the presence of a lipid bilayer, it folds into an amphipathic a-helix structure, which facilitates peptide-lipid bilayer binding. Based on the studies of other 2793-Pos Board B223 cationic AMPs, it was hypothesized that piscidin-1 (P1) and piscidin-3 (P3) What Vesicle Leakage Reveals about Antimicrobial Activity (and What It would fold into their a-helical state and bind strongly to LPS, breaking apart Doesn’t) its large aggregates. Using isothermal calorimetry to explore the thermody- Sara Hovakeemian1, Runhui Liu2, Samuel H. Gellman2, Heiko Heerklotz1. namics of binding, it was shown that both peptides bound strongly to LPS through 1UofT, Toronto, ON, Canada, 2University of Wisconsin, Madison, WI, USA. an exothermic reaction. Furthermore, fluorescence-dequenching studies were The mode of action of antimicrobial peptides and their mimics is often assessed carried out using Fluorescein isothiocyanate labeled LPS (FITC-LPS), demon- by vesicle leakage experiments, and most of these compounds are believed to act strating piscidin’s ability to separate FITC-LPS aggregates. Finally, both pep- by membrane permeabilization. This work aims at improving the interpretation tides were induced by LPS to be approximately 97% helical from their native of vesicle leakage data in general, and at understanding and optimizing the random coil state, as shown by circular dichroism. Based upon these results, it fungicidal activity of nylon-3 polymers. We have studied the membrane permea- is clear that significant interactions occur between LPS and piscidin, warranting bilizing properties of the cationic homopolymer poly-NM (Liu, R et al. JACS, future exploration of the effect of piscidin on LPS recognition by immune cells. 2013, 135, 5270), which displays significant antifungal activity, and two related cationic/hydrophobic binary copolymers using the lifetime-based leakage assay 2796-Pos Board B226 of calcein-loaded vesicles. We compared the results with biological activities Studying the Mechanisms of Hybrid Peptides Containing Permeabalizing against Candida albicans. Poly-NM induces all-or-none leakage of vesicles and Cell Penetrating Domains 1 2 that are made from yeast polar lipid extract (YPLE), at the polymer’s MIC Maria A. LaBouyer , Donald E. Elmore . 1Dept. of Biological Sciences, Wellesley College, Wellesley, MA, USA, against C. albicans (3 mg/mL). At this and higher concentrations, leakage re- 2 quires a lag time but then proceeds to 100%. Concerted activity tests imply Dept. of Chemistry, Wellesley College, Wellesley, MA, USA. that the activity of the polymer does not involve detergent-like effects. Both Antibiotic resistant pathogens cause 2 million infections and 23,000 deaths in vesicle leakage and antimicrobial activity against C. albicans spheroplasts are the U.S. every year. Bacteria’s adaptability to the current drugs on the market independent of the presence of a detergent, octyl glucoside (OG). Negligible ac- necessitates alternative therapeutics, such as antimicrobial peptides (AMPs). tivity is found against zwitterionic vesicles or red blood cells. All these charac- AMPs exhibit non-specific bactericidal activity against both gram positive teristics provide a consistent, detailed picture of membrane leakage induced by and negative bacteria, selectively killing prokaryotes over eukaryotes. These electrostatic lipid clustering. The cationic/hydrophobic binary copolymer 40:60 peptides function by two main mechanistic categories: 1) permeabilization, MM:CO shows a fundamentally different pattern. Vesicle leakage is transient where the peptide compromises the membrane causing intercellular leakage, (limited to <100%) and graded, unspecific between zwitterionic and YPLE ves- and 2) translocation, where the integrity of the membrane is maintained while icles, additive with detergent action, and correlates poorly with biological activ- the peptide enters the cell and disrupts intercellular processes. Our current ity. This activity profile suggests action by membrane asymmetry stress. We study examines the effect of combining AMPs that utilize different antimicro- conclude that comprehensive leakage experiments can identify the mode of bial mechanisms together to form a hybrid peptide. Based on previous work in action for model membrane disruption, and we hypothesize that the correlation our lab considering the mechanism of hipposin, we predicted that hybrid pep- between vesicle leakage and antimicrobial activity is good for some types of tides made from combining permeabilizing and translocating peptides would membrane leakage but not for others. generally follow a permeabilizing mechanism. In particular, we considered the activity of hybrid peptides made from parasin, a previously characterized 2794-Pos Board B224 AMP known to work via permeabilization, and DesHDAP1, a designed peptide Virus-Mimicking Polymer Molecular Brushes Are Potent Antibiotics with known to translocate through bacterial membranes. We attached DesHDAP1 to Double Selectivity both the N- and C-termini of Parasin, both with and without an alanine linker Yunjiang Jiang1, Wan Zheng1, Hairong Ma2, Hongjun Liang1. between the peptides. The activity and mechanism of these hybrid peptides was 1MT, Colorado School of Mines, Golden, CO, USA, 2Department of Physics, considered using radial diffusion assays, propidium iodide uptake assays, and Drexel University, Philadelphia, PA, USA. confocal microscopy. Confocal microscopy integrated the formation of larger Evolution of antibiotics-resisting pathogens has become one of the greatest bacterial spheroplasts and the utilization of a membrane specific dye to test challenges in the battle of bacterial infection. Inspired by the structures of for peptide-membrane co-localization. These enhancements have improved im- bacteria-invading viruses and antimicrobial peptides, we hypothesize that in age quality and aid in reliable interpretations of translocation versus membrane addition to a balance of amphiphilicity and electropositivity, the nanoscale ar- localization. Overall, the hybrid peptides did demonstrate significant permeabi- chitecture is an essential determinant that dictates how membrane-active anti- lizing activity and were at least as active as the parent peptides. 552a Wednesday, February 11, 2015

2797-Pos Board B227 j.jmb.2013.07.013). Foremost amongst these is acyl transfer from lipid constit- Binding of Daptomycin to Lipid Bilayers is not Significantly Altered by the uents of the membrane to acceptor sites on the peptide, generating lipidated Inclusion of Lysyl-Phosphatidylglycerol peptides and lyso-lipids. As an example, addition of the peptide melittin to a Tala Khatib1, Hannah Lineberry1, Heather Stevenson1, Bayer S. Arnold2, two-component lipid membrane leads to the potential formation of up to 20 Michael R. Yeaman2, Antje Pokorny1. different peptide species and 8 different lyso-lipids, many of which have 1Chemistry and Biochemistry, Univ. North Carolina Wilmington, been detected, some in significant quantities. The lipidation by-products, Wilmington, NC, USA, 2Division of Adult Infectious Diseases, University of lyso-lipids, may also serve as acyl group donors for further lipidation. This pro- California at Los Angeles Medical Center, Los Angeles, CA, USA. cess is not restricted to peptides; some proteins and low molecular weight com- In many bacterial cytoplasmic membranes, including that of Staphylococcus pounds exhibit the hallmarks of this lipidation activity. In a proof-of-principle aureus, approximately 20 mol% of the diacylphosphatidylglycerol fraction in study, we have examined the lipidation profiles of candidate proteins and the cytoplasmic membrane is esterified to the amino acid lysine. This secondary shown that they closely resemble the fatty acyl composition of the membrane modification leads to the formation of a net-cationic membrane lipid, which re- in which they are generated, supporting the hypothesis that their lipidation duces the negative surface charge of the bacterial membrane. Elevated levels of arises from membrane lipid precursors. At the other extreme, low molecular lysyl-phosphatidylglycerol (LPG) are associated with increased bacterial resis- weight compounds have been found to undergo similar lipidation processes. tance to a number of cationic antibacterial peptides, including the clinically Current work to understand the factors that dictate this reactivity, and the con- important calcium-dependent lipopeptide antibiotic daptomycin. Inclusion of sequences of lipidation, will be discussed. lysylated phospholipids in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphogly- 2800-Pos Board B230 cerol (POPG) has been shown to effectively protect lipid vesicles against pHLIPÒ Targeting and Delivery of PNA to Silence MicroRNA in Tumor leakage of contents induced by the alpha-helical antimicrobial peptides RP-1, Cellsx mastoparan X, and cecropin A. However, peptide binding to the lipid bilayer Donald M. Engelman1, Christopher J. Cheng2, Raman Bahal3, was only significantly reduced at LPG fractions exceeding 30 mol%. Here, Imran A. Babar4, Zachary Pincus5, Francisco N. Barrera6, Connie Liu7, we investigated whether binding of daptomycin to lipid vesicles containing Alexander Svoronos1, Demetrios T. Braddock8, Peter M. Glazer3, mixtures of POPG, POPC and LPG is affected in ways similar to that of W. Mark Saltzman9, Frank J. Slack7. alpha-helical peptides. We find that daptomycin binds calcium ions in aqueous 1Dept Molec Biophys/Biochem, Yale University, New Haven, CT, USA, solution with an apparent dissociation constant on the order of 10 mM. In gen- 2Departments of Molec, Cell and Devel Biol, Biomed Eng, Molec eral, binding of daptomycin to lipid bilayers depends strongly on calcium and Biophys/Biochem., Yale University, New Haven, CT, USA, 3Department of the fraction of POPG in the mixture. However, when 20 mol% of POPG are Therapeutic Radiology, Yale University, New Haven, CT, USA, replaced by LPG, daptomycin binding is only minimally affected. We also 4601 Lexington Avenue, 54th Floor, OrbiMed Advisors LLC, New York, show that daptomycin does not cross the lipid bilayer into the vesicle lumen NY, USA, 5Departments of Developmental Biology and Genetics, in giant lipid vesicles. These data suggest that clinical daptomycin resistance Washington University, Saint Louis, MO, USA, 6Dept. of Biochemistry and in S. aureus strains is caused by mechanisms beyond an LPG-mediated reduc- Cellular and Molecular Biology, University of Tennessee–Knoxville, tion in daptomycin binding. Knoxville, TN, USA, 7Dept Molec/Cell/Devel Biol, Yale University, New Haven, CT, USA, 8Department of Pathology, Yale University, New Haven, CT, USA, 9Dept biomed eng, Yale University, New Haven, CT, USA. Proteins, Lipids, and Small Molecules MicroRNAs (miRNAs) are short non-coding RNAs that, when expressed in different tissue and cell types, suppress the expression of complementary genes. 2798-Pos Board B228 Certain miRNAs, called oncomiRs, play a causal role in the onset and mainte- Phosphatidylserine, a Lipid Present at the Outer Membrane Leaflet of nance of cancer when overexpressed. Tumors that depend on these miRNAs are Cancer Cells, Hinders the Insertion of pHLIP, a Potential Cancer Cell said to display oncomiR addiction. Inhibition of oncomiRs using antisense Marker oligomers (i.e. antimiRs) is an evolving therapeutic strategy. The efficacy of Haden Scott. current antimiR technologies in vivo is, however, hindered by barriers to target- BCMB, University of Tennessee, Knoxville, TN, USA. ing and delivery into tumor cells. The pH-Low Insertion Peptide (pHLIP) has shown the ability to insert into pHLIPs are peptides that insert across cell membranes in acidic environments, membranes when acidic conditions are present. pHLIP can be found in three delivering cargoes into the cytoplasm, and releasing them by disulfide cleav- different states: a random coil in pH 8 solution devoid of lipids (state I), a sur- age. We have used a pHLIP as an antimiR delivery platform that targets the face bound form when lipids are present (state II), and a transmembrane helix in acidic tumor microenvironment, evades systemic clearance by the liver, and fa- acidic conditions (state III). From this ability of insertion based on pH, pHLIP cilitates cell entry via a nonendocytic pathway. We found that the disulfide has been shown to effectively target diseases in which the extracellular pH of attachment of peptide nucleic acid (PNA) antimiRs to the inserting end of a the tissue has become acidic. Cancer is an example of such a disease, and it has pHLIP peptide produced a construct that could transport these antimiRs across been shown that pHLIP can target cancerous cells. One concern with this tar- plasma membranes under acidic conditions such as those found in solid tumors geting is that some cancerous cells are known to display phosphatidylserine (pH ~6). This conjugate effectively inhibited the miR-155 oncomiR in vitro and (PS) in the outer leaflet of the their plasma membranes, as opposed to normal in vivo by targeting lymphoid tumors in mouse models. In a large B-cell lym- cells. However, little is known about how pHLIP’s tumor targeting is influ- phoma mouse model, silencing miR-155 reduced tumor burden, prevented enced by this increased PS content. Here, we study how pHLIP properties lymphocyte metastases, and derepressed targets of miR-155 identified using are influenced by the presence of PS by using phosphatidylcholine (PC) large RNA-seq analysis, with no apparent toxicity to the mice. This study introduces unilamellar vesicles (LUV’s) that contain PS. Circular dichroism data shows a new paradigm in the targeted delivery of polar molecules, such as antimiR that the characteristic three states are maintained in the presence of PS. Our re- PNAs, as anticancer drugs, which can have broad applicability in the field of targeted drug delivery. sults show that pHLIP has higher affinity to PC than to PS. We also found that x when LUV’s contain PS, the insertion pKa decreases compared to LUV’s only Nature, 2014, accepted. Supported by NIH grants CA133890, CA131301, containing PC with a midpoint at 0.950.4 percent. This decrease in insertion GM073857, ES005775, CA148996, HL007974, and the Yale Comprehensive pKa might limit pHLIP’s targeting of mildly acidic tumors. We added sodium Cancer Center. chloride to screen any electrostatic repulsion; however, no change in the pKa was observed. This suggests that the effect of PS on the binding and insertion 2801-Pos Board B231 of pHLIP is not merely due to electrostatic repulsion. Membrane Cholesterol Association and Structure of Two Leukotoxin Peptides 2799-Pos Board B229 Cayla M. Miller, Angela C. Brown, Jeetain Mittal. Understanding Molecular Complexity in Protein and Peptide-Lipid Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, Systems PA, USA. Hannah M. Britt, Vian S. Ismail, Aruna S. Prakash, Jackie A. Mosely, The study of membrane effects on protein structure and functioning is relevant John M. Sanderson. to understanding many native cell processes as well as the cytotoxic effects of Dept. of Chemistry, Durham University, Durham, United Kingdom. some proteins. The cholesterol recognition/interaction amino acid consensus Peptide addition to lipid membranes has been shown in some cases to lead to (CRAC) motif is a primary structure pattern that is common among many pro- significant molecular complexity, principally as a consequence of the chemical teins which interact with cholesterol, a key membrane component. However, processes that occur subsequent to the addition (http://dx.doi.org/10.1016/ the CRAC pattern alone is not sufficient to determine the cholesterol-binding Wednesday, February 11, 2015 553a ability of a protein. Of two regions which fit the CRAC criteria from a leuko- states. Indeed, the multi-state behavior of GWALP23-H12 and GWALP23- toxin LtxA produced by a pathogenic bacterium, only one is responsible for the H13 between pH 1.5 and pH 3 resembles closely that of GWALP23-R12 at cholesterol binding of the toxin. Molecular dynamics simulation of peptides neutral pH (J. Am. Chem. Soc. 132, 5803). The dramatic change in the behavior corresponding to both regions reveals that, despite the peptides sharing similar of each peptide suggests a pKa value of less than 3 to yield the neutral His imid- structural characteristics in a solution environment, this comparison is lost near azole side chain when buried in a lipid bilayer. Chemical exchange of the a cholesterol-bearing bilayer. Near such a bilayer, the cholesterol-binding C2 imidazole proton for deuterium introduces a probe which potentially sequence shows a significant loss of secondary structure upon association allows for direct observation of the His ring by solid-state 2H NMR over a range with the membrane. Furthermore, these results were not observed near a pure of conditions. Multiple His residues further alter the peptide properties, as phospholipid bilayer, indicating that this behavior is specific to cholesterol- GWALP23-H12,13 appears to aggregate in DLPC and DOPC bilayers over a containing membranes. range of pH conditions. Similar patterns are observed with GWALP23- H12,14; yet the 2H quadrupolar splittings for the b ¼ 90 and b ¼ 0 membrane 2802-Pos Board B232 orientations suggest different helix dynamics. Further aspects of the pH depen- Cholesterol Accessibility Sensing by Perfringolysin O Derivatives is dence of transmembrane helices having one or two histidine residues are under Linked to Changes in the Size of the Oligomer investigation. Benjamin B. Jonhson1, Robert J.C. Gilbert2, Alejandro P. Heuck1. 1Biochemistry and Molecular Biology, University of Massachusetts, 2805-Pos Board B235 Amherst, MA, USA, 2Division of Structural Biology, Wellcome Trust Centre Influence of Cholesterol on Single Arginine-Containing Transmembrane for Human Genetics, University of Oxford, Oxford, United Kingdom. Helical Peptides Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that Jordana K. Thibado, Ashley N. Martfeld, Denise V. Greathouse, requires cholesterol in the target cell membrane for binding. Binding of PFO to Roger E. Koeppe. membranes is modulated by cholesterol accessibility. No PFO binding occurs Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. until the accessibility of cholesterol at the membrane surface reaches a certain Polar amino acids within the helical core of a transmembrane sequence, threshold. Amino acid modifications at the tip of the PFO C-terminal domain although sparse, are essential to the structure and function of many membrane (or domain 4) changes the threshold for how much cholesterol is required to proteins. In order to examine polar residues in lipid bilayer membranes, it is trigger binding. These PFO derivatives are excellent candidates to develop useful to employ a peptide framework such as GWALP23 (acetyl-GGALWLA probes to image cholesterol in cells and to study cholesterol accessibility on LALALALALALWLAGA-amide). GWALP23 and related model peptides different cellular membranes. fold into helices and adopt defined tilted orientations that can be observed in The molecular mechanism for how cholesterol regulates PFO binding remains bilayer membranes by means of solid-state NMR spectra from particular labels. elusive. We have explored the effect of domain 4 modifications in the ability of For example, it is convenient to include specific 2H-labeled alanine residues the toxin to form arcs and rings on membranes using electron microscopy. Our within the helical core of the peptide. GWALP23 is a favorable host peptide data revealed that the amount of cholesterol in the membrane required to trigger for single residue replacements due to the limited dynamic averaging of PFO binding correlates with changes in the size and shape of the formed PFO NMR observables such as the deuterium quadrupolar splittings of the alanine oligomers. side chains. The goal of this project is to study the influence of arginine residues and pH on helix behavior in cholesterol-containing bilayers. GWALP23-R14 2803-Pos Board B233 was incorporated into DOPC bilayers with varying amounts of cholesterol Free Energies for Trans-Membrane Pore Formation in the Presence of (0-20%). Although 10% cholesterol has little effect on the orientation of Arginine-Rich Peptides from Molecular Dynamics Simulations GWALP23-R14 in DOPC bilayers, solid-state 2H NMR spectra reveal a Neha Awasthi, Jochen S. Hub. marked difference in peptide behavior when 20% cholesterol is present in Structural Molecular Biology, Georg-August University, Goettingen, the bilayers. Multiple peaks in the 2H NMR spectra of GWALP23-R14 (Ala 50% 100% Germany. d4:11 and 13 ) in DOPC bilayers with 20% cholesterol at high pH sug- Antimicrobial peptides are unique and diverse group of molecules, where the gest a multi-state behavior of the peptide. Changes in the magnitudes of the 2H amino acid composition, cationic charge and size enables them to attach and quadrupolar splittings furthermore suggest that 20% cholesterol may alter the insert into plasma membrane to form pores. These trans-membrane pores even- helix tilt even at lower pH. The results reveal a sensitivity of peptide helix prop- tually lead to the cell death of the microbe. Several mechanisms were proposed erties to cholesterol. for the activity of antimicrobial peptides, but a quantitative understanding of those mechanisms has remained elusive. Therefore, we modeled the formation 2806-Pos Board B236 of a trans-membrane pore in the presence of a cationic Arginine-rich model Influence of a Potentially Destabilizing Central Tryptophan on Transmem- peptide using coarse-grained and atomistic molecular dynamics (MD) simula- brane Helix Domains tions. Free energies for trans-membrane pore formation were calculated as a Vasu Suresh Kumar, Ashley N. Martfeld, Denise V. Greathouse, function of peptide concentration and lipid composition. We find that electro- Roger E. Koeppe. statics and presence of counter-ions; esp. for the case of charged lipids and pep- Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. tides, play a critical role in free energy calculations. We address the following Synthetic model peptides, such as GWALP23 (acetyl-GGALW5LALALALA questions: a) does insertion of a peptide or several of them, influence the free LALALW19LAGA-amide), provide a favorable ‘‘host’’ framework for inves- energy of pore-formation; and b) is pore-formation a required step, or is the tigations of the influence of chosen ‘‘guest’’ amino acids. For example, it is of increased disorder of the lipids sufficient to insert a peptide? Finally, we present interest to know the consequences of having a third, centrally located, trypto- a quantitative comparison between the free energy of trans-membrane pore in phan (Trp) within the hydrophobic core of a well characterized, anchored, the absence and presence of N-Arginine peptides, with the aim to provide a transmembrane helix. It is crucial to note that the orientation and rotation quantitative description of the activity of antimicrobial peptides on lipid of GWALP23 are sensitive to single-residue replacements, in part because membranes. the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. 2804-Pos Board B234 J. 101, 2939). A Trp residue was introduced in the 12th or 13th position of Response of GWALP Transmembrane Peptides to Incorporation of GWALP23, and specific deuterated alanine labels (2H-Ala) were included Buried Histidine Residues as probes within the core helical sequence. The 2H quadrupolar splittings Ashley N. Martfeld, Denise V. Greathouse, Roger E. Koeppe. from solid-state NMR spectra of GWALP23-W12 and GWALP23-W13 Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. show that the peptide remains helical and retains a dominant preferred tilted To investigate histidine residue replacements in lipid bilayer membranes, we transmembrane orientation (similar to GWALP23) in lipid bilayer mem- have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA- branes of DOPC, DLPC, and DMPC. Modified Gaussian and semi-static amide) as a favorable host peptide framework. We inserted His residues into treatments of the dynamics yield similar conclusions. While a central Trp position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorpo- at position 12 or 13 does not alter the characteristics of bilayer-spanning rated specific 2H-Ala labels within the helical core sequence. Solid-state 2H GWALP23, incorporation of the peptide helix into the bilayer membrane NMR spectra of GWALP23-H12 reveal a marked difference in peptide behavior becomes more difficult. The properties of W4, 5 GWALP23 are also being between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 investigated, for comparison with the highly dynamic Y4, 5 and the less dy- and GWALP23-H13 exhibit well-defined tilted transmembrane orientations namic F4, 5 peptides. Deuterium labels at Ala3 and Ala21 will allow assess- in both DOPC and DLPC bilayer membranes. Under acidic conditions ment of possible fraying of the ends of selected helices in differing lipid GWALP23-H12 and GWALP23-H13 are highly dynamic and exhibit multiple membrane environments. 554a Wednesday, February 11, 2015

2807-Pos Board B237 Peptide ...... Sequence 15 Ionization-Dependent Behavior of Transmembrane Helices that Incorpo- RRWWALP15 . . . . acetyl-GRRWWLALALWWRRA -NH2 13 rate Glu or Tyr Residues RRWWALP13 . . . . acetyl-RRWWLALALWWRR -NH2 11 Venkatesan Rajagopalan, Denise V. Greathouse, Roger E. Koeppe. RWALP11 ...... acetyl-GRWLALALWRA -NH2 9 Chemistry and Biochemistry, University of Arkansas, FAYETTEVILLE, RWALP9 ...... RWLALALWR -NH2 AR, USA. Our results indicate that despite having the same core length, the peptides GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) is a exhibit different conformations, different interactions with lipids, and different constructive low-dynamic model peptide for investigations of single-residue in- antimicrobial activities. While the 13-mer is a-helical in neutral and anionic fluence on protein-lipid interactions and the properties of membrane-spanning lipid mixtures, the conformations of the shorter 11-mer and 9-mer vary consid- helices (J. Biol. Chem. 285, 31723). To investigate the pH dependence, ioniza- erably depending on the lipid environment. Fluorescence spectroscopy sug- tion behavior and orientational constraints when potentially negatively charged gests that the tryptophan residues of all peptides are located at the glutamic acid side chains are present, we have substituted a single Leu residue membrane-water interface, with slightly varying depths of insertion into the with Glu at different positions and incorporated specific 2H-Ala labels in the lipid bilayer. Despite small perturbations to the phospholipid head groups, core GWALP23 or Y5GWALP23. Solid state NMR experiments show well solid-state 31P NMR spectra indicate that the lipids remain primarily in a defined 2H-Ala quadrupolar splittings for Y5GWALP23-E14 over the pH range bilayer phase. 2H NMR spectra reveal that the two shorter peptides aggregate, of 6 to 10, suggesting that the peptide helix is well oriented in DLPC and DOPC while the two longer (and more helical) peptides align in neutral and anionic lipid bilayers. Spectral changes are evident above pH 11 (in ether-lipid bila- lipid mixtures. Although all four peptides exhibit similar antimicrobial activ- yers), but the titration of the Glu is uncertain because the Tyr residue itself ities, some variation is observed against gram negative and gram positive bac- appears to titrate around pH 11.5. GWALP23-E14 shows no spectral changes terial strains. over the pH range 6.0 to 11.5, yet a change in quadrupolar splittings is observed at pH 13. The combined results suggest that the Glu residue may titrate with a 2810-Pos Board B240 Characterization of Maximin 3 Structure and Membrane Leakage pKa near 12. In bilayers of DLPC and DOPC, GWALP23-E16 shows similar Brian Herbst, Jillian Glatz, Elizabeth Middleton. trend and is suggested to have a pKa of around 12. The rather modest shifts in the 2H quadrupolar splittings, nevertheless, suggest that the orientation of Chemistry and Biochemistry, Purchase College, State University of New the transmembrane helix actually may change rather little at high pH. It is York, Purchase, NY, USA. conceivable that the close proximity of either E14 or E16 to W19 could provide Maximin 3 is a 27 amino acid cationic antimicrobial peptide that is derived stability to the neutral peptide helix and perhaps influence the results. We addi- from the skin secretions of the toad Bombina maxima. Maximin 3 has strong tionally are investigating the possibilities for helix unwinding near the ends of activity against a variety of bacteria (Gram-positive and Gram-negative), fungi, these peptides. and viruses and is thought to cause toxicity by interaction with the plasma membrane. We previously quantified the binding affinity of Maximin 3 for lipid 2808-Pos Board B238 vesicles that mimic both bacterial and mammalian membranes and found that Detection of Helix Fraying in Designed Transmembrane Alpha Helices the peptide interacts more strongly with bacterial than mammalian models, in Armin Mortazavi, Venkatesan Rajagopalan, Denise V. Greathouse, part due to the negative charge of these membranes. Roger E. Koeppe. In our current work we used Fo¨rster resonance energy transfer (FRET) and fluo- Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. rescence leakage assays to further characterize this interaction. Specifically, we Transmembrane helices of integral membrane proteins often have aromatic res- found that the end-to-end distance of the peptide as determined by FRET is a idues flanking them. The aromatic residues favor locations within the mem- consistent with an extended -helical conformation, both in solution and brane–water interface of a lipid bilayer and may serve as anchors to aid the when bound to bacterial and mammalian model membranes. Under the same stabilization of a tilted transmembrane orientation. To further understand experimental conditions, we found that Maximin 3 is capable of inducing the influence of Tyr, Trp or Phe residues upon the properties of helical mem- leakage of rhodamine from vesicles that mimic bacterial membranes. These re- brane proteins (see Biochemistry 53, 3637), we have investigated the possibil- sults are consistent with the hypothesis that the mechanism of Maximin 3 ity of partial unwinding near the ends of selected transmembrane helices. For toxicity towards bacteria is due to a direct interaction between Maximin 3 this purpose, we substituted positions 4 and 5 with either two Phe or two Ala and the outer or inner bacterial membrane. We further suggest that Maximin 4,5 4 5 19 3 adopts an extended a-helical conformation in the leakage-inducing state, residues to generate F GWALP23 (acetyl-GGAF F (LA)6LW LAGA-etha- 4,5 4 5 19 though it is not yet clear if the peptide is monomeric or oligomeric under these nolamide) or A GWALP23 (acetyl-GGAA A (LA)6LW LAGA-ethanola- mide), respectively. By incorporating specific 2H-Ala labels at positions 3 conditions. and 21, we are able to compare the influence of interfacial Phe and Ala on 2811-Pos Board B241 the unwinding of the helix ends. Solid state NMR spectra of macroscopically Characterization of Membrane Interactions of Antimicrobial Lactoferri- aligned bilayer samples show well defined 2H-Ala quadrupolar splittings for 4,5 4,5 cin Peptides with Central Residue Substitutions both F GWALP23 and A GWALP23, suggesting that the peptide helices Amanda Lowe, Denise V. Greathouse. are well oriented in DLPC, DMPC and DOPC lipid bilayers. We are also University of Arkansas, Fayetteville, AR, USA. able to estimate the helix tilt from deuterium labeling of the core alanine resi- The rise in antibiotic-resistant bacteria has led to an active search for new and dues. Geometric Analysis of Labeled Alanines (GALA) then shows unwinding 4,5 4,5 more effective antimicrobial drugs. A hexapeptide (LfB6: RRWQWR-NH2) at the terminals for both F GWALP23 and A GWALP23. It is conceivable derived from the iron-binding protein lactoferrin exhibits antimicrobial activity that the helix fraying may be critical for the stability of the transmembrane (Tomita, Acta Paediatr Jpn, 1994, 36:585). A related heptapeptide produced in helix orientation in the lipid bilayer membranes. our lab, with 4 positively charged arginines and 2 methylated tryptophans (RRMeWQMeWRR-NH2; MeTrp-LfB7), exhibits enhanced activity against 2809-Pos Board B239 gram negative and gram positive bacteria. Substitutions of the central gluta- Comparing Peptide-Lipid Interactions and Antimicrobial Activities of mine (Gln4;Q) residue that may alter peptide conformational flexibility are Peptides with Similar ‘‘Core’’ Lengths But Variable Arginine and Trypto- now being investigated. When Gln4 was changed to glycine (Gly;G) or proline phan Residues (Pro;P), significant changes in peptide-membrane interactions were observed, Sara E. Whitlock, Roger E. Koeppe II, Denise A. Greathouse. although the antimicrobial activity was not increased. We now examine the Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. effects of replacing Gln4 with gamma amino butyric acid (GABA), to introduce Antimicrobial peptides offer a potential alternative therapy option for the more flexibility; or D-Pro-Gly, to constrain the backbone into a b-hairpin turn increasing problem of microbial drug resistance. To address this issue, ‘‘hybrid’’ (Stanger and Gellman, J. Am. Chem. Soc. 1998, 120:4236). The increased peptides with total lengths intermediate between the surface-acting antimicro- positive ellipticity at ~230 nm observed in the CD spectrum of the D-Pro- bial lactoferrin-derived LfB6 (RRWQWR-NH2) and the inert model transmem- Gly peptide in anionic membranes suggests significant Trp-Trp interactions. brane a-helical peptide RWALP23 (acetyl-GRALW(LA)6LWLARA-NH2) Tryptophan fluorescence emission spectra indicate that peptides with either have been designed with varying numbers of arginines, tryptophans and net GABA or D-Pro-Gly substitutions are more deeply buried in anionic mem- positive charge, but with the same ‘‘core’’ length of five residues. The branes. Although the GABA and D-Pro-Gly peptides were both more active lipid interactions of the hybrid LfB-RWALP peptides having between 9 and against gram negative (E. coli), compared to gram positive (S. aureus) bacteria, 15 residues were investigated by circular dichroism, tryptophan fluorescence, they were not as active as the Gln4 peptide. Investigations of the peptide-lipid and 31P and 2H solid-state NMR spectroscopy, and their antimicrobial activities interactions are being continued by means of solid-state 2H and 31P NMR assayed. spectra. Wednesday, February 11, 2015 555a

2812-Pos Board B242 the antiviral potency and extension of the in vivo half-life of two new HIV Promotion of Coalescence in Bicellar Mixtures by an SP-B Fragment fusion inhibitors: HIVP3 (C34-PEG4-cholesterol) and HIVP4 ([C34-PEG4]2- Chris Miranda, Valerie Booth, Michael R. Morrow. cholesterol). Given the importance of lipophilicity and selective affinity for Physics and Physical Oceanography, Memorial University of Newfoundland, different lipid domains, the aim was to evaluate the interaction of these St. John’s, NL, Canada. HIV fusion inhibitor peptides with biomembranes model systems and human Maintenance of a functional surfactant layer requires the transfer of material blood cells, in order to clarify where and how they are located. This study between bilayer reservoirs and the surface-active layer. It is believed that allowed the understanding of the mechanism of action of these peptides at SP-B facilitates the interlayer contact and mixing implicit in such activity. the molecular level, and which strategies may be followed to increase their SP-B’s role in promoting these contacts was investigated though the use of bi- fusion inhibition efficacy. Membrane partition, dipole potential and surface layered micelle mixtures containing short- and long-chain lipids. Upon warm- pressure measurements indicated that HIVP3 and HIVP4 interact preferen- ing, these bicellar mixtures progressively coalesce into more extended tially with cholesterol-rich liquid ordered membranes. HIVP3 and HIVP4 structures and are thus an interesting system in which to study the capacity are able to interact with human erythrocytes and peripheral blood mononu- of polypeptides to promote interactions between lipid structures. 2H NMR clear cells to a similar degree as previously described for the simpler drug was used to identify the capacity of perturbation by an SP-B fragment (SP- C34-cholesterol. However, the pocket binding domain(PBD) of both HIVP3 B63-78) upon bicellar mixtures of DMPC-d54/DMPG/DHPC and DMPC-d54/ and HIVP4 is more exposed to the aqueous environment than in C34- DMPC/DHPC in nominal 3:1:1 molar ratios. In the presence of mixtures con- cholesterol. The efficient blocking of HIV entry results from the synergic taining anionic lipids (DMPC-d54/DMPG/DHPC), SP-B63-78 (concentration effect between the membranotropic behavior and the enhanced exposure of approximately 10% of lipid weight) was found to lower the temperature at the PBD[1]. Maximizing antiviral activity requires finding the proper balance which coalescence to extended lamellar structures occurred. Conversely, of membrane affinity and exposure of the peptide moiety, through variations when anionic lipids were replaced with zwitterionic lipids (DMPC-d54/ in the lipid-binding domain, PEG spacer region, and number of peptide moi- DMPC/DHPC), SP-B63-78 did not perturb the temperature at which the transi- eties in the construct. tion to extended lamellar structures occurred. These results indicate that the [1] Augusto MT et al.(2014)Improvement of the HIV fusion inhibitor C34 interaction of SP-B63-78 with model membranes is dependent upon the presence efficacy by membrane anchoring and enhanced exposure.J Antimicrob Chemo- of anionic lipids and further suggest a mechanism by which full length SP-B ther,69:1286-1297 may interact with membranes. 2815-Pos Board B245 2813-Pos Board B243 Strength, Not Depth: An Exploration of Differential Membrane Binding Lipid Composition Influences the Insertion and Folding of pHLIP Peptides Kinetics of Synaptotagmin-1 and Synaptotagmin-7 C2 Domains Alexander G. Karabadzhak1, Dhammika Weerakkody2, Oleg A. Andreev2, Joshua V. Vermaas, Emad Tajkhorshid. Yana K. Reshetnyak2, Donald M. Engelman1. University of Illinois at Urbana-Champaign, Urbana, IL, USA. 1MB&B, Yale University, New Haven, CT, USA, 2Physics, University of Synaptotagmins (Syt) play an essential role in mediating Ca2þ-dependent Rhode Island, Kingston, RI, USA. vesicle fusion, and are believed to function by binding its Ca2þ-sensing C2 The study of polypeptide insertion into biological membranes can inform our domains to anionic phospholipids and thereby bring the vesicle near the understanding of membrane protein stability and folding, and has potential membrane into which the vesicle will fuse. Many Syt isoforms exist, and practical applications. This has been hard to investigate, since peptides hydro- despite their close structural similarity, the binding kinetics of each isoform phobic enough to form transmembrane helices are likely to be insoluble in to membranes can vary widely. We have performed an extensive set of aqueous buffers, and soluble peptides are unlikely to insert. pH-Low Insertion molecular dynamics simulations on Syt-1 and Syt-7 C2 domains to charac- Peptides (pHLIPs) provide an opportunity to study insertion since they are sol- terize the factors that determine the rapid unbinding for Syt-1 and the slow uble monomers that bind to bilayer surfaces at neutral pH, and can be triggered unbinding of Syt-7. Through repeated atomistic binding simulations of to form monomeric transmembrane helices in acidic conditions. Syt-1 and Syt-7 C2 domains to anionic membranes, enabled by our enhanced This set of unusual properties allows the study of spontaneous insertion and exit dynamics membrane mimetic model (HMMM), we found that while the of a transmembrane polypeptide by changing the pH of its microenvironment. penetration depth of the different loop regions are unchanged, Syt-7 C2 do- Previous studies showed that pHLIP inserts into a POPC liposomes through mains form many more contacts with anionic head groups. These additional rapid formation of an interfacial helix (~ 0.1s), followed by a slow insertion contacts both strengthen and lengthen duration of the interaction between pathway. The time-course of pHLIP insertion can be changed by varying the Syt-7 and the membrane, including contacts between loop 2 of Syt. These number of protonatable groups at the inserting end of the peptide, which results provides the first atomistic picture of differential binding of different need to be moved across the bilayer. isoforms of Syt. We have employed various biophysical methods: fluorescence spectroscopy, anisotropy, CD, OCD, and stopped-flow fluorescence, to study the pHLIP inser- 2816-Pos Board B246 tion into bilayers composed of different monounsaturated lipids (diC(14:1)PC, How Synaptotagmin I, N-BAR and F-BAR Domains Generate Membrane diC(16:1)PC, diC(18:1)PC, diC(20:1)PC, and diC(22:1)PC). We found that Curvature pHLIP can form a TM helix in bilayers of different thickness. The kinetics Zhe Wu, Hang Yu, Anton Arkhipov, Ying Yin, Klaus Schulten. of pHLIP insertion vary, and correlate with bilayer thickness and fluidity. University of Illinois Urbana Champaign, Urbana, IL, USA. Further, pHLIP association with bilayer surfaces also depends on the lipid Protein-induced membrane curving governs many cellular processes, including composition. The activation energy of pHLIP insertion increases with the cell division, growth and cell-cell communication. In order to unravel the membrane thickness but the process of inserted helix formation does not signif- mechanism for how membrane curvature occurs driven by the proteins, one icantly depend the membrane type. A model for membrane-associated inser- needs to have detailed molecular pictures on the key membrane-protein inter- tion/folding is discussed. This work was supported by grants from National actions and protein-protein interactions. Computer simulation serves as a great Institute of Health, GM073857, and CA133890. tool in providing such molecular pictures and thus in rationalizing how proteins curve membrane. For example, it is known that neurotransmitter release in- volves a Caþþ ion-regulated fusion process between synaptic vesicles and Protein-Lipid Interactions III the presynaptic membrane, but little is known for how such small ions function. With the help of computer simulation, we realized the Caþþ-sensor protein, 2814-Pos Board B244 synaptotagmin I, undergoes a conformational transition after binding to both Enhanced HIV Fusion Inhibitors Efficacy Requires Membrane Affinity the Caþþ and membrane that differs from the membrane-free crystal/NMR and Exposure of the Pocket Binding Domain of C34 Derivatives structure. The new synaptotagmin conformation has its C-terminal helix to Marcelo T. Augusto1, Axel Hollmann1, Miguel A.R.B. Castanho1, interact with the presynaptic membrane, and thereby, causes the presynaptic Matteo Porotto2, Antonello Pessi3, Nuno C. Santos1. membrane to curve and facilitates membrane-vesicle fusion. Another example 1Instituto de Medicina Molecular, Lisbon, Portugal, 2Departments of in showing the power of computer simulation lies in resolving how N-BAR and Pediatrics and of Microbiology and Immunology, Weill Medical College of F-BAR domains sculpt a flat membrane. As these domains form a lattice in the Cornell University, New York, NY, USA, 3PeptiPharma, Rome, Italy. sculpting process, we identified the optimal lattice type and key protein-protein Viral fusion inhibitors block the fusion between the membranes of an envel- interactions within the lattice. The whole process of membrane tubulation from oped virus and a target cell, therefore preventing the entry of the viral content. a flat membrane was resolved from the simulations, and agreements were found It has been demonstrated that the combination of cholesterol-tagging and between the lattice structures observed via cryo-electron microscopy and the dimerization of the C34 peptide sequence resulted both in an increase of simulations. 556a Wednesday, February 11, 2015

2817-Pos Board B247 ing reactions. The results from the EAM analysis are in agreement with the Reconstitution and Characterization of Na/K-ATPase in Model Lipid flexible surface model (FSM), which describes elastic coupling of the mem- Membranes brane lipids to integral membrane proteins through a balance of curvature Tripta Bhatia. and hydrophobic forces in lipid-protein interactions [4]. Our study also pro- FKF, Memphys, University of Southern Denmark, Odense M, Denmark. vides insight into the thermodynamic parameters that govern rhodopsin-like We have mapped the morphology and orientation of the Na/K ATPase inside GPCR activation in native membrane lipid environments. [1] A.V. Struts model membranes and made a detailed quantifications of the membrane lateral et al. (2011) PNAS 108, 8263-8268. [2] A.V. Struts et al. (2014) Meth. Mol. structure. Further work to characterize the lateral organization and function of Biol. (in press). [3] E. Zaitseva et al. (2010) JACS 132, 4815-4821. [4] M.F. the pump in membranes and vice-versa, is in progress. Brown (2012) Biochemistry 51, 9782-9795. 2818-Pos Board B248 2820-Pos Board B250 Detergent-Free Extraction of the Reaction Center from Rhodobacter Inside the Cell under Oxidative Stress: Protein Assembly at Mitochondrial sphaeroides into Native Nanodiscs. Nanodisc Size Matters! Membranes and its Consequences Stefan Scheidelaar1, David Swainsbury2, Martijn Koorengevel1, Martin Lidman, Artur Dingeldein, Ilona Dudka, Farhana Nasrin, Hans Meeldijk3, Eefjan Breukink1, Rienk van Grondelle4, Michael Jones5, Gerhard Gro¨bner. J. Antoinette Killian1. Chemistry, Biophysical Chemistry, Umea˚, Sweden. 1Membrane Biochemistry & Biophysics, Utrecht University, Utrecht, Intracellular oxidative stress is one major factor leading to apoptosis accompa- Netherlands, 2School of Biochemistry, Bristol University, Bristol, United nied by the permeabilization of the mitochondrial outer membrane; a process Kingdom, 3Electron Microscopy Utrecht, Utrecht University, Utrecht, causing release of apoptotic factors such as cytochrome c. Upon onset of intra- Netherlands, 4Division of Physics and Astronomy, Department of cellular stress phospholipids can become oxidized. These oxidized phospho- Biophysics, VU University Amsterdam, Amsterdam, Netherlands, 5School of lipids (OxPls) can severely alter the properties of these mitochondrial Biochemistry, Bristol University, Bristol, United Kingdom. membranes, and can therefore have i) a direct effect on the membrane properties It has been discovered that styrene-maleic acid (SMA) copolymers are able to and its perforation, ii) can change aggregation behavior of amyloidogenic pro- solubilize membrane proteins directly from their native membrane in the form teins in contact with these membranes iii) can have an indirect effect by altering of nanodiscs [1]. Using the SMA technology, we purified and characterized re- the function of mitochondrial membrane-coupled Bcl-2 proteins (such as the action centers (RCs) from the purple bacterium Rhodobacter sphaeroides [2]. anti-apoptotic Bcl-2 or the apoptotic Bax), with Bcl-2 potentially involved in Our most significant findings were (i) that the SMA copolymer efficiently sol- SOD1 mediated toxicity [A]. We therefore devised a model system that mimics ubilizes membranes of this bacterium, (ii) that it allows preservation of the oxidative stress conditions by incorporating oxidized phospholipids (OxPls) into local lipid environment of the solubilized RCs, (iii) that the protein is even mitochondria-like liposomes, and studied the OxPls’ impact on basic lipid mem- more stable in these ‘‘native nanodiscs’’ than in the native membrane, and brane systems and subsequently on protein-membrane interplays. In addition we (iv) that the size of the RC containing nanodiscs is significantly larger than also carried out a lipidomics profiling of cancer tissues overexpressing Bcl-2 that of protein-free nanodiscs. protein to provide information about apoptosis-preventing lipid patterns. We next investigated what physical properties of the polymer and lipids deter- mine the efficiency and kinetics of solubilization and how they affect the size of 2821-Pos Board B251 the nanodiscs. In particular, we tested the effect of SMA copolymer length, hy- Elucidation of a Raft-Partitioning Motif in Transmembrane Proteins drophobicity, and charge state (varying pH) using different lipid compositions. Joseph H. Lorent, Blanca B. Diaz-Rohrer, Kevin J. Spring, Ilya Levental. In complementary assays, we determined how the cross-sectional diameter of a Integrative Biology and Pharmacology, UT Health Science Center at protein affects the size of a nanodisc by using purified constructs of covalently Houston, Houston, TX, USA. linked oligomeric RCs of different size. Eukaryotic plasma membranes (PMs) are believed to possess lateral lipid do- Our study contributes to the fundamental knowledge about the mode of action mains termed lipid rafts, which form functional platforms for membrane sorting of SMA and thereby to the general applicability of native nanodiscs as host for and cell signalling. The function of these domains depends on their selective membrane proteins and protein complexes of different size. recruitment of specific membrane proteins. However the underlying structural 1. Knowles et al., 2009, JACS, 131, 7484-7485 features governing protein partition into lipid rafts remain unknown. In live 2. Swainsbury & Scheidelaar et al. 2014, Angewandte Chemie, 53, http:// cells, the nanoscopic size and subsecond lifetime of rafts presents great difficulty dx.doi.org/10.1002/anie.201406412 for measuring their properties and composition. Intact PMs isolated as Giant Plasma Membrane Vesicles (GPMVs) phase separate into two microscopic, sta- 2819-Pos Board B249 ble lipid domains, with one of these domains possessing greater lipid order, Membrane-Lipid Mediated Rhodopsin Signaling Involves an Ensemble of lower diffusivity and enriching for canonical raft components. This microscopic Conformational Substates raft phase separation thus presents an optimal system for measuring protein par- Udeep Chawla1, Blake Mertz2, Eglof Ritter3, Franz Bartl4, titioning between coexisting membrane domains. Using GPMVs, we demon- Michael F. Brown1,5. strate that a protein’s transmembrane domain (TMD) is a central determinant 1Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, of raft affinity. A major factor governing raft partition was the length of the 2Chemistry, West Virginia University, Morangtown, WV, USA, TMD, with longer TMDs preferring the thicker raft domains. Further, by system- 3Experimentelle Biophysik, Humboldt-Universita¨t zu Berlin, Berlin, atic mutation of the TMD of LAT (Linker for activation of T-cells), we were able Germany, 4Charite´-Universita¨tsmedizin, Institut fu¨r Medizinische Physik und to determine amino acid sequences which are necessary and sufficient for raft Biophysik, Berlin, Germany, 5Department of Physics, University of Arizona, partitioning and which highly resemble the a-helix oligomerization motif Tucson, AZ, USA. GxxxG. This resemblance suggested that TMD oligomerization is the structural G-protein-coupled receptors (GPCRs) constitute about 50% of the known drug basis for the discovered raft-partitioning motif. We confirmed this hypothesis by targets. Using rhodopsin as a model GPCR, we study the role of membrane measure sequence-dependent TMD oligomerization by fluorescence lifetime lipids in the rhodopsin activation. Absorption of light by rhodopsin leads to a microscopy (FLIM) combined with Fo¨rster resonance energy transfer (FRET). series of conformational changes that establishes an equilibrium between inac- Finally, we demonstrate the biological significance of our observations by tive Meta-I and an ensemble of activated Meta-II states [1,2]. Using UV-visible showing that raft partitioning determines sorting between organellar mem- and FTIR spectroscopy, we show that the distribution of late conformational branes. For the entire panel of TMD-variants, we observed a strong quantitative substates during rhodopsin activation is lipid-mediated as explained by an relationship between raft association and sorting to the plasma membrane, with ensemble activation mechanism (EAM). Lipid bilayer composition (head non-raft mutants being sorted into the lysosomes for degradation. groups and acyl chain lengths) and membrane protein-lipid interaction govern the EAM through biasing conformational substates. Rhodopsin recon- 2822-Pos Board B252 stituted in DOPC backshifts the equilibrium to the Meta-IIa substate, whereas The Mechanism of the Disintegration of Phospholipid Membranes with mixed-chain POPC membranes favor the inactive Meta-I state [3]. The Human Monoacylglycerol Lipase (hMGL) wavenumber-dependent analysis of the FTIR-difference spectra yields a Vitalii I. Silin1, Ioannis Karageorgos2, David J. Vanderah1, Nikolai Zvonok3, 2 4 distribution of pKa and alkaline endpoint values, consistent with an ensemble John Marino , Alexandros Makriyannis . of substates for each lipid bilayer-rhodopsin system. A phenomenological 1IBBR_SG, University of Maryland, Rockville, MD, USA, 2NIST, Henderson-Hasselbalch function was fitted to the pH titration curves to derive Gaithersburg, MD, USA, 3Northeastern University, Boston, MA, USA, thermodynamic parameters from the spectral analysis. Our thermodynamic 4Center for Drug Discovery, Northeastern University, Boston, MA, USA. studies show that activation of rhodopsin is accompanied by an entropy gain In human cancer cells, hMGL is the principal regulator of fatty acids, con- compensating the unfavorable enthalpy increase, analogous to protein unfold- trolling the hydrolysis of cellular lipids and the production of free fatty Wednesday, February 11, 2015 557a acid-derived oncogenic lipid messengers that promote cancer cell migration characterized the 3D structure, activity, and behavior of NA-CATH, a 34 amino and invasiveness. hMGL is a membrane proximal protein that belongs to the acid CAMP from the Naja Atra snake. NMR studies reveal a single straight serine hydrolase family involved in the endocannabinoid signaling system amphipathic a-helix with a disordered tail. Using one and two phase liposomes, (ECBSS). As a major part of the ECBSS that is composed of two main cannabi- we have found that lipid composition and the presence of phase separation noid receptors CB1 and CB2 and endogenous ligands, hMGL exclusively hydro- affect the activity and behavior of NA-CATH. Fluorescence leakage and re- lyzes 2-arachidonoylglycerol (2-AG), making it a particularly critical modulator quenching assays indicate that the presence of phase separation increases for the endocannabinoid transmission. hMGL also plays important roles in the leakage activity and the anionic lipid locale affects the leakage mechanism. metabolism of lipid storage. The key roles of hMGL in tuning homeostatic ECBSS and in supporting aggressive tumorogenesis make this protein a prom- 2825-Pos Board B255 ising therapeutic target for treating cancer as well as managing pain and treating A Combined Effect of Proteins SP-B and SP-C and Membrane Curvature inflammatory, neurodegenerative, and immunological disorders. on Cholesterol Partition in Lung Surfactant Membranes: Answers from Using a custom built device that allows simultaneous surface plasmon reso- Fluorescence 1 2 2 1 nance (SPR) and electrochemical impedance spectroscopy (EIS) measurements Nuria Roldan , Thomas K.M. Nyholm , Peter Slotte , Jesus Perez-Gil , 1 in real-time, we report for the first time, that hMGL introduces significant de- Begon˜a Garcia Alvarez . 1Biochemistry and Molecular Biology I, Faculty of Biological Science. fects into pre-formed, intact, tethered phospholipid bilayer membranes 2 (tBLMs) composed of phosphatidylcholines and other lipids. To probe the Complutense University of Madrid, Madrid, Spain, Department of ˚ mechanism of hMGL’s ‘destructive activity’ to tBLMs we investigated wild- Biosciences/Biochemistry, Abo Akademi University, Artillerigatan, Finland. type and mutant hMGLs with respect to concentration, inhibitors, and pH. Cholesterol is the major neutral lipid present in lung surfactant, a lipid-protein Our data shows that MGL’s lid domain, lytic activity center, and hydrophobic complex responsible for the stabilization of the air-liquid interface in mamma- pocket all play important roles in this process. lian lungs, which is essential for the breathing function. It has been proposed This work supported by NIDA grant DA-3801 that cholesterol content is tightly regulated in surfactant and that protein SP-C could have a role in this respect. To further analyse the affinity of surfac- 2823-Pos Board B253 tant membranes for cholesterol in the absence or presence of proteins, we have Effect of Lipid Composition on the Affinity and Binding of Dimeric used cyclodextrin as a cholesterol solubilising agent and a fluorescent analog of Tubulin to Membranes Studied using Surface Plasmon Resonance, cholesterol, cholestatrienol (CTL), whose partition can be followed in mem- Neutron Reflectivity, Electrophysiology, and AC Electrical Methods brane vesicles with different lipid/protein compositions and sizes. Our results David P. Hoogerheide1,2, Oscar Teijido Hermida2, Tatiana K. Rostovtseva2, suggest that there is a direct effect of vesicle size/curvature on the affinity of Sergey M. Bezrukov2, Hirsh Nanda1,3. membranes for CTL. This is translated into lower partition coefficients (Kx) 1Center for Neutron Research, National Institute of Standards and for membranes with higher curvatures, possibly caused by an increased expo- Technology, Gaithersburg, MD, USA, 2Eunice Kennedy Shriver National sure of CTL in more curved bilayers. Surfactant hydrophobic proteins SP-B Institute of Child Health and Human Development, National Institutes of and SP-C were found to have a curvature-independent effect on CTL mobility, Health, Bethesda, MD, USA, 3Physics Department, Carnegie Mellon as well as in the mobility of other probes such as transparinaric acid- University, Pittsburgh, PA, USA. sphingomyelin (tPA-SM) or diphenylhexatriene-phosphatidylcholine (DPH- Dimeric tubulin has emerged as an important regulatory factor of the perme- PC) in membranes of the same composition. Furthermore, in the absence of ability of voltage-dependent anion channel (VDAC) in the mitochondrial outer SP-B, SP-C-containing liposomes were found to spontaneously split into 30 membrane, with implications for mitochondrial energetics as well as the War- nm vesicles increasing membrane curvature regardless lipid composition, burg effect observed in cancers. Previously, single-channel studies revealed which correlated with a reduction of Kx compared to pure lipid systems. Taken that the on-rate of the VDAC-tubulin interaction is strongly dependent on the all together, our results indicate that curvature alters cholesterol accessibility in lipid environment. To understand the nature of the binding of this abundant surfactant lipid bilayers and that lung surfactant proteins disturb membranes in cytosolic water-soluble protein to lipid membranes, we have employed an array cholesterol containing domains, which may have important physiological con- of biophysical techniques using unsupported planar lipid membrane and teth- sequences for cholesterol mobilization in lung surfactant membranes and films. ered bilayer lipid membrane (tBLM) platforms. Surface plasmon resonance (SPR) of tBLMs shows that tubulin at concentrations less than 100 nM binds 2826-Pos Board B256 irreversibly to DPhPC and 1:1 DOPC:DOPE membranes. The binding rate is pH-Induced Reorganization and Membrane Insertion of the Diphtheria significantly larger in high salt concentrations, suggesting a hydrophobic inter- Toxin T-Domain Studied by Spr and Neutron Reflectometry 1 1,2 1,2 3 action between tubulin and lipid membrane. No binding to DOPC membranes Rebecca Eells , Frank Heinirch , Mathias Lo¨sche , Mykola V. Rodnin , Alexey S. Ladokhin3. is observed under the same conditions. Electrochemical impedance spectros- 1 copy (EIS) of tBLMs reveals that only DPhPC membranes are strongly per- Department of Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2National Institute of Standards and Technology, Center for Neutron turbed by the binding of tubulin. Neutron reflectivity (NR) measurements on 3 tBLMs give structural information regarding the penetration of the tubulin Research, Gaithersburg, MD, USA, Department of Biochemistry and into the membranes. Electrophysiological measurements of the tubulin/ Molecular Biology, KUMC, Kansas City, KS, USA. VDAC interaction on the unsupported bilayers confirm the irreversibility of Diphtheria toxin (DT) is secreted by Corynebacterium diphtheria as a polypep- tubulin binding to DPhPC membranes and membranes with significant tide chain that consists of three domains which facilitate entry into host cells via DOPE content. Second harmonic analysis of planar lipid membranes’ response the endosomal pathway: the receptor binding domain, the translocation domain to ac excitation suggests that tubulin binding causes significant rearrangements (T-domain), and the catalytic domain. Receptor binding triggers the inclusion of the lipid headgroups. We conclude that tubulin binds to and modifies the of DT into endosomes where acidification induces conformational changes in structure of lipid membranes even at nanomolar concentrations. Considering the T-domain that lead to its membrane insertion and result in the transfer of that there is up to 10 mM of free dimeric tubulin in cells, our results suggest the catalytic domain into the cytosol. However, the structural basis of this a new broad regulatory role of dimeric tubulin in vivo. pH-induced reorganization and membrane insertion remains unclear. Here we report binding studies of the T-domain using surface plasmon resonance 2824-Pos Board B254 and low-resolution structures using neutron reflectometry. We focus on three Membrane Interactions with NA-CATH different experimental conditions that were previously identified to arrest the Robin Samuel1, Haijuad Du1, Michael Massiah1, Barney Bishop2, protein at the membrane surface in distinct intermediate states along the inser- Susan Gillmor1. tion pathway. The wild type T-domain was studied on bilayers containing 1George Washington University, Washington, DC, USA, 2George Mason either 10% or 50% anionic lipids, known to stabilize the interfacial intermedi- University, Washington, DC, USA. ate and the fully inserted state, respectively (Kyrychenko et al, Biochemistry The ‘‘catastrophic threat’’ of antibiotic resistance has prompted research into 2009). We also studied the triple mutant H322Q/H323Q/H372Q on bilayers biological methods of combating bacterial infection. One such pervasive strat- containing 50% anionic lipids, known to populate a partially inserted interme- egy employs cationic antimicrobial peptides, CAMPs. These peptides use their diate (Rodnin et al, Biophys. J. 2011). Neutron reflection reveals similar structure to target and disrupt bacterial membranes. They specifically attach to membrane-bound structures for the wild type T-domain on 10% negatively PG (phosphatidylglycerol) and CL (cardiolipin) rich membranes, a common, charged bilayers and the triple-Q mutant on 50% negatively charged bilayers. anionic lipid of the outer bacterial leaflet. Most have shown broad spectrum ac- Under both conditions, the protein is primarily located in the outer lipid leaflet. tivity, adequate potency, and minimal resistance. Considering these peptides For wild type T-domain on 50% negatively charged bilayers, however, a have been active against pathogens for millions of years and have not devel- different membrane-bound structure is observed in which the protein spans oped any broad resistance, they are of particular interest to study. We have both leaflets. 558a Wednesday, February 11, 2015

2827-Pos Board B257 lular oxidative stress is one major factor leading to apoptosis accompanied by Reconstitution of the Coat Protein Complex II Induces Morphological the permeabilization of the mitochondrial outer membrane; a process causing Changes on Artificial Membranes release of apoptotic factors such as cytochrome c. Upon onset of intracellular Sebastian Daum1, Daniela Kru¨ger1, Lea Dietrich1, Mona Groß1, stress, phospholipids can become oxidized in their unsaturated fatty acid re- Annette Meister2, Kirsten Bacia1. gion. These oxidized phospholipids (OxPls) can severely alter the properties 1ZIK HALOmem, Martin-Luther-University Halle-Wittenberg, Halle, of these mitochondrial membranes, and can therefore have i) a direct effect Germany, 2Cryo Electron Microscopy Group, Martin-Luther-University on the membrane properties and its perforation and ii) can have an indirect Halle-Wittenberg, Halle, Germany. effect by altering the function of membrane-associated Bcl-2 proteins (such In eukaryotic cells, the transport of cargo from the endoplasmic reticulum (ER) as the anti-apoptotic Bcl-2 and the apoptotic Bax). We therefore devised a to the Golgi apparatus is conducted by protein-coated membrane vesicles. The model system that mimics oxidative stress conditions by incorporating oxidized stepwise formation of the coat protein complex II (COPII) on ER membranes is phospholipids (OxPls) into mitochondria-mimicking vesicles, and studied the initiated by the small GTPase Sar1. Following GDP/GTP exchange, which is OxPls’ impact on Bax-membrane interactions. Conformational changes in catalyzed by the guanine nucleotide exchange factor (GEF) Sec12, Sar1 un- the protein upon contact with the lipid membranes were monitored using far- dergoes a conformational change upon which an amphipathic helix inserts UV circular dichroism (CD) spectroscopy. Differential scanning calorimetry into the proximal leaflet of the ER membrane. Membrane-bound Sar1p subse- (DSC) and solid-state magic angle spinning nuclear magnetic resonance quently recruits the inner and outer coat subcomplexes Sec23/Sec24 and Sec13/ (MAS NMR) spectroscopy was used to study membrane organization. In a bio- Sec31 to complete the COPII coat. This process leads to marked changes in physical study combining CD with surface plasmon resonance techniques we membrane curvature. We apply in vitro reconstitution studies with purified also investigated the putative interaction of solubilized full-length human yeast proteins on giant unilamellar vesicles (GUVs) as an artificial membrane Bcl-2 with Bax. There, we found a direct Bcl-2 interaction with Bax in the pres- system to examine the COPII vesicle formation process. Using confocal micro- ence of sub-CMC concentration of detergent and could observe a high affinity scopy for visualization, we observe the formation of rigid tubes protruding from between both partners (KD 35.8 nM). the GUVs under conditions where GTP hydrolysis is prevented. Cryo-electron microscopy shows tubules that apparently fail to fission into separate vesicles. 2830-Pos Board B260 Moreover, we can distinguish individual stages in the formation of the COPII The Interaction of Hsp70 with Phosphatidylserine Membranes is Mediated coat on the bilayer. Altering the lipid composition, we observe different mem- by a Highly Positive Region of the Molecule brane morphologies. Our in vitro investigation of the COPII complex shows Victor Lopez1,2, David M. Cauvi2,3, Nelson Arispe4, Antonio De Maio2,5. 1Initiative for Maximizing Student Development Program, University that GTP hydrolysis is not essential for binding of the coat to the membrane 2 but may have a role in vesicle fission. of California, San Diego, La Jolla, CA, USA, Center for Investigations of Health and Education Disparities, La Jolla, CA, USA, 3Department of 2828-Pos Board B258 Surgery, University of California, San Diego, La Jolla, CA, USA, Solubilization of Membranes by Styrene Maleic Acid (SMA) Results in 4Department of Anatomy, Physiology and Genetics, Uniformed Services Formation of Nanodiscs with Retention of Native Lipid Composition University, Bethesda, MD, USA, 5Departments of Surgery and Neuroscience, Juan J. Dminguez Pardo. University of California, San Diego, La Jolla, CA, USA. MBB, University Utrecht, Utrecht, Netherlands. Heat shock proteins (hsp) participate in many cellular processes during normal Purification of membrane proteins has been a real headache for the scientific physiological conditions. In particular, they are involved in protein folding and community during the last decades. Recently, a new purification method has are referred to as molecular chaperones. During stress conditions, hsp partici- emerged using the SMA (styrene-maleic acid) co-polymer, which is able to sol- pate in the repair and recovery from an insult and confer protection from sub- ubilize membranes into small bilayer discs. Its importance lies mostly in the sequent stresses. In addition, they are released into the extracellular milieu presumed ability of retaining the native lipid environment of the membrane where they act as signaling molecules directed at activating the immune system proteins, preventing misfolding and/or loss of activity. However, it cannot be to avoid the propagation of the insult. Hsp70, the major inducible form of the excluded that SMA preferentially solubilizes specific lipids, thereby modifying hsp family, does not contain any consensus secretory signal that predicts its the extent to which the native lipid environment is indeed retained. export via the classical ER-Golgi secretory pathway. The proposed mechanism To find out whether preferential solubilization by the polymer occurs, we per- for the release of Hsp70 requires an initiated translocation into the plasma formed an extensive biophysical study using model membranes of synthetic membrane. However, Hsp70 does not contain any hydrophobic domain that lipids with well-defined compositions. Solubilized and non-solubilized material can predict its insertion into lipid membranes. Using an in vitro liposome inser- was obtained by adding limiting amounts of SMA polymer to the phospholipid tion assay, we have determined that Hsp70 displays selectivity for negatively vesicles. The mixture was centrifuged and lipid analysis was performed on the charge lipids and its insertion is enhanced by a decrease in the membrane supernatant, containing the nanodiscs, and the non-solubilized fraction in the fluidity. The region that is inserted into the lipid membrane has been mapped pellet. This procedure was performed for mixtures of different phospholipids, toward the C-terminus end, which contains the peptide binding domain. Lipid in which we varied the acyl chain length, unsaturation, head group charge recognition/insertion requires a highly positive region on the molecule. Finally, and lipid phase. The results showed no preferential solubilization in any of the addition of ADP, but not ATP, reduced membrane insertion, suggesting that these lipid mixtures, the only exception being a raft-like system (DOPC/SM/ the interaction with the lipid bilayer is dependent on the conformation of the cholesterol), where it was shown that SMA is incapable of solubilizing SM/ molecule. cholesterol-rich domains into nanodiscs. Supported by NIH R01 GM098455. The results highlight the potential of SMA polymer as a strong alternative for membrane protein purification, in which the native membrane lipid composi- 2831-Pos Board B261 tion is generally retained. Interestingly, the behavior of the raft-like systems Elucidating the T Cell Receptor Transmembrane Organization via Multi- upon addition of SMA resembles the behavior of detergent-resistant mem- Scale Molecular Dynamics Simulations Antreas C. Kalli1, Andre Cohnen2, Oreste Acuto2, Mark S.P. Sansom1. branes (DRMs) when membranes are treated with detergent, paving the way 1 2 for novel approaches of lipid raft isolation. Biochemistry, University of Oxford, Oxford, United Kingdom, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom. 2829-Pos Board B259 The T cell antigen receptor (TCR) plays a key role in the adaptive immunity of The Effect of Oxidized Lipids on the Interplay of Bcl-2 and Bax Proteins at all vertebrates. It is able to identify with high sensitivity, specificity and rapidity Mitochondrial Membranes peptide antigens from invading microbial pathogens presented by MHC proteins Martin N. Lidman1, Artur Dingeldein1, Marcus Wallgren2, (peptide-MHC) and signals via multiple intracellular pathways to prepare Anders Pedersen3,Go¨ran Karlsson3, Sarka Pokorna4, Martin Hof4, adequate countermeasures to fend them off. The TCR has a complex structural Gerhard Gro¨bner1. organization consisting of the TCR ab heterodimer, which recognizes peptide- 1Chemistry, Faculty of Science and Technology, Umea˚, Sweden, 2Medical MHC, and it is non-covalently associated with three dimers (the CD3 εg, εd and Biochemistry and Biophysics, Faculty of Science and Technology, Umea˚, zz) that regulate signal transduction. Despite advances in the field, the topology Sweden, 3Faculty of Science, Gothenburg, Sweden, 4J. Heyrovsky´ Institute of of TCR-CD3 subunit organization is still largely hypothetical. Molecular dy- Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, namics simulations can now be applied with confidence to study the assembly Czech Republic. of transmembrane (TM) segments of membrane proteins in model lipid bilayers. Mitochondria play a crucial role in the intrinsic apoptotic pathway. The Bcl-2 In this study we probe the assembly of the TCR ab TM heterodimer and its in- family proteins, which interact with the mitochondrial outer membrane to teractions with the CD3 accessory subunits at both atomistic and coarse-grained modulate membrane permeability, are key regulators of this pathway. Intracel- resolution using a multi-scale simulation approach. Our results suggest a Wednesday, February 11, 2015 559a dynamic structural model for the TCR ab TM complex and how it associates 2835-Pos Board B265 with the CD3 zz subunit. Additionally, the interaction of the TCR with its Structural Comparison of Membrane-Bound Retroviral Gag Proteins lipid environment may also regulate the organization of the TCR-CD3 TM Marilia Barros1, Robert A. Dick2, Siddhartha A.K. Datta3, Volker M. Vogt2, complex. We will also discuss simulation of the TCR TM region in complex Alan Rein4, Mathias Lo¨sche5,6, Hirsh Nanda7,1. lipid bilayers that resemble plasma membranes. Our results suggest preferential 1Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2Molecular interactions of the TCR TM region with cholesterol and anionic lipids in the Biology and Genetics, Cornell University, Ithaca, NY, USA, 3HIV Drug bilayer. Resitance Program, National Cancer Institute, National Institutes of Health, Fredrick, MD, USA, 4HIV Drug Resitance Program, National Cancer 2832-Pos Board B262 Institute, National Instutes of Health, Fredrick, MD, USA, 5Physics, Carnegie Evolutionary Avoidance of Transmembrane Embedded Arginines is due Mellon University, PIttsburgh, PA, USA, 6Center for Neutron Research, to Slowed Folding Kinetics National Institute of Standards and Technology, Gaithersburg, MD, USA, Ashlee M. Plummer1, Harris D. Bernstein2, Karen G. Fleming1. 7 1 2 NIST Center for Neutron Research, National Institute of Standards and Biophysics, Johns Hopkins University, Baltimore, MD, USA, National Technology, Gaithersburg, MD, USA. Institute of Diabetes and Digestive and Kidney Diseases, National Institute of he Gag polyprotein is essential for the assembly of retroviral particles. It is ex- Health, Bethesda, MD, USA. pressed in the cellular cytoplasm and targets the surface of the plasma mem- Conserved transmembrane (TM) arginines are essential for the transport func- brane, where assembly occurs. All Gag proteins from various retroviral tion of many membrane proteins, such as voltage-gated ion channels. However, species contain structured domains - the membrane-binding matrix (MA), the among the lipid-facing residues in TM regions, the statistical propensity of capsid (CA) and the genome-binding nucleocapsid (NC) domains - that are arginines is extremely low. The minimal occurrence of TM arginines is often separated by unstructured linker regions. Cryo-EM of immature virions shows attributed to the thermodynamic cost of inserting an arginine into a hydropho- assemblies on the plasma membrane in which Gag polyproteins are in an bic lipid environment. But the energetic cost of TM arginine insertion measured extended conformation, apparently stabilized by lateral interactions between by the Moon-Fleming Hydrophobicity scale is only 3 kcal mol-1, implying that their CA domains. However, the structural dynamics are complex. In a recent Arg should be allowed in the bilayer. To investigate the discrepancy between study with neutron reflection (NR), we showed that in HIV-1 Gag, the MA-CA this moderate thermodynamic cost and the evolutionary avoidance of TM argi- linker is so flexible that the polyprotein binds a model membrane in a back- nines, we utilized Outer Membrane Phospholipase A (OmpLA) as a host for folded structure in the absence of nucleic acid, and that a transformation into lipid-facing arginine mutations, at three different depths in the bilayer. We an extended conformation is triggered upon nucleic acid binding (Datta probed the effects of the introduction of TM arginines on the secondary struc- et al., J. Mol. Biol. 406, 2011, 205). Here we investigate the membrane- ture and enzymatic activity of OmpLA and found that Arg has a minimal binding of murine leukemia virus (MLV) and Rous sarcoma virus (RSV) impact on the structure and function of OmpLA. We also determined that a Gag proteins, which also include unstructured linkers between their MA and TM Arg does not affect the thermodynamic binding of OmpLA to the periplas- CA domains. Both wild-type MLV and RSV Gag bind membranes in vitro in mic chaperones, SurA and Skp. Molecular dynamics simulations revealed that folded conformations, as shown by NR, indicating structural flexibility of their all TM Args studied form hydrogen bonds with water and phospholipid head MA-CA linkers. In contrast, an MLV mutant, Dp12, in which the MA-CA groups. Kinetics of folding into large unilamellar vesicles of varying head linker has been eliminated, binds the membrane in an extended conformation. group compositions reveal that the R-variants exhibit slow folding, even in An investigation of the RSV mutant, D104-220, that is analogous to MLV- the presence of the OMP assembly machinery protein, BamA. Slow kinetics Dp12, is currently in progress. These studies suggest that the disordered linker are additionally observed for in vivo folding of these R-variants. In addition regions in retroviral Gag proteins may function to prevent unproductive assem- to a thermodynamic cost for TM arginine insertion our results indicate that a bly processes in the host cytosol or on the membrane in the absence of viral pronounced kinetic barrier may play a role in the evolutionary pressure against genome. TM arginines. 2836-Pos Board B266 2833-Pos Board B263 Probing Protein-Lipid Interactions at the Single Molecule Level Site Directed Spin Label EPR Spectroscopy of Influenza a M2 Protein Tina R. Matin1, Krishna P. Sigdel1, Linda L. Randall2, Gavin M. King1,2. Kathleen Howard, Bryan Green, Shenstone Huang, Sang Woo Kim. 1Physics and Astronomy, University of Missouri Columbia, Columbia, Swarthmore College, Swarthmore, PA, USA. MO, USA, 2Biochemistry, University of Missouri Columbia, Columbia, M2 is a membrane protein critical to the life cycle of influenza A. We have MO, USA. capitalized on the expanding body of biochemical and high-resolution struc- Cell membranes consist mostly of lipids and embedded proteins, forming a tural data available for the M2 protein to design and interpret site-directed shell around the cell that controls information and material fluxes into and spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) ex- out of the cell. Protein-lipid interactions thus play a crucial role in numerous periments on the conformation and dynamics of the homotetrameric M2 protein cellular mechanisms. Understanding these interactions would provide basic embedded in lipid bilayers. We have obtained data for three different M2 con- biophysical knowledge in addition to advance pharmaceutical developments, structs (M2TM 22-46, M2TMC 23-60 and full length M2 protein) spin-labeled many of which target membranes. Despite their significance, protein-lipid in- at multiple sites within the transmembrane and C-terminal domains. CW and teractions are not fully understood and have not yet been fully investigated pulsed EPR spectra show evidence that M2 adopts multiple conformational from a mechanistic standpoint. In this study we investigated synthesized pep- states in bilayers, and that cholesterol content dictates the relative populations tide constructs that insert into lipid bilayers via force spectroscopy. The con- of the states. Furthermore comparison of full-length M2 protein and a M2TMC structs used here are based on SecA, a membrane-associated ATPase of the 23-60 peptide demonstrates that the C-terminal juxtamembrane region in both general secretory system. We investigated the first 10 amino terminal residues constructs forms an amphipathic membrane surface helix. of SecA (SecA2-11) in three different arrangements to provide control exper- 2834-Pos Board B264 iments and guide interpretation. In one arrangement, two copies of SecA2-11 Studying the Membrane-Bound Conformation of Alpha-Synuclein using a were covalently linked in series to the atomic force microscope tip, in another, Model Transmembrane Peptide System in a Lipid Bilayer they were linked in parallel. In all cases the constructs were brought into close Graham P. Lobel, Alice R. Vienneau, Casey H. Londergan. proximity of a supported POPC lipid bilayer and the mechanical interaction be- Chemistry, Haverford College, Haverford, PA, USA. tween the peptide and lipid was recorded using single-molecule dynamic force Alpha-synuclein is intrinsically disordered in solution but adopts a partially spectroscopy. Investigating chemically similar yet geometrically different con- alpha-helical structure when membrane bound; we are currently investigating structs provides important benchmarks for the interpretation of future studies this structure using a cyanylated cysteine vibrational probe at various sites on involving longer constructs such as full length SecA. the aS membrane binding domain. In order to quantitatively understand the 2837-Pos Board B267 depth dependence of the SCN probe in a lipid bilayer, four variants of a Single-Molecule Diffusion Measurements Indicate Independent Mem- poly-L transmembrane helical peptide were designed and synthesized with brane Insertion by the Tandem C2 Domains of Synaptotagmin 7 cysteine residues at different depths in a membrane bilayer. A nonaqueous cy- Joseph Vasquez, Kan Chantranuvatana, Daniel Giardina, Jefferson Knight. anylation and purification protocol was developed to introduce the probe Chemistry, University of Colorado Denver, Denver, CO, USA. group on these very hydrophobic model peptides. Vibrational spectroscopy The synaptotagmin (Syt) family of proteins is characterized by the presence of was carried out on the four variants in a model lipid system using both solu- tandem C2 domains, C2A and C2B, which sense Ca2þ to trigger vesicle fusion tion/vesicle and oriented, immobilized samples. These data will help to eluci- during exocytosis. The widely studied Syt1 is central to rapid neurotransmitter date the membrane interactions of both aS and other membrane-bound release, while Syt7 is involved in slower secretion of large dense-core vesicles proteins. and has C2 domains that dock much more tightly to target membranes. Despite 560a Wednesday, February 11, 2015 recent progress in understanding mechanisms of vesicle fusion, the molecular E. coli polar lipids or 2:1 POPE/POPG, doped with 0.3% of AF488-NHS ester mechanisms of synaptotagmin C2AB membrane docking remain incompletely labelled POPE. Following multiple freeze/thaw cycles to form multilamellar characterized. For example, the two C2 domains of Syt1 are reported to cooper- vesicles, the proteoliposomes are extruded through polycarbonate filters of atively insert into target membranes, but specific interdomain contacts have not 30, 100 and 400 nm pore diameter resulting in reproducibly distinct size distri- been identified. To test whether the two C2 domains from Syt7 interact on a butions. Reconstitution follows the Poisson distribution resulting in liposomes planar lipid bilayer, lateral diffusion constants of fluorescent-tagged C2A, containing either 0, 1, 2 or more protein molecules. The apparent protein occu- C2B, and C2AB domains from human Syt7 were measured on PC:PS (3:1) bi- pancy into liposomes depends on protein:lipid density, efficiency of fluorescent layers using total internal reflection fluorescence microscopy with single- labelling, liposome surface area and the subunit stoichiometry of the protein particle tracking. The Syt7 C2AB tandem exhibits a lateral diffusion constant complex. Liposome size distributions are determined by cryo-electron micro- half the value of the isolated single domains, and does not change when addi- scopy whereas the protein density and fluorescent-labeling is controlled during tional residues are engineered into the C2A-C2B linker. This is the expected the reconstitution step. We measure the first three terms in the Poisson distri- result if C2A and C2B are separated when membrane-bound; theory predicts bution: F0 (unoccupied liposomes), F1 (single occupancy) and F2 (double that C2AB diffusion would be faster if the two domains interact. Furthermore, occupancy) by single molecule imaging of the fluorescent proteoliposomes to ensemble stopped-flow measurements of membrane dissociation kinetics also measure protein/lipid co-localization and photobleaching of protein conjugated support an absence of interdomain interactions, as EDTA-induced dissociation fluorophores. We test our model with a CLC-ec1 engineered monomeric kinetics of the C2AB tandem are similar to the isolated C2A domain and remain construct, the native CLC-ec1 homodimer and tetrameric KcsA Kþ channel. unchanged when rigid or flexible linker extensions are included. Together, the The methods outlined in this study can be used to determine the subunit stoi- results suggest that the two C2 domains of Syt7 bind independently to mem- chiometry of unknown purified membrane protein complexes in a variety of branes. Ongoing efforts seek to perform analogous measurements with Syt1, liposome environments. whose C2 domains have much shorter membrane-bound lifetimes. 2838-Pos Board B268 Mechanosensation Single Molecule Diffusion Studies of PTEN: Insights into Membrane Binding 2840-Pos Board B270 Rakesh K. Harishchandra1, Anne-Marie M. Bryant1, Abigail C. Cornwell1, Nanobiomechanics and Mechanotransduction of Sensory Neurons Mathias Lo¨sche2, Alonzo H. Ross3, Arne Gericke1. Laura Andolfi1, Marco Lazzarino1, Valentina Masciotti1, Yanmei Qi2, 1 Department of Chemistry and Biochemistry, Worcester Polytechnic Jing Hu2. 2 Institute, Worcester, MA, USA, Department of Physics, Carnegie Mellon 1IOM-CNR, Trieste, Italy, 2Centre for Integrative Neuroscience, Tu¨bingen, 3 University, Pittsburg, PA, USA, Department of Biochemistry and Molecular Germany. Pharmacology, University of Massachusetts Medical School, Worcester, A detailed knowledge of mechanical parameters such as cell elasticity, stiffness MA, USA. and viscoelasticity is essential for understanding the mechanisms that control PTEN, a tumor suppressor gene that encodes a dual specificity phosphatase that the mechanotransduction in mechanosensory neurons (MSN). Indeed, in order dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3), is one to tune and maximize their sensitivity, MSN should be neither too rigid nor to of the most frequent genes deleted or mutated in a wide variety of tumors. compliant; moreover they are expected to show different elasticity as a function PTEN acts as an antagonist to phosphoinositide-3-kinase (PI3K) signaling, of the typology of mechanical stimulus they should record. However a precise thereby affecting various cellular processes such as cell proliferation and sur- correlation between MSN mechanical properties and mechanotransduction vival. The activity of PTEN is regulated by dynamic shuttling between the mechanism is still missing, and the sensory mechanical transduction, necessary cytoplasm and the plasma membrane. The membrane association of PTEN for the senses of touch and pain, remains poorly understood. strongly depends on the composition and lateral distribution of the lipids in Indentation measurements by atomic force microscopy (AFM) enable to inves- the membrane. Several biophysical techniques have been used to characterize tigate and quantify in vitro the softness of living MSN thanks to its ability to PTEN-membrane interaction. Here, we use single-molecule total internal measure low forces (pN) and nanometer scale displacement. Moreover, the reflection fluorescence microscopy (TIRFM), which is a powerful tool to study integration of AFM with fluorescence microscopy opens up the possibility to the molecular mechanism of membrane targeted proteins. The single-molecule relate the involvement or activation of either cytoplasmatic structures or trans- TIRFM allows us to observe single PTEN molecules as they dynamically asso- membrane proteins with variations of cell mechanical properties and, as result, ciate/dissociate and laterally diffuse along the lipid bilayer membrane. PTEN their role in the modulation of mechanosensory neurons activity. lipid binding is investigated on supported lipid bilayers of binary and ternary In this study we performed AFM indentation measurements to evaluate the lipid mixtures of PC with physiological relevant levels of PS, PI(4,5)P2 and mechanical properties of wild type and genetically modified proteins of the PI. We tracked individual PTEN molecules and statistically determine the stomatin system of dorsal root ganglia (DRG). We found a decrease of cell lateral diffusion and dwell time of PTEN on heterogeneous lipid bilayers. elasticity in DRG neurons where stomatin system is genetically modified. We find significant differences in PTEN dynamic behavior when bound to The role of cell elasticity in mechanotransduction regulation of mechanosen- the different membrane environments. Furthermore, to gain insight into the mo- sory neurons is discussed. lecular mechanisms of PTEN membrane association, we compared the lipid binding of wt PTEN, an N-terminally truncated PTEN-(D1–15 AA) that lacks 2841-Pos Board B271 the PI(4,5)P2 binding site, and the recently discovered, PTEN-L with a 173 AA Cross-Linked Matrix Rigidity and Soluble Factors Induce Differentiation N-terminal extension. We find profound differences in the dynamic behavior of via Distinct but Overlapping Pathways these PTEN derivatives at the membrane. It has recently been suggested that Irena L. Ivanovska, Joe Swift, Kyle Spinler, P.C. Dave P. Dingal, PTEN associates as a dimer with the membrane. We find that the tendency Dennis E. Discher. to form membrane bound dimers varies among these PTEN variants. Biophysical Eng’g Lab, University of Pennsylvania, Philadelphia, PA, USA. Stem cell differentiation is regulated by both soluble factors and the physical 2839-Pos Board B269 properties of extracellular matrix, but the extent to which differentiation path- A Single-Molecule Imaging Based Method for Estimating Subunit Stoichi- ways are distinct or overlap is often unclear. Here, the micromechanical stiff- ometry of Purified Membrane Protein Complexes in Liposomes ness of the collagenous bone surface together with broad compositional Rahul Chadda1, Larry Friedman2, Ankita Chadda1, Mike Rigney2, correlations with collagen-I across many soft tissues suggests enzymatic Luci-Kolmakova Partensky2, Jeff Gelles2, Janice L. Robertson1. cross-linking of matrix correlates with nucleoskeletal protein lamin-A, with a 1Department of Molecular Physiology and Biophysics, University of Iowa, retinoid receptor RARG, and with induction toward osteogenesis. Collagen Iowa City, IA, USA, 2Department of Biochemistry, Brandeis University, films just 2 nm thick on mica were stiffened or not by transglutaminase Waltham, MA, USA. cross-linking and used as minimal culture substrates for Mesenchymal stem Estimating the number of subunits of a purified novel membrane protein is cells (MSCs). Cells pulling on pristine nano-films visibly deformed and aligned challenge. There are various methods for determining stoichiometry accurately with the collagen fibrils, but on cross-linked films, cells spread isotropically as in detergent, but often we are interested in the protein structure in the native if adhering to a substrate of greater effective stiffness. Cell nuclei also spread solvent environment - the lipid bilayer. Here we describe a robust and widely and stiffened, with an increase of lamin-A, nuclear localization of RARG, and applicable method to estimate the number of subunits in a purified membrane upregulation of key early and late osteogenic factors. RARG antagonists also protein sample using single molecule fluorescent imaging. First, the multimeric increased lamin-A, and enhanced osteogenesis on rigid substrates in vitro as protein complex is solubilized in detergent and purified. Second, it is quantita- well as in xenografts of MSCs in mice. A model of the underlying Mechano- tively labelled with Cy5-maleimide and reconstituted into liposomes made of chemical Gene Circuit couples the sensitivity of stem cells to both insoluble Wednesday, February 11, 2015 561a and soluble factors, while a proteomic comparison underscores both differences binding sites at the linker region between them. Recent sequence analyses and overlaps in differentiation pathways. and structures of cadherins revealed unusual calcium-free inter-repeat linkers in some protocahderins and other non-classical cadherins. Bound calcium 2842-Pos Board B272 ions have been shown to provide structural rigidity to cadherins, thus the pres- Altered Contractile Machinery in Airway Epithelial Cells in Response to ence of unusual sites may confer higher flexibility and perhaps affect the ter- Cigarette Smoke tiary and quaternary arrangement of cadherins that harbor them. Analysis of Corrine Kliment, Vasudha Srivastava, Douglas Robinson, Ramana Sidhaye. the protocadherin-15 sequence shows unusual calcium-binding sites in some Medicine, Johns Hopkins University, Baltimore, MD, USA. of its inter-repeat linkers. Here we present the x-ray crystal structure of repeats Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death EC8-10 refined at 3.3 A˚ resolution, which shows an EC9-10 calcium-free linker in the US with cigarette smoke being the primary insult leading to disease pro- that alters the linear arrangement of protocadherin-15’s EC repeats. We suggest gression. While the lung epithelium is the site that makes initial and primary con- that several unusual features of these repeats affect the overall elastic response tact with the inhaled cigarette smoke, we do not understand the role of of protocadherin-15 that is relevant for tip link function in sensory perception. cytoskeleton and cell mechanics in the airway response to cigarette smoke. We know that cytoskeletal proteins, such as myosin II, actin, alpha-actinin, 2845-Pos Board B275 the catenins, and E-cadherin, are involved in cell-cell adhesion formation and Force-Free Transition from Closed to Open MscL: A Molecular Dynamics barrier function of the lung. Furthermore, E-cadherin levels decrease in primary Study epithelial cells from COPD patients. Therefore, we hypothesized that cigarette Natalie E. Smith, Ben Corry. smoke drives key cytoskeletal protein changes that lead to alterations in barrier Research School of Biology, The Australian National University, ACT, function and thereby influence the development of chronic lung disease. To test Australia. this, we studied major cytoskeletal (actin and nonmuscle myosin II isoforms) The mechanosensitive channel of large conductance (MscL) is exceptionally and cell adhesion proteins (E-cadherin) in normal human bronchial epithelial important in bacterial cells as when the cellular pressure becomes too high cells (NHBE, collected directly from human patients) or 16HBE cells (primary and the membrane tension rises, these channels rapidly open allowing an efflux human bronchial epithelial cell line) grown on an air-liquid interface. Using a of cellular contents that prevents the cell from bursting. As there are no homo- Vitrocell smoke chamber, we found that in response to acute smoke exposure logues of this channel in humans, it is seen as a promising new target for anti- (four cigarettes in 24 hrs), myosin IIB and actin assembled into apical stress fi- biotics, something that is critically important given the increasing resistance to bers, which are not normally found in airway epithelial cells. Cigarette smoke existing drugs. While MscL has been studied for many years, exactly how it exposure also resulted in decreased total E-cadherin levels as well as decreased senses membrane tension to go from a closed to an open state is still unknown, E-cadherin at the basolateral surface. Thus, cigarette smoke promotes dramatic however, it has been observed that mutations in the gating region strongly acute cytoskeletal changes that likely lead to altered cell mechanical properties affect the tension required to open the channel. We have applied molecular dy- (tension, elasticity), which in turn lead to reduced epithelial barrier function and namics to study wild type MscL and three channel mutants, G22E, G22S and our data suggest that epithelial barrier function is critical in dictating chronic tis- G22N-G26N that each have lower gating tension thresholds than wild type. sue responses that contributes to COPD development and progression. In the absence of any external force we have observed specific and interesting 2843-Pos Board B273 differences in the propensity of each channel to transition towards the open Primary Cilia Length is Critical to Cellular Mechanotransduction state. On the timescale sampled for each mutant channel (0.5-2.25 ms), we Milos Spasic, Christopher Jacobs. have observed sub-conductant states of MscL allowing both the measurement Biomedical Engineering, Columbia University, New York, NY, USA. of an appreciable current and atomistic detail of how the transition from a Mechanotransduction is an essential cellular function in a variety of tissues closed to a partially open channel occurs. This provides a model for how including bone, kidney, and endothelia. The primary cilium is a single immotile mechanical forces can be converted into a physiological response. organelle protruding from the surface of these cells, and has repeatedly been demonstrated as a critical mechanotransducer in these cell types. When the pri- 2846-Pos Board B276 mary cilium is impaired, these cells have an abrogated response to mechanical Patch Clamp Characterisaton of the Effect of Cardiolipin on the Bacterial stimulation. Here, we demonstrate a method to enhance cellular mechanotrans- Mechanosensitive Channels of Small (MscS) and Large (MscL) duction, by increasing primary cilia length. To elongate primary cilia, we Conductance 1 1 1,2 treated MLO-Y4 osteocytes with fenoldopam, lithium chloride, or vehicle con- Pietro Ridone , Yoshitaka Nakayama , Boris Martinac , 3,4 trol for 16 hours. We then subjected these cells to oscillatory fluid flow for 1 Andrew R. Battle . 1Victor Chang Cardiac Research Institute, Darlinghurst, 2010, Australia, hour at 1 Hz and 1 Pa wall shear stress. Immediately following flow, cells 2 were lysed, and mRNA expression was analyzed. Cells with longer cilia dis- St Vincent’s Clinical School, The University of New South Wales, Kensington, 2052, Australia, 3Griffith Health Institute, Griffith University, played increased expression of osteogenic markers cyclooxygenase-2 and 4 osteopontin, compared to vehicle control, suggesting that these cells are Gold Coast Campus, 4222, Australia, School of Biomedical Sciences, more mechanosensitive. To discern the role of fenoldopam on cilia length The University of Queensland, St Lucia, 4072, Australia. from other cellular processes, we treated cells with IFT88 siRNA_IFT88 is crit- The bacterial mechanosensitive channels MscS and MscL respond to mem- ical for primary cilia formation_which resulted shorter cilia and an abrogated brane tension by opening when the bacterium experiences hypoosmotic shock response to fluid flow. Fenoldopam was able to restore cilia length in cells conditions to prevent cell lysis [1]. Environmental factors such as cholesterol with impaired cilia formation, and rescued flow-induced osteogenic signaling. [2] and cations/anions [3] also affect the gating behaviour of these channels. Together, these data suggest that cells with longer cilia are more mechanosen- We have previously shown that addition of the negatively charged lipid cardi- sitive, and that cellular mechanotransduction can easily be modulated by olipin to POPE/POPC membranes cause rapid and flickery behaviour of MscS pharmacologically lengthening primary cilia. Primary cilia-mediated mechano- [4]. Here, in an expanded study to include MscL, we compare the gating ki- transduction is a critical function in an array of cell types, and numerous netics and pressure sensitivitiy of the channels with and without cardiolipin diseases, such as polycystic kidney disease and Bardet-Biedl Syndrome, are in both azolectin and mixtures of pure lipids DOPE/DOPC. In azolectin lipo- characterized by impaired cilia function. This work suggests a potential thera- somes, mixtures of 10% cardiolipin abolish hysteresis of MscS, but MscL re- peutic strategy to combat such conditions. mains largely unaffected, indicating it may stabilise the closed state of MscS. Compared to the azolectin, mixtures of DOPE/DOPC abolish the hysteresis 2844-Pos Board B274 gating of MscS even in the absence of cardiolipin and addition of cardiolipin Structure of an Inner-Ear Protocadherin-15 Fragment with an Atypical increases the opening and closing thresholds of both MscS and MscL. These Calcium-Free Linker results suggest that cardiolipin shows a significant effect on the mechanosensi- Raul Araya-Secchi, Marcos Sotomayor. tive gating of both MscS and MscL. Chemistry and Biochemistry Department, The Ohio State University, References: Columbus, OH, USA. 1. Martinac B, Curr Top Membr. 2007 58, 25 Tip links are protein filaments essential for hearing and balance. They convey 2. Nomura T, Cranfield CG, Deplazes E, Owen DM, Macmillian A, Battle AR, force to and gate inner ear hair cell transduction channels to mediate sensory Constantine M, Sokabe M, Martinac B. PNAS 2012, 109, 8770 perception. Cadherin-23 and protocadherin-15 form tip links through a 3. Cox, C., Nomura, T., Ziegler, CS, Campbell AK, Wann KT, Martinac B. calcium-dependent heterophilic interaction of their extracellular domains, Nature Communications 2013, 4, 2137 which are comprised by multiple modules termed extracellular cadherin 4. Battle AR, Nomura T, Martinac B, Biophys J 2011, 100 S1, 278 ‘‘EC’’ repeats. These EC repeats are similar but not identical to each other in Supported by a JSPS Fellowship to Y.N., APP1079398 grant from NHMRC to terms of sequence and structure, often featuring highly-conserved calcium- B.M. and Griffith University Project grant to A.B. 562a Wednesday, February 11, 2015

2847-Pos Board B277 roles of somatosensory neurons in light touch perception. Here, we explored Structural and Biophysical Characterization of Inner Ear Tip Link functional specialization of somatosensory ganglia from animals which have Variants taken the sense of touch to the extreme - tactile foraging ducks. These animals Yoshie Narui, Marcos Sotomayor. have acutely mechanosensitive bill innervated by trigeminal (TG) neurons, and Chemistry and Biochemistry, The Ohio State University, Columbus, as such provide an opportunity to study general principles of mechanotransduc- OH, USA. tion from an unconventional standpoint. We found that, in contrast to species At the molecular level, human hearing relies upon the tip-to-tip interaction of without tactile specialization, the majority (85%) of duck TG neurons are two unique non-classical cadherins, protocadherin 15 (Pcdh15) and cadherin 23 large-diameter myelinated mechanoreceptors expressing the mechano-gated (Cdh23). Together, these two proteins form a filament called the tip link that ion channel Piezo2. Electrophysiological analyses showed that mechanosensi- connects neighboring stereocilia of mechanosensitive hair cells. Sound waves tivity of duck TG neurons has been optimized in three ways. Compared to cause the stereocilia to deflect applying a force to the tip link and opening a mouse cells, duck neurons exhibit (i) lowered threshold of mechano- nearby transduction channel. Disruption of the tip link caused by loud sound activation, (ii) elevated signal amplification gain, and (iii) prolonged kinetics or chemical treatments eliminates transduction currents and illustrates that of inactivation, all of which increase the amount of depolarizing charge tip link integrity is critical for mechanosensing. Recent studies have found entering the cell upon mechanical stimulation. Thus, duck TG neurons have that remodeling of the tip link after disruption is a dynamic process, which augmented intrinsic ability to convert mechanical force into excitatory ionic leads to the formation of atypical complexes that may incorporate alternatively current, which explains the acute mechanosensory properties of the duck bill. spliced variants or isoforms of Pcdh15. Our current work focuses on under- Our studies emphasize a key role of the intrinsic mechanosensory ability of standing these unusual tip links and comparing them with the prototypical tip somatosensory neurons in touch physiology, reveal an evolutionary strategy link. Here, we present the crystal structure of a new complex formed from utilized by vertebrates to hone tactile perception, and suggest a novel model Cdh23 and isoform 2 of Pcdh15 refined at 3.5 A˚ resolution. While the molec- system to study the sense of touch at the cellular and molecular level. ular structure reveals subtle differences between the two complexes, the bind- Schneider ER, Gracheva EO, Bagriantsev SN et al, PNAS 2014 (e-pub ing affinity between Cdh23 and isoform 2 of Pcdh15 is notably different from Sept 22). Cdh23 and isoform 1 of Pcdh15 as observed in analytical size exclusion chro- matography. These results clearly demonstrate that alternative heterotypic tip 2850-Pos Board B280 link structures form stable protein-protein interactions in vitro and provide Is Cryptochrome a Primary Sensor of Extremely Low Frequency no evidence to support the existence of homotypic Pcdh15-Pcdh15 tip links. Magnetic Fields in Childhood Leukemia? Patricia L. Bounds1,2, Niels Kuster1,3. Additional studies will focus on other Pcdh15 isoforms and determine how 1 changes in the tip link structure alter the mechanical properties of the filament. Foundation for Research on Information Technologies in Society (IT’IS), CH-8004 Zurich, Switzerland, 2Department of Chemistry and Applied 2848-Pos Board B278 Biosciences, Swiss Federal Institute of Technology, CH-8093 Zurich, Probing the Mechanosensitivity of Piezo1 Channels Switzerland, 3Deptartment of Information Technology and Electrical Charles D. Cox1, Boris Martinac1,2. Engineering, Swiss Federal Institute of Technology, CH-8092 Zurich, 1Victor Chang Cardiac Research Institute, Sydney, Australia, 2St Vincent’s Switzerland. Clinical School, University of New South Wales, Australia. Extremely low frequency magnetic fields (ELF MF) are classified as possibly Piezo channels are a new family of cation-selective mechanosensitive ion chan- carcinogenic to humans, but the biophysical mechanisms of a causal relationship nels, which have been shown to be integral in numerous physiological pro- remain unclear. A cryptochrome-based radical pair mechanism (RPM) has been cesses. Central to these physiological roles is the ability to sense mechanical invoked as the primary MF sensor in animal magnetoreception to explain effects force. Whether this force is transmitted directly from the lipid bilayer or from MF strengths in the nT range. Model studies of the RPM in aprotic solution from tethering to the cytoskeleton and/or ECM is unknown. The ‘gold standard’ require cryogenic temperatures and MFs in the mT range to elicit marginal re- for testing the ‘inherent’ mechanosensitivity of ion channels has become puri- sponses, implying that physiological responses evoked by radical pairs in biolog- fication and subsequent reconstitution of mechanosensitive channel proteins ical milieu are unlikely. We explore ideas about how signals from transient into liposomes of known lipid composition. This is experimentally difficult radical pairs in cryptochrome might be transduced and amplified in mutagenic and time consuming and is a process that has to be determined empirically responses. The cryptochrome-based RPM involves blue light activation of the for individual channel proteins. An underused paradigm for the study of chan- flavin adenine dinucleotide (FAD) cofactor followed by electron transfer (ET) nel mechanosensitivity is the production of cytoskeleton deficient membrane from a conserved triad of tryptophan residues. However, intramolecular ET ‘blebs’ [1]. In order to study the inherent mechanosensitivity of Piezo1 chan- involving additional conserved aromatic residues in cryptochrome likely extends nels using this paradigm we used transiently transfected HEK293 cells. Herein beyond the canonical triad. Further, ascorbate, which is present at millimolar we show that; a) membrane blebs can be formed by the addition of both Hypo- concentrations in leukocytes, is likely to transfer an electron to the ultimate and Hyper-osmolar solutions of sodium gluconate in HEK293 cells, b) this amino acid radical formed during one-electron reduction of FAD. The ascorbyl treatment induces significant cell death after 2 hours as determined by trypan radical has been proposed as a potential radical pair partner in the RPM. blue exclusion assays, c) these resulting blebs are deficient in a major cytoskel- The flavin semiquinone radical may be reoxidized to the resting state via ET etal component (actin) and d) Piezo1 channels can be activated in this environ- to O2 to form O2 . Increased levels of the ascorbyl radical and O2 would ment and have a lower pressure threshold of activation. This has important contribute to oxidative stress pathways in the cell. Oxidative stress responses implications with respect to Piezo1 channels being gated according to the force in cancer are thought to be mediated in part by the mitogen activated protein from lipids concept [2]. In addition we also assess the affect of mutating a num- kinase (MAPK) signaling pathway. We have presented evidence for alteration ber of aromatic residues in the most highly conserved region of the Piezo of MAPK activation in response to ELF MF. sequence on the mechanosensitivity of the channel. Acknowledgement: This project has received funding from the European References: Union’s Seventh Programme for research, technological development, and [1] Zhang, Y., et. al., (2000) J. Physiol.(London) 523, 117-130. demonstration under grant agreement No. 282891. [2] Anishkin, A., et. al., (2014) PNAS 111(22): 7898-7905. Supported by APP1047980 grant from the National Medical and Health 2851-Pos Board B281 Research Council of Australia. A Structure-Function Approach to Understanding the Dual Functions of the Plant Mechanosensitive Ion Channel MSL10 2849-Pos Board B279 Grigory Maksaev, Kira Veley, Elizabeth Haswell. Sensing Force by Trigeminal Neurons of Acutely Mechanosensitive Birds Biology Dept, Washington University in Saint Louis, Saint Louis, MO, USA. Eve R. Schneider, Marco Mastrotto, Willem J. Laursen, Vincent P. Schulz, MscS-Like (MSL) 10 is a member of the MscS superfamily of mechanosensi- Jena B. Goodman, Owen H. Funk, Patrick G. Gallagher, Elena O. Gracheva, tive ion channels and one of 10 MSL proteins in the model flowering plant Sviatoslav N. Bagriantsev. Arabidopsis thaliana. Unlike Escherichia coli MscS, MSL10 contains 6 trans- Yale School of Medicine, New Haven, CT, USA. membrane helices, and only its C-terminal TM helix shows homology with Mechanosensation is a fundamental way animals interact with the environment, EcMscS’s pore-lining domain. MSL10 has been shown to provide a mechano- but it remains the least well understood at cellular and molecular level. Somato- sensitive activity in plant cells and Xenopus oocytes. However, its structural sensory ganglia of the standard laboratory species house a highly diverse pop- organization and function in plants are just beginning to be elucidated. We ulation of neurons, where low-threshold mechanoreceptors - the neurons that have shown that MSL10 is involved in one or more signal pathways that do innervate light touch receptors in the skin - represent only a small fraction. not require its ion conducting ability. Instead, MSL10’s intracellular N-termi- This heterogeneity significantly impedes progress in understanding functional nus was shown to have a regulatory function in the induction of programmed Wednesday, February 11, 2015 563a cell death, depending on its phosphorylation state. Attempting to uncouple at the furrow, compared to their interphase dynamics. This mobility shift did MSL10’s ion channel and regulatory functions in plants, we are performing not arise from a single biochemical event, but rather from global inhibition of an analysis of several key mutations in the putative pore-lining domain. Several protein dynamics by mechanical stress-associated changes in cytoskeletal GOF mutants indicate the critical role of hydrophobic residues of TM6 in chan- structure. We further expanded our genetic and biochemical understanding nel activation. We also have identified LOF mutants with specific changes in of this mechanosensory system using a proteomics approach to identify rele- particular channel properties, such as single channel conductance and tension vant protein-protein interactions. We identified that, in addition to binding to sensitivity when tested in Xenopus oocytes. We hypothesize that the bulky each other, both cortexillin I and IQGAP2 also interact with myosin II under F553 and F563 residues of MSL10 mediate force transduction from the mem- conditions that prevent myosin II-F-actin binding. This validates the high brane to the channel’s pore. This may occur via interaction with hydrophobic crosstalk occurring between various mechanosensitive elements. Mechanical residues of other TMs (like EcMScS F68 and L111) or directly with the fatty tuning of contractile protein dynamics provides robustness to the cytoskeletal acid tails of membrane lipids. Testing these mutants in in planta assays will pro- framework responsible for regulating cell shape and contributes to the fidelity vide insight into the role played by the MSL10 in plant signaling and may iden- of cytokinesis. tify its interacting partners. In summary, our work is beginning to reveal a dual role of MSL10 in plant physiology and the molecular mechanisms governing its 2854-Pos Board B284 behavior and functions. Effects of Physical Loading on Adipogenic Differentiation in 3T3-L1 Preadipocytes 2852-Pos Board B282 Jongyun Choi, Jeongkun Lee, Yeong-Min Yoo, Chi Hyun Kim. Fluid Shear Induces Proarrhythmic Ca2D Wave and Alters Atrial Ca2D Department of Biomedical Engineering, Yonsei University, Wonju, Korea, Signaling: A Role of Autocrine Activations of P2-Purinergic/Type 2 Republic of. Receptor Signaling Adipose tissue is subjected to various types of physical loading. Previous Joon-Chul Kim1, Ju Chen2, Sun-Hee Woo1. studies show that adipocytes have mechanosensitive properties and are affected 1College of Pharmacy, Chungnam National University, Daejeon, Korea, by mechanical stimulation. In this study, we investigated the effects of fluid Republic of, 2School of Medicine, University of California San Diego, La shear stress on adipogenic differentiation using 3T3-L1 preadipocytes. The Jolla, CA, USA. study included 3 groups: (1) No Loading, (2) Loading-Immediate harvest, Cardiac myocytes are subjected to fluid shear force (FSF) during each contrac- and (3) Loading-Post 2 day harvest. In the loading groups, dynamic fluid shear tion and relaxation. Under pathological conditions, such as valve disease, heart stress of maximum 1 Pa at 1 Hz was applied to the preadipocytes for 30 mins. failure and hypertension, FSF in cardiac chamber increases due to high blood GAPDH, ERK, p-ERK, C/EBPb, PPARg proteins were assessed by Western volume and pressure. We have previously shown that FSF induces proarrhyth- blot analysis. Loading resulted in increased p-ERK and reduced C/EBPb ex- mic longitudinal global Ca2þ waves and premature beat in rat atrial myocytes pressions compared to the No Loading group. These changes were more signif- (Biophys J, 2014; 106 (2): 320a). In the present study, we further explored un- icant when the cells were harvested post 2 day loading. However, loading did derlying cellular mechanisms for the FSF-induced longitudinal global Ca2þ not result in significant changes in PPARg expression. C/EBPb plays a major waves and their effects on Ca2þ signaling in rat atrial myocytes. A FSF of role in promoting the differentiation process of preadipocytes and also induces ~16 dyn/cm2 was applied onto entire single myocyte using pressurized fluid PPARg which shows enhanced expressions in the terminal differentiation stage puffing. Confocal Ca2þ imaging was performed to measure local and global of adipocytes. These results indicate that fluid shear stress may suppress adipo- Ca2þ signals. Proarrhythmic longitudinal Ca2þ waves in atrial cells under genic differentiation through C/EBPb inhibition. Greater decrease in C/EBPb FSF were resistant to the blockers for stretch-activated cation channel, Naþ- when cells were harvested post 2 day loading suggests that changes in Ca2þ exchange or Ca2þ-activated cation channel (TRPM4), and to removal C/EBPb are not immediate with loading and therefore longer duration may of external Ca2þ. Interestingly, this FSF-induced atrial Ca2þ wave was elimi- be necessary to observe changes in PPARg expression. Further experiments nated by inositol 1,4,5-trisphosphate receptor (IP3R) blockers, tetracaine, or with increased harvest time as well as experiments using 3T3-L1 preadipocytes by phospholipase C inhibitor, and were absent in the type 2 IP3R (IP3R2) of more advanced differentiation stage are underway. knockout cells. Furthermore, the FSF-induced atrial Ca2þ waves were sup- pressed by blockade of P2-purinergic receptors or by inhibition of gap junction 2855-Pos Board B285 that can secrete ATP. In field-stimulated cells, FSF immediately enlarged Ca2þ Characterization of Biomechanical Properties of Primary Endothelial transients with larger increase in non-junctional Ca2þ release relative to junc- Cells Exposed to Shear Stress 1 1 1 2 tional release. This FSF-induced enhancement in Ca2þ release was soon fol- Nickolas Boroda , Andrew K. Wong , Pierre Llanos , Shahin Rafii , 2þ Sina Y. Rabbany1,2. lowed by significant reductions in Ca transient and sarcoplasmic reticulum 1 2þ 2þ Bioengineering Program, Hofstra University, Hempstead, NY, USA, Ca content. Our data suggest that IP3R2-mediated Ca release, triggered 2 by autocrine activation of P2-purinergic signaling, may generate proarrhythmic Department of Medicine, Weill Cornell Medical College, New York, Ca2þ wave, thereby altering atrial Ca2þ signaling. NY, USA. Mechanotransduction, the process where cells convert biophysical signals into 2853-Pos Board B283 chemical signals, directs the behavior and function of endothelial cells (ECs) The Dynamic Interplay between Cleavage Furrow Proteins in Cellular because of their unique position on the luminal surface of blood vessels. ECs Mechanoresponsiveness are in direct contact with blood and can sense changes in shear stress caused Vasudha Srivastava1, Irina Tchernyshyov2, Jennifer Van Eyk2, by alterations in flow rate. To investigate the biophysical implications of this Douglas N. Robinson1. response we recapitulated physiological levels of shear stress in vitro using 1Cell Biology, Johns Hopkins University, Baltimore, MD, USA, 2Medicine, two systems, imaged and measured the consequent cytoskeletal changes with Johns Hopkins University, Baltimore, MD, USA. immunofluorescence, and quantified cortical stiffness distributions. We devel- Cell shape changes associated with processes like cytokinesis and motility oped a sampling method for Atomic Force Microscopy (AFM) stiffness mea- proceed on several second time-scales. However, they are derived from mo- surements by which force-indentation data is collected in a uniform grid lecular events occurring much faster, including protein-protein interactions, (force map) over multiple cells within a monolayer. In this way, the sampling filament assembly, and force generation. How these fast dynamics define bias resulting from the selection of force-indentation measurements was cellular outcomes remain unknown. While accumulation of cytoskeletal ele- reduced. Hertzian contact modeling for AFM was used to specifically isolate ments is often portrayed as being driven by signaling pathways, mechanical the cortical stiffness of the cell using a pyramidal indenter and small indentation stresses also direct proteins. A myosin II-based mechanosensory system con- depths (100 nm). Nonparametric statistics were used to compare the distribu- trols cellular contractility and shape during cytokinesis and under applied tions of moduli from a given experiment, as the data distribution was not stress. In Dictyostelium, this system tunes myosin II accumulation under me- Gaussian. Actin filament alignment was quantified using Hough Transforms chanical stress by feedback through the actin network, particularly through the on immunofluorescence images and increased when cells were exposed to shear crosslinker cortexillin I. Cortexillin-binding IQGAP proteins are major regu- stress. The medians of stiffness distributions within sheared EC monolayers lators of this system. We examined the dynamic interplay between these key increased in magnitude when compared to static monolayers, yielding a cytoskeletal proteins using fluorescence recovery after photobleaching and method-dependent increase in cortical stiffness of up to 60%. This demonstrates fluorescence correlation spectroscopy, defining the short time-scale dynamics that physiological levels of shear stress dramatically affect ECs morphology in a of these players during cytokinesis and under mechanical stress. Actin and its way that cannot be simulated under static conditions. We concluded that the polar cortex-enriched crosslinkers showed sub-second recovery, while equato- biophysical milieu of a cell’s physiological microenvironment must be rially enriched proteins including cortexillin I, IQGAP2, and myosin II recov- recapitulated in order to represent characteristic cellular mechanical properties ered in 1-5 seconds. Mobility of these equatorial proteins was greatly reduced in vitro. 564a Wednesday, February 11, 2015

2856-Pos Board B286 application of negative pressure to the patch pipette suggesting that the mem- Remodeling of Caveolae Mediates Stretch-Induced Increase of L-Type brane tension resulting from the gigaohm seal formation was sufficient to acti- Calcium Current in Rat Mesenteric Artery vate TRPC6. These results demonstrate that TRPC6 expressed in the plasma Kyung chul Shin1, Sang Woong Park1, Hyunji Park1, Jin-Yeon Park2, membrane of HEK293 cells is mechanosensitive. Future studies will focus on Young-Sun Kang2, Dong Jun Sung3, Hyung-Sik Kim4, Soon-Cheol Chung4, purifying and incorporating the TRPC6 channel protein into liposomes to estab- Jae Gon Kim5, Hana Cho5, Young Min Bae1. lish if TRPC6 is intrinsically mechanosensitive. 1Department of Physiology, KU Open Innovation Center, Research Institute of Medical Science, Konkuk University School of Medicine, Chungju, 2858-Pos Board B288 Chungbuk 380-701, Korea, Republic of, 2Department of Biomedical Science The N-Terminal Domain Acts as an Anchor during the Gating Cycle & Technology, Laboratory of innate immunity, Konkuk University, Seoul, of MscL 3 Navid Bavi1, Takeshi Nomura2, Qinghua Qin3, Boris Martinac1. Korea, Republic of, Division of Sport Science, College of Science and 1 4 Molecular cardiology and Biophysics Division, Victor Chang Cardiac Technology, Konkuk University, Chungju, Korea, Republic of, Department 2 of Biomedical Engineering, Research Institute of Biomedical Engineering, Research Institute, Darlinghurst, NSW, Australia, Department of Molecular 5 Cell Physiology and Bio-Ionomics, Prefectural University of Medicine, Konkuk University, Chungju, Korea, Republic of, Department of 3 Physiology and Samsung Biomedical Research Institute, Sungkyunkwan Kyoto, Japan, Research School of Engineering, The Australian National University School of Medicine, Suwon, Korea, Republic of. University, Canberra, Australia. 2þ This study utilizes a finite element (FE) model to probe the gating mechanism of Background: Voltage-dependent L-type Ca channel (VDCCL) of vascular smooth muscle is facilitated by mechanical stimuli, which is thought to the mechanosensitive channel of large conductance (MscL). When compared to contribute to the myogenic contraction of resistant arteries. However, the molecular dynamics simulations, FE computations are generally more appli- cable due to their much longer simulation times as well as larger size scales. molecular mechanisms underlying the VDCCL activation is controversial. Aim and Methods: We examined the hypothesis that the remodeling of caveo- Herein we use an FE model of MscL, which has been developed as an initial tem- plate to provide not only information about the gating cycle of this channel but lae and its downstream signaling contributes to the facilitation of VDCCL by mechanical stretch in rat mesenteric arterial smooth muscle cells using also to provide a structural framework for a mechanistic understanding of the patch-clamp technique and biochemical analysis. gating of other MS channels at the continuum level. The FE model, unlike mo- Results: Hypotonic membrane stretch (HMS) reversibly increased VDCC lecular dynamics simulations, utilized membrane tension corresponding to L midpoint activation of MscL close to that seen in patch-clamp experiments current. HMS also induced caveolar reorganization as well as the phosphoryla- < tion of caveolin-1. Methyl-b cyclodextrin (MbCD) also reorganized caveolae ( 12 mN/m). For MscL in the open state, our model indicates that the N-termi- and increased VDCC . Subsequent application of HMS did not further increase nus and TM1 of each subunit become aligned to form a single helix. Previous L mutagenic work suggests that the N-terminus of MscL, which connects the the VDCCL, indicating stretch and cyclodextrin increased VDCCL via common pathway. Both the HMS and MbCD phosphorylated c-Jun N-terminal protein TM1 helix to the lipid bilayer at an angle of ~95 in the closed state, is imperative for mechanosensation of the channel. In agreement with experimental data, dele- kinase (JNK), of which inhibition prevented the HMS-induced VDCCL facili- tation. Caveolin-1 knockdown with siRNA blocked HMS-induced JNK tion of the N-terminal domain from our model increases the tensional force 2þ required for the full opening of MscL. Moreover, we observed that during gating, phosphorylation and VDCCL activation. High KCl (70 mM)-induced [Ca ]i increase was markedly facilitated by HMS and this facilitation was sig- the channel gate moves from the inner leaflet region towards the bilayer mid- nificantly attenuated by caveolin-1 knockdown. Finally actin cytoskeletal plane as the membrane thins. Since we do not see this upward movement of disruption with cytochalasin-D blocked HMS-induced phosphorylations of the gate when the N-terminal is removed from our model this further implies caveolin-1 and JNK and facilitation of VDCC and [Ca2þ] increase. that N-terminal region acts as an anchor in the gating process of MscL. L i Supported by the University International Postgraduate Award (UIPA) to N.B. Conclusion: These results indicate hypotonic stretch activates VDCCL by actin-cytoskeleton-dependent caveolar reorganization. Phosphorylation of and APP1079398 grant from NHMRC to B.M. caveolin-1 seems to be closely associated with the caveolar remodeling. Acti- 2859-Pos Board B289 vation of JNK is induced by caveolar remodeling and transduces this signal to The Tension-Activated Channels in the Cytoplasmic Membrane of Vibrio VDCCL activation. These results define a novel function for caveolae, Cholerae cavelolin-1 and JNK in stretch-induced activation of VDCCL. Ian Rowe1, Simona Patange2, Vladislav Belyy3, Anthony Yasmann4, Sergei Sukharev4. 2857-Pos Board B287 1Biochemistry, University of Maryland, College Park, MD, USA, Mechanosensitivity of TRPC6 Ion Channels 2Biophysics, University of Maryland, College Park, MD, USA, 3Biophysics, Yury A. Nikolaev1,2, Paul R. Rohde1, Charles D. Cox1, Derek R. Laver3, University of California, Berkeley, Berkeley, CA, USA, 4Biology, University Boris Martinac1,4. of Maryland, College Park, MD, USA. 1Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Pathogenic Vibrio cholerae easily transitions between fresh water reservoirs Institute, Sydney, Australia, 2School of Biomedical Sciences and Pharmacy, and host intestines, having exceptional adaptation to abrupt osmotic changes. Universety of Newcastle, Newcastle, Australia, 3School of Biomedical Mechanosensitive (MS) channels in the inner membrane act as tension- Sciences and Pharmacy, Universety of Newcastle, Sydney, Australia, sensitive release valves, ejecting osmolytes with water during osmotic down- 4St Vincent’s Clinical School, Universety of New South Wales, Sydney, shock, allowing the bacteria to maintain an optimal turgor within the cell and Australia. avoid lysis. We report the absolute tensions associated with the opening of The transient receptor potential (TRP) ion channel family is a diverse group of MscS- and MscL-like channels (7.3 and ~11 mN/m, respectively), determined channels gated by various physical and chemical stimuli. One of the members, by imaging and simultaneous patch-clamping V. cholerae spheroplasts in transient receptor potential canonical-6 (TRPC6), is a calcium permeable cation whole-cell mode. By cloning the MS channel homologs from V. cholerae channel (PCa/PNa ~ 6) located in the T-tubules of ventricular cardiomyocytes. and studying them in E. coli spheroplasts we determined that the major contrib- Abnormal TRPC6 activity has been implicated in cardiac hypertrophy, a mal- utors to these mechanoelectrical responses are VcMscS, a 287 aa protein 52% adaptive process likely triggered by increased mechanical stress. As a result, identical to EcMscS, a ‘long’ 569 aa homolog VcMscS-L (36% identical to it is important to elucidate the gating mechanism of TRPC6. In addition to acti- EcMscS), and a single VcMscL (136 aa, 65% identity to EcMscL). We found vation by diacylglycerol (DAG) TRPC6 has been reported to be also activated by that the lateral tension in the membrane, which drives gating of these channels, mechanical force, though this has not been supported by other studies. In this can be modulated by asymmetric stress induced by intercalation of exogenous study, we investigated the mechanosensitivity of TRPC6 overexpressed in amphipathic substances. CAI-1, a quorum sensing molecule involved in HEK293 cells using the patch clamp technique. Three TRPC6 constructs were signaling biofilm formation in stressed Vibrio populations has strong amphipa- utilised: N- terminal fusion GFP, C-terminal fusion GFP and non-GFP fusion thicity. Whether CAI-1 permeates through the membrane directly or requires TRPC6. We have successfully transfected these constructs into HEK293 cells specific transporters remains unknown. In response to asymmetric addition of with a 60% transfection efficiency rate using LipofectamineÒ 3000 reagent. CAI-1 we observed shifts of activation curves of V. cholerae MS channels Confocal images of C-GFP TRPC6 demonstrated that the channel is localised toward higher pressure. Measurements of lateral pressures created by different in the plasma membrane. However, N-GFP and non-GFP TRPC6 both distrib- concentrations of CAI-1 in native membranes and in lipid monolayers gave uted uniformly throughout the cells suggestive of impaired trafficking. In our estimations of the membrane association constant as ~106 M-1. This suggests patch clamp experiments we recorded TRPC6 single channel activity in cell- that CAI-1 does not require transporters and permeates through the membrane attached and whole-cell configurations. By applying negative pressure (suction) directly. Thus, tension-activated channels which act as osmolyte release valves of 50 mmHg to the patch pipette we were able to record single channel activity in the cytoplasmic membrane of Vibrio cholerae can be used as lateral pressure of TRPC6 channels. Moreover, the channels were spontaneously active without gauges to detect partitioning of lipophilic signaling molecules. Wednesday, February 11, 2015 565a

2860-Pos Board B290 the upper part of the filament and two protocadherin-15 molecules forming the The Electrophysiology of Mechanosensitive Channels in Pseudomonas lower end. The interaction between cadherin-23 and protocadherin-15 is medi- aeruginosa ated by their N-terminal tips. Missense mutations that modify the interaction Ugur Cetiner, Sergei Sukharev, Ian Donald Rowe, Christina Mayhew, interface impair binding and lead to deafness. Molecular dynamics simulations Andriy Anishkin. predict that the tip-link bond is mechanically strong enough to withstand forces Department of Biology, University of Maryland, College Park, MD, USA. in hair cells, but its experimentally determined strength is unknown. We have Pseudomonas aeruginosa is an opportunistic pathogen that causes infections developed molecular tools to facilitate single-molecule force spectroscopy on mainly in immuno-compromised organisms and is characterized with excep- the tip link bond. Self-assembling DNA nanoswitches are functionalized with tional adaptability to a broad range of habitats, from soil and freshwater to med- the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sor- ical equipment and mammalian tissues. Part of its adaptive potential is the tase under conditions that preserve protein function. These tip link nanoswitches ability to adjust its turgor pressure under conditions of drastically varied osmo- are designed to provide a signature force-extension profile. This molecular signa- larity. The osmolyte release system in this Gram-negative bacterium, like in ture allows us to identify single-molecule rupture events in pulling experiments. E. coli, comprises the high-threshold tension-activated channel MscL and six channels from the MscS family. In the present work we have developed the giant spheroplast system for direct patch-clamp recording from the cytoplasmic Calcium Signaling II membrane of P. aeruginosa and characterized mechanoelectrical responses in 2863-Pos Board B293 the wild-type strain (PA14) using pressure protocols, revealing a complex Probing the Functional Coupling Interface between STIM1 and Orai1 adaptive behavior involving several channel species. We have cloned and func- Xizhuo Wang1,2, Eunan Hendron1, Yandong Zhou2, Jun-Ichi Goto3, tionally characterized PaMscL (137 aa) and two MscS-like channels, PaMscS-1 Katsuhiko Mikoshiba4, Yoshihiro Baba5, Tomohiro Kurosaki5, (278 aa) and PaMscS-2 (283 aa) expressed in E. coli giant spheroplasts. The Youjun Wang6, Donald L. Gill2. ~2 nS PaMscL exhibited short open dwell times and was found non- 1Temple University, Philadelphia, PA, USA, 2Pennsylvania State University, selective. Both PaMscS-1 (~1 nS) and PaMscS-2 (~0.5 nS) have a slight anionic Hershey, PA, USA, 3Yamagata University, Yamagata, Japan, 4RIKEN Brain preference with similar pressure midpoint ratios P MscS/ P MscL of ~0.5. 0.5 0.5 Science Institute, Saitama, Japan, 5Osaka University, Osaka, Japan, 6Beijing Under a standard 10 s pressure-step protocol, PaMscS-1 displays a 30% Normal University, Beijing, China. tension-dependent inactivation, whereas PaMscS-2 under similar conditions þ Store operated Ca2 entry (SOCE) plays a crucial physiological roles and in- shows a 60% inactivation, with extremely slow (5-10 min) recovery. Sequence volves two key proteins, STIM and Orai. Triggered by store-depletion, ER analysis and homology models reveal all components necessary for inactivation STIM proteins aggregate at ER-PM junctions and directly activate PM Orai conserved between P. aeruginosa and E. coli MscS. PaMscL and PaMscS-1 þ Ca2 channels. Few SOCE inhibitors have been described that specifically target perfectly rescue an MS channel-free E. coli strain (MJF465) from abrupt the STIM/Orai machinery. Recently, we revealed a previously described SOCE osmotic shock, whereas PaMscS-2 does not, possibly due to lower conductance modifier, DPB162-AE, potently and specifically blocks STIM1-induced Orai1 and expression level. The electrophysiology of P. aeruginosa gives a better channel activation. We narrowed the DPB162-AE site of action to the STIM- understanding of the components and mechanisms of environmental stability Orai activating region (SOAR) of STIM1. DPB162-AE does not affect SOAR- of this opportunistic pathogen easily changing its habitats. dimerization nor does it prevent the SOAR-Orai1 interaction. However, it potently blocks SOAR-mediated Orai1 channel activation, yet its action is not 2861-Pos Board B291 as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which pre- The N-Terminal Domain of Bacterial Mechanosensitive MscL Acts as a vents both physical and functional coupling to Orai1, we reveal DPB162-AE Mechanosensor: Molecular Dynamics Study rapidly restores Orai-binding of this SOAR mutant but only slowly restores its 1 2 Yasuyuki Sawada , Masahiro Sokabe . activation of Orai1 channel-mediated Ca2þ entry. With the same SOAR mutant, 1Physiology, Nagoya University Graduate School of Medicine, Nagoya, 2 2-APB induces rapid physical and functional coupling to Orai1, but channel acti- Japan, Mechanobiology lab., Nagoya University Graduate School of vation is transient. In STIM2, the equivalent residue to Phe-394 is Leu-485 result- Medicine, Nagoya, Japan. ing in STIM2’s lower efficacy for Orai channel activation. This difference may The bacterial mechanosensitive channel MscL is constituted of homopentamer explain the unusual specificity of DPB162-AE in blocking the action of STIM1 of a subunit with two transmembrane inner and outer (TM1, TM2) a-helices, but not STIM2. We infer that the actions of both 2-APB and DPB162-AE are and its 3D structure of the closed state has been resolved. The major issue of directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, MscL is to understand the gating mechanism driven by tension in the mem- DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncou- brane. To address this question, molecular dynamics (MD) studies have been pler. DPB162-AE provides an important pharmacological tool and a useful mech- performed, however, as they do not include MscL-lipid interactions, it remains anistic probe for the function and coupling between STIM1 and Orai1 channels. unclear which amino acids sense membrane tension and how the sensed force induces channel opening. Thus we performed MD simulations for the opening 2864-Pos Board B294 of MscL embedded in the lipid bilayer. Among amino acids in TM2 facing the STIM1 and STIM2 Proteins Regulation of Endogenous Store-Operated bilayer, Phe78 showed exceptionally strong interaction with lipids. Upon mem- Calcium Channels in HEK293 Cells brane stretch, Phe78 was dragged by lipids, leading to an opening of MscL. Alexey Shalygin, Olga Zimina, Vera Kamaletdinova, Anton Skopin, Thus Phe78 was concluded to be the major tension sensor. Neighboring Lyuba Glushankova, Galina N. Mozhayeva, Elena Kaznacheyeva. TM1s cross and interact with each other near the cytoplasmic side through hy- Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, drophobic interaction between Leu19-Val23 in one TM1 and Gly22 in the Russian Federation. neighboring TM1, forming the most constricted hydrophobic part of the pore The endoplasmic reticulum calcium sensors STIM1 and STIM2 are key mod- called gate. Upon membrane stretch, the helices are dragged by lipids at ulators of store-operated calcium entry. They play a major role in physiological Phe78 and tilted, accompanied by the outward sliding of the crossings, leading functions in the norm and pathology, however, available data on native STIM2- to expanding of the gate. In this study, we newly modeled the Eco-MscL with regulated plasma membrane channels are scarce: it is in only a few studies that the N-terminal (S1) helices running parallel to the cytoplasmic membrane STIM2-induced CRAC currents have been recorded. The STIM1 protein has instead of forming the tight bundle proposed previously and determined the been shown to regulate not only CRAC but also TRPC channels, but it has re- role of the S1 helices in channel opening. As a result, the newly modeled mained unclear whether STIM2 is capable of regulating store-operated non- MscL opened faster than the previous one. Furthermore, some amino acids CRAC channels. Here we present experimental evidence for the existence of in the S1 interact with lipids rigidly, suggesting that the S1 acts as a mechano- endogenous non-CRAC STIM2-regulated channels. In single-channel patch- sensor to transmit force for accelerating the gate opening. clamp experiments on HEK293 cells, store-operated Imin channels were induced by selective activation of native STIM2 proteins or STIM2 overexpres- 2862-Pos Board B292 sion. It was found that STIM1 activation blocked store-operated mode of Imin Single-Molecule Force-Spectroscopy of Inner Ear Proteins activation. Changes in the ratio between active STIM2 and STIM1 proteins Mounir A. Koussa1, Wesley P. Wong2, David P. Corey1. could switch Imin channels regulation between store-operated and store- 1Neurobiology, Harvard University, Boston, MA, USA, 2BCMP, Harvard independent modes. University, Boston, MA, USA. We have previously characterized electrophysiological properties of different Inner-ear mechanotransduction relies on tip links, fine protein filaments made of Ca2þ influx channels coexisting in HEK293 cells. The results of this study cadherin-23 and protocadherin-15 that convey tension to mechanosensitive show that STIM1 and STIM2 differ in the ability to activate these store- channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought operated channels: Imin channels are regulated by STIM2, TRPC3- to form a heterotetrameric complex, with two cadherin-23 molecules forming containing INS channels are induced by STIM1, and TRPC1-composed Imax 566a Wednesday, February 11, 2015 channels are activated by both STIM1 and STIM2. These new data about cross- dent membrane proteins which sense the Ca2þ content of the ER via their talk between STIM1 and STIM2 and their different roles in store-operated luminal EF-hands. After partial or complete store depletion STIM-proteins mul- channel activation are indicative of an additional level in the regulation of timerize and trigger store-operated calcium entry (SOCE) by directly gating store-operated calcium entry pathways. Orai channels localized at the plasma membrane. Here, we report the identifica- This study was supported by the Russian Scientific Foundation 14-14-00720 (to tion and characterization of a novel STIM2 splice variant, named STIM2.1, E.K. and A.S.); the program ‘‘Molecular and Cellular Biology’’ of the RAS (to which differs in a single additional exon consisting of only 8 amino acids located G.N.M and L.G.); the Scientific School Support Program SS-1721.2014.4 within the STIM2 channel activating domain (CAD). We show the novel variant (to G.N.M.); the Dynasty Foundation; and the Russian Foundation for Basic STIM2.1 is present in a variety of primary cells and cell lines although its rela- Research. tive expression varies in regard to the known variant (STIM2.2) and depends on the activation state and cell type. In contrast to STIM2.2, STIM2.1 is unable to 2865-Pos Board B295 gate Orai channels. Coexpression of STIM2.1 together with STIM1 displays STIM1-STIM2 Interactions Modulate Store-Operated Calcium Entry reduced SOCE when compared to STIM2.2 coexpression. Splice variant specific Krishna Subedi, Hwei Ling Ong, Indu Ambudkar. knockdown of STIM2.1 in naı¨ve human CD4þ T cells increases SOCE whereas MPTB, NIH, Bethesda, MD, USA. 2þ 2þ specific down-regulation of STIM2.2 decreases basal calcium as well as SOCE, Store-operated Ca entry (SOCE) provides local and global Ca signals that suggesting that STIM2.1 acts as a negative regulator of STIM mediated SOCE. regulate numerous cellular functions. The stromal interaction proteins STIMs) 2þ Biochemical experiments are being conducted to delineate the functional defects reside within the ER and function as sensors of the ER-[Ca ]. STIM1 and of STIM2.1. STIM2 share a high sequence identity and yet, both have different functions 2þ in the store-operated calcium entry (SOCE) pathway. Following ER-Ca store 2868-Pos Board B298 depletion STIM1 aggregates and translocates to the ER-plasma membrane (ER- Roles of the Orai1 C-Terminus and N-Terminus in 2-APB-Induced STIM1 PM) junctional regions where it binds to and gates plasma membrane Orai1. Coupling STIM2 also clusters in response to stimulation but is a relatively weak activator Yandong Zhou1, Youjun Wang2, Xizhuo Wang1, Natalia A. Loktionova1, of Orai1. In the present study, we investigated the role of STIM2 in SOCE and Xianming Wang1, Donald L. Gill1. show that loss of STIM2 decreases the sensitivity of SOCE activation by the 1Cellular and Molecular Physiology, Penn State University, Hershey, PA, agonist. Knock down of endogenous STIM2 did not affect CCh-induced intra- 2 2þ 2þ USA, Beijing Key Laboratory of Gene Resources and Molecular cellular Ca release but changed the pattern [Ca ]i signals due to SOCE. Sus- Development, College of Life Sciences, Beijing Normal University, Beijing, tained elevations in cells were changed to an oscillatory pattern with loss of 2þ China. STIM2 whereas SOCE-dependent [Ca ]i signals were abolished with The small molecule, 2-aminoethoxydiphenyl borane (2-APB) has complex STIM1 knock down. Further, STIM2 was required for clustering STIM1 in effects on store-operated Ca2þ entry (SOCE). Higher levels (50 mM) strongly ER-PM junctional domains at physiological [CCh], where there is relative 2þ inhibit STIM1-miedated Orai1 channel activation. However, using the poorly less depletion of internal Ca stores. Further analysis revealed critical regions active STIM1 C-terminal domain (S1CT) 235-685), 50 mM 2-APB greatly in STIM2 that determine its interaction with STIM1 and attenuate agonist- enhances binding to and activation of Orai1 channels. We assessed which parts activation of SOCE. Together, our data demonstrate that STIM2 has a critical 2þ of the Orai1 channel were required for this coupling interaction. Using the Orai1 role in SOCE and agonist-stimulated Ca signaling, as it escorts STIM1 to the C-terminal truncation of Orai1 (removal of residues 267-301), 50 mM 2-APB ER-PM junctional domains where STIM1-Orai1 channels are assembled. caused neither binding between S1CT and truncated Orai1 nor Ca2þ entry. 2866-Pos Board B296 We replaced the missing Orai1 C-terminus with the FKBP-rapamycin binding Remodeling of the Cytoskeleton and Regulation of Store-Operated (FRB) domain of the mTOR protein, and added the 12-kDa FK506- and rapamycin-binding protein (FKBP12) to the carboxyl end of S1CT. There was Calcium Entry 2þ Lorena de Souza, Timothy Lockwich, Hwei Ling Ong, Kwong Tai Cheng, no Ca entry even when S1CT and truncated Orai1(1-266) were physically teth- ered through rapamycin-induced FRB/FKBP12 interactions. However, the addi- Indu S. Ambudkar. 2þ MPTB, NIDCR - NIH, Bethesda, MD, USA. tion of 2-APB now recovered full Ca entry. Thus, the C-terminus of Orai1 Store-operated calcium entry (SOCE) is a critical Ca2þ entry and is activated (267-301) is not specifically required and can be substituted by a simple binding by ER-Ca2þ store depletion while refilling the stores leads inactivation of the interaction between FRB/FKBP12. We also revealed using FRET and fluores- process. STIM1 is the main Ca2þ sensor protein in the ER that responds to cence imaging, that the 267-301 C-terminus of Orai1 attached to the CFP- store depletion, aggregates and translocates to ER-PM junctional domains, tagged PM-localization construct, was alone sufficient to trap S1CT upon the where it interacts with, and activates, the channels mediating SOCE, such addition of 50 mM 2-APB. Finally, we identified a single residue (Leu-79) in as Orai1. However, little is known about other proteins that interact with the N-terminus of Orai1 that is critical for the effects of 2-APB on SOCE. The STIM1 to facilitate the regulation of SOCE. In this study, we identified 155 L79N Orai1 mutant completely abolished the activation effect of 50 mM specific STIM1 binding partners using a shotgun proteomic approach on 2-APB on S1CT, but only partially prevented the action of store-induced STIM1 immunoprecipitated complex from HSG cell lysates. In order to deter- STIM1 activation of Orai1. Our data provide new insights into our understanding mine quantitative changes in the STIM1 proteome during stimulation status, of 2-APP-induced activation of STIM1/Orai1 coupling. mixtures of lysates from control cells and stimulated cells with Tg were analyzed using a SILAC (Stable Isotope Labeling by Amino acids in Cell 2869-Pos Board B299 culture) approach. Our analysis reveal several interesting changes in the Menthol-Induced Changes in Mesenchymal Stem Cell Differentiation STIM1 proteome upon stimulation. As a result of the SILAC findings, we Juan C. Henao, Adriana Grismaldo, Yolima P. Torres. found that cytoskeletal interacting/remodeling proteins such as CDC42, Departamento de Nutricio´n y Bioquı´mica, Pontificia Universidad Javeriana, ARP2, N-WASP, and zyxin interact with STIM1 and Orai1. In addition to Bogota´, Colombia. inducing changes in cell morphology and actin cytoskeleton, some of these Channels from the Transient Receptor Potential (TRP) family are involved in also significantly impact SOCE. Together, our data suggest that cytoskeletal changes in intracellular calcium concentration. TRPM8 is a member of this remodeling has an important role in the assembly of the Orai1-STIM1 com- family expressed in different tissues that participates in the activation of plex and regulation of SOCE. signaling cascades involved in thermosensation and proliferation. Recent studies have shown that changes in intracellular calcium concentration are 2867-Pos Board B297 associated with proliferation and differentiation of mesenchymal stem cells A Novel STIM2 Splice Variant Functions as a Break for STIM Mediated (MSC). However, it is unknown whether TRPM8 channels are expressed in Activation of Orai Calcium Channels MSC and if modulation of these channels affects the proliferation and differen- Anna-Maria Miederer1, Dalia Alansary1, Gertrud Schwaer1, Martin Jung2, tiation of this kind of cells. The expression of TRPM8 channels was evaluated Barbara A. Niemeyer1. in adipose-derived human mesenchymal stem cells that have been deeply 1Biophysics, Homburg, Germany, 2Biochemistry, Homburg, Germany. studied due to its potential in regenerative medicine. The results obtained by Ca2þ signaling depends on a tight regulation of the intracellular Ca2þ concentra- RT-PCR, western blot and flow cytometry showed the presence of TRPM8 tion. Alterations in basal Ca2þ can lead to various diseases and likely contribute transcript and protein in adipose-derived human mesenchymal stem cells. to development of abnormal growth. Different regulators such as calmodulin Additionally, it was found that regulation of the TRPM8 activity by menthol and Ca2þ pumps limit cytosolic [Ca2þ] and their down-regulation by siRNA although does not affect cell viability, it decreases the differentiation of lead to an increased basal [Ca2þ]. Another important regulator is the stromal MSC to adipocytes. These results strongly suggest that TRPM8 channels partic- interaction molecule 2 (STIM2) that shows a reduction in basal [Ca2þ] following ipate in the regulation of mesenchymal stem cells differentiation. Supported by knock down. The two known isoforms of STIM, STIM1 and STIM2, are ER resi- Pontificia Universidad Javeriana, Grant ID 3937. Wednesday, February 11, 2015 567a

Intracellular Calcium Channels and Calcium ing tissue in sham. xyt scanning served to characterize Ca sparks at the focal plane, relative to the xyz distribution of mitochondria. Mitochondrial z-stacks Sparks and Waves II were processed using standard procedures, while a xyt spark detector sharing features with published algorithms [Kong et al (2008) Biophys J. 2870-Pos Board B300 95(12):6016-24; Ba´nya´sz et al. (2007) Biophys J. 92(12):4458-65] was created. MICU1 and MICU2 Operate Together to Regulate the Uniporter Spark frequency was plotted as a function of the distance between each spark’s Kimberli J. Kamer1, Vamsi K. Mootha2,3. center of mass (at first detection) and its closest outer mitochondrial membrane 1 Chemistry and Chemical Biology, Harvard University, Cambridge, MA, (OMM). 2 USA, Molecular Biology, HHMI, Massachusetts General Hospital, Boston, Control experiments in healthy permeabilized cells showed that, in our condi- 3 MA, USA, Systems Biology, Harvard Medical School, Boston, MA, USA. tions, ROS were produced by mitochondria. In healthy and diseased myocytes, The mitochondrial calcium uniporter is regulated by the calcium binding, most sparks occurred within 0.5 microns of an OMM. However, it was found EF hand-containing proteins MICU1 and MICU2. These proteins reside in that the relationship between spark frequency and spark center-OMM distance the intermembrane space and physically and functionally interact with the did not change with MI. Yet in experiments in intact cells, scavenging of uniporter channel. While MICU1 and MICU2 are paralogous proteins, we mitochondrial ROS affected spark production in MI predominantly in RyRs now establish that their roles are complementary and nonredundant. Both at a distance from the T-tubules. These results might indicate that ROS pro- proteins appear to be involved in inhibiting calcium uptake when calcium duction by mitochondria likely affects all RyRs due to their ubiquitous nature levels are low, while allowing calcium transport when levels rise. Mutations but that further regulatory factors of RyR activity may be related to the pres- to the EF hand domains suggest that this inhibition is accomplished by the ence of an intact sarcolemma. apo state (calcium free) of MICU1 and MICU2. Finally, the association of MICU2 with MCU, the pore-forming subunit, appears to require MICU1, but 2873-Pos Board B303 MICU1 interacts with MCU even in the absence of MICU2. Taken together, Statin Induced Myopathy: A Role for Mitochondrial Ca2D and No in our results suggest that MICU1 and MICU2 play complementary roles in the Enhanced Sarcoplasmic Reticulum Ca2D Leak regulation of the uniporter. Sabine Lotteau, David MacDougall, Derek Steele, Sarah Calaghan. 2871-Pos Board B301 Faculty of Biological Sciences, School of Biomedical Sciences, Leeds, Activation of Mitochondrial SK Channels in Cardiomyocytes Derived United Kingdom. from Hypertrophic Hearts Attenuates Ca2D-Dependent Arrhythmia by The need to understand the mechanism of statin-induced myopathy is Reducing Mitochondrial ROS Production Thereby Stabilizing RyRs increasing as the cardiovascular risk threshold for statin prescription is TaeYun Kim, Weiyan Li, Karim Roder, Radmila Terentyeva, Gideon Koren, reduced across the globe. Here we determine for the first time the effect of chronic statin treatment in vivo (simvastatin 40 mg/kg/day by oral gavage Bum-Rak Choi, Dmitry Terentyev. 2þ Medicine, Brown University and Rhode Island Hospital, Providence, over a 4 week period) on Ca homeostasis in intact (non-permeabilised) type II rat skeletal muscle fibres, and investigate a role for mitochondria RI, USA. 2þ Small conductance Ca2þ-activated Kþ (SK) channels were recently dis- and NO. In fluo-4 loaded FDB fibres, sarcoplasmic reticulum (SR) Ca leak (indexed in the presence of the SERCA inhibitor cyclopiazonic acid) covered in mitochondria inner membrane. Activation of these voltage-inde- < pendent channels was found to be protective against ischemia-reperfusion was significantly higher (P 0.05) following statin treatment. This could be explained by the observed increase (P<0.001) in the frequency and dura- injury by reducing intracellular levels of reactive oxygen species (ROS). 2þ 2þ tion of Ca sparks/embers in statin fibres. Interestingly, inhibition of the Considering submicromolar [Ca ] sensitivity of SKs we hypothesized that 2þ these channels may play an important role in cardiac hypertrophy. To inves- mitochondrial Ca uniporter by Ru360 in statin fibres normalised spark fre- quency and duration to control levels (P<0.05), suggesting that mitochondrial tigate the role of mSK channels in cardiac hypertrophy we used a rat model þ þ Ca2 uptake contributes to statin effects on SR Ca2 release. NOS inhibition of pressure-overload induced by ligation of ascending aorta (TAB). Using < confocal Ca2þ imaging and patch-clamp we found that inhibition of SK with L-NAME had a greater impact (P 0.05) on NO (indexed with DAF-2) in statin fibres compared with controls, suggesting increased NOS activity channels with cell-permeable inhibitor UCL-1468 (1 uM) increased frequency < of spontaneous Ca2þ waves (SCWs) and delayed afterdepolarizations with statin treatment. This is consistent with reduced expression (P 0.05) (DADs) in TAB ventricular myocytes (TCMs). Conversely, membrane- of GAPDH-normalised and 3 in gastrocnemius muscle from permeable SK enhancer CyPPA (10 uM) attenuated pro-arrhythmic SCWs statin treated animals; caveolins are the main constitutive inhibitors of nNOS and eNOS. Together these data show for the first time that statin and DADs in TCMs. Furthermore, inhibition of mSKs enhanced; and activa- þ treatment enhances SR Ca2 leak in intact muscle fibres. We propose that tion reduced ROS production by mitochondria in TCMs measured with Mito- 2þ Sox. Monobromobimane assay demonstrated that increased oxidation of Ca -dependent mitochondrial ROS production and NO modify RyR to effect ryanodine receptors (RyRs) in TCMs was reversed by CyPPA. Experiments this leak. Defining the cellular processes that underlie statin induced myo- in permeabilized myocytes showed that CyPPA was unable to completely pathy is the first step in the development of co-therapies to improve statin reverse increase in spark frequency in TCMs. However, incubation of compliance. TCMs with CyPPA restored SR Ca2þ content, suggestive of substantial Sponsored by the British Heart Foundation. improvement in RyR function. Optical mapping experiments of TAB hearts using dual Ca2þ and membrane potential imaging revealed that incubation 2874-Pos Board B304 with membrane-permeable SK activator NS309 (2 uM) improved aberrant Rieske Iron-Sulfur Protein-Dependent Mitochondrial ROS-Mediated Ca2þ homeostasis and abolished DADs and VT/VF induced by beta- Dissociation of FKBP12.6/RyR2 Complex Plays an Essential Role in adrenergic stimulation. These data suggest that pharmacological activation Pulmonary Hypertension of mSK channels in hypertrophy protects from Ca2þ-dependent arrhythmia Yong-Xiao Wang, Yun-Min Zheng. via reduction of mitochondrial ROS, and thereby reduction of oxidized RyR Center for Cardiovascular Sciences, Albany Medical College, Albany, leading to its stabilization. NY, USA. An abnormal increase in intracellular calcium concentration ([Ca2þ]i, i.e., þ 2872-Pos Board B302 Ca2 signaling) in pulmonary artery smooth muscle cells (PASMCs) has Altered Ryanodine Receptor Function in Ischemic Heart Disease: Is there been generally believed to play an essential role in the development of pul- a Role for Mitochondria? monary artery contraction and remodeling, thereby leading to devastating Demetrio J. Santiago, Eef Dries, Ilse Lenaerts, Karin R. Sipido. pulmonary hypertension. However, it is unclear which and how ion channels þ Experimental Cardiology, KU Leuven, Leuven, Belgium. may mediate the abnormal increase in Ca2 signaling in PASMCs and Ventricular myocytes remodel after myocardial infarction (MI). Myocytes associated pulmonary hypertension. Herein we have for the first time found þ hypertrophy, mitochondria are affected and the density of T-tubules reduces, that RyR activity and Ca2 release are significantly increased in PASMCs while their distance to ryanodine receptors (RyRs) and mitochondria increases. from mice with hypoxia-induced pulmonary hypertension, a most common Reactive oxygen species (ROS) production may increase and modulate RyR type of pulmonary hypertension in clinic. Consistent with the increased function within microdomains. We investigated the hypothesis that distance RyR functions, SMC-specific RyR2 knockout (RyR2-/-) mice neither þ to mitochondria may be an independent determinant of RyR activity, in partic- show the increased RyR activity and Ca2 release nor develop pulmonary ular after MI. hypertension in mice following hypoxic exposure. Subcutaneous infusion We studied permeabilized myocytes, where the influences of sarcolemma, of the RyR blocker tetracaine produces similar inhibitory effects. Our bulk [Ca] and [ROS] in sham vs. MI are minimized. Cells were isolated biochemical studies demonstrate that the endogenous RyR2 stabilizer from 6-weeks post-MI pig hearts, from the area adjacent to the MI and match- (inhibitor) FK506 binding protein 12.6 (FKBP12.6) is dissociated from 568a Wednesday, February 11, 2015

RyR2 in PASMCs from mice with pulmonary hypertension. FKBP12.6 2877-Pos Board B307 D D knockout promotes, while subcutaneous infusion of the FKBP12.6 stabilizer Effects of Arrhythmogenic Mutations on Ca2 -Induced Ca2 Release S107 prevents, the development of pulmonary hypertension. Intravenous in- Activities of Type 2 Ryanodine Receptors jection of lentiviral shRNAs specific for mitochondrial Rieske iron-sulfur Nagomi Kurebayashi1, Takashi Murayama1, Junji Suzuki2, protein (RISP) gene blocks the increased generation of PASMCs reactive Kazunori Kanemaru2, Masamitsu Iino2, Takashi Sakurai1. oxygen species (ROS), dissociation of FKBP12.6/RyR2 complex, and 1Dept. of Pharmacology, Juntendo University School of Medicine, Tokyo, pulmonary hypertension in mice. Taken together, we conclude that RISP- Japan, 2Dept. of Pharmacology, The University of Tokyo, Tokyo, Japan. þ dependent mitochondrial ROS production may cause FKBP12.6/RyR2 Type 2 ryanodine receptor (RyR2) is a Ca2 release channel on the sarco- complex dissociation, RyR2 hyperfunctions and Ca2þ release in PASMCs, plasmic reticulum and plays a pivotal role in excitation-contraction coupling which leads to pulmonary hypertension. Presumably, specific interventions in heart. RyR2 is the major target for arrhythmogenic diseases, e.g., catechol- targeted at RyR2, FKBP12.6 and RISP may become new and effective aminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic therapeutic strategies to treat pulmonary hypertension and other relevant right ventricular cardiomyopathy (ARVC). To date, over 150 mutations have vascular diseases. been identified in the RyR2 gene of CPVT and ARVC patients. It is widely believed that CPVT and ARVC mutations cause hyperactivation of Ca2þ 2875-Pos Board B305 release and result in ventricular arrhythmia by abnormal Ca2þ homeostasis in Calcium Signaling among RyR2s within a Calcium Release Unit during a cardiac muscle. The RyR2 channel is known to be regulated by both cyto- Calcium Spark plasmic Ca2þ (Ca2þ induced Ca2þ release: CICR) and luminal Ca2þ (store Didier X.P. Brochet1, Gang Wang2, W. Jonathan Lederer1, Heping Cheng2. 2þ 1 2 overload induced Ca release: SOICR). It is well documented that the SOICR Physiology, BioMET, Baltimore, MD, USA, Peking University, Beijing, threshold and termination levels are affected in the disease-associated muta- China. 2þ tions, whereas it remains unclear how these mutations affect CICR. CICR Although Ca sparks were discovered more than two decades ago, very little shows biphasic Ca2þ dependence against cytoplasmic Ca2þ, thus the activity is known about the interactions among the different RyR2s within a dyad 2þ 2þ 2þ 2þ can be determined by three parameters: sensitivity to activating Ca , sensi- (i.e. a Ca release unit, CRU) during a Ca spark. Examination of Ca tivity to inactivating Ca2þ, and the gain (i.e., peak activity). In this study, we signaling within cardiac ventricular myocytes by targeting the nanoscopic þ expressed RyR2 channels carrying CPVT and ARVC mutations in HEK cells ‘‘subspace’’ of cardiac dyads (using a triadin or junctin-targeted Ca2 sensor) 2þ 2þ 2þ and tested their Ca homeostasis by live-cell Ca imaging using gene- was carried out in conjunction with simultaneous visualization of Ca dy- encoded Ca2þ indicators for optical imaging (GECO) and calcium-measuring namics within the cytosol (using rhod-2) and within the sarcoplasmic reticu- 2þ organelle-entrapped protein indicators (CEPIA). In addition, three parameters lum (using Fluo-5N). This combination of Ca signals enabled us to unravel of CICR activity was determined with [3H]ryanodine binding assay. Our results the sequence of activation of the different RyR2s within a dyad during a Ca2þ 2þ suggest that the disease-associated mutations divergently affects the parameters spark. A Ca spark is first initiated by the activation of a single RyR2 on the of CICR depending on the sites for mutation. The underlying molecular mech- edge of the array of RyR2s that will in turn activate several RyR2s within the anism will be discussed. array which activation will propagate by local CICR to the rest of the array. The order of activation of the RyR2s is stochastic, meaning that every Ca2þ 2878-Pos Board B308 sparks are different. Furthermore, the fewer RyR2s that were activated within DHBP Block of Ryanodine Receptor Channels the cluster at the CRU, the smaller the width of sparks. Increasing the Ca2þ Yuanzhao Lv, Julio A. Copello. buffer capacity with EGTA will both narrow the Ca2þ spark width and also Pharmacology, Southern Illinois University, Springfield, IL, USA. decrease the frequency. In this context, Quarky Ca2þ release events (QCRs) 1,10-diheptyl-4,40-bipyridinium dibromide (DHBP), a divalent organic cation, correspond to the activation of one or a very few RyR2s. Taken together, was reported to inhibit muscle contraction, which was attributed to inhibition þ these results suggest that the intra-CRU communication between RyR2s of ryanodine receptor (RyR) mediated Ca2 release from the sarcoplasmic during a Ca2þ spark may be very important for our understanding of diseases reticulum (SR). Still, single RyR channel studies are lacking and the mecha- associated with altered Ca2þ signaling such as arrhythmogenesis and heart nism by which DHBP blocks RyRs is unknown. We reconstituted RyRs from failure. skeletal muscle and heart SR microsomes into planar bilayers. We observed with DHBP addition to the cytosolic surface of RyRs (clamped at Vm ¼ 2876-Pos Board B306 0 mV) decreased channel conductance (IC50 ~ 1 mM). The percentage block Bioorthogonal Calcium Modulation by Direct Intracellular Access using in channel conductance elicited by DHBP was invariable in the range 30 Nanostraws to þ30 mV. DHBP did not affect RyR gating characteristics, which was Alexander Xu, Amin Aalipour, Sally Kim, Nicholas Melosh. measured under Ca2þ conditions that produced low, moderate or high Po Stanford University, Stanford, CA, USA. and in the presence/absence of physiological modulators (ATP, Mg2þ). Ionic calcium plays a ubiquitous role in cell signaling. Calcium signaling uses DHBP added to luminal and cytosolic sides of the RyRs, resulted in additive numerous mechanisms for regulation including coordinated transport across effects on channel conductance. DHBP also induced inhibition of RyR- cell membranes, internal sequestration and storage, and various specific and mediated SR Ca2þ release from skeletal SR microsomes. A similar block of nonspecific buffers. These mechanisms make the generation of ectopic calcium RyRs conductance was observed with other bipyridinum derivatives, none signals difficult, especially if minimal cell perturbation is required. Neverthe- of which affected SERCA ATPase activity. Previous reports in the literature less, manipulating calcium effectively is necessary to study how the cell signals indicated block of RyRs by DHBP in cells required lower drug concentrations, using calcium. Patch clamp pipettes can control intracellular ionic content in suggesting a possible cellular mechanism of DHBP accumulation. In sum- single cells for a limited time. To increase the number of cells studied, chemical mary, DHBP blocks RyRs Ca2þ conductance resembling the action of poly- methods for controlling intracellular calcium can be used such as phorbol amines and organic cations. DHBP properties make it an attractive ester stimulation or thapsigargin release of calcium storage. These indirect experimental probe for understanding the role of RyR conductance in Ca2þ methods can conflate different signaling pathways with calcium signaling. release from intracellular Ca2þ stores (Supported by American Heart Associ- To isolate intracellular calcium from other cell functions, techniques which ation and Eskridge Foundation). do not rely on biological mechanisms of release are needed, such as calcium photouncaging, which uses external light. Here, we propose an alternate strat- 2879-Pos Board B309 egy to introduce calcium to cells in a bioorthogonal fashion to minimize the Filling the Gap Between Calcium Sparks and Waves: Automatic Detection biological footprint. Based on a gap junction-like architecture, we fabricated and Classification of Local Calcium Releases in Cardiac Pacemaker Cells a system of supported nanotubes, called nanostraws. Due to their high aspect Alexander V. Maltsev, Michael D. Stern. ratio (10:1), the nanostraws penetrate into the cell to allow fluidic exchange Laboratory of Cardiovascular Science, National Institute on Aging, NIH, between the cell and an external reservoir, which we use to deliver calcium. Baltimore, MD, USA. We focus on generating temporal signal patterns and controlling the quantity Local calcium releases (LCRs) observed in cardiac pacemaker cells have a of calcium delivered to the cell. To demonstrate the bioorthogonality of nano- complex spatiotemporal structure that has never been studied. We developed straw calcium modulation, we also show that calcium signals can be elicited a computer algorithm for automatic detection and classification of LCRs in through normal stimulation methods. This technique represents a microfluidic simulations of rabbit sinoatrial-node cells (using our recent 3D-model) to get technique to manipulate intracellular calcium which requires no chemical new insights into pacemaker cell operation, specifically, the role of sarco- modification of cells, and the fluidic nature of the delivery can be leveraged plasmic reticulum calcium pumping rate (Pup). to expand the delivery cargo to other biological ions such as zinc or more Identified release events that share a common intensity level are categorized complex biomolecules. as a release cluster, i.e. a complex release with multiple intensity peaks. Wednesday, February 11, 2015 569a

These complex LCRs tend to live longer and propagate farther via calcium- imated by only three layers of diffusively linked intracellular voxels: sub- induced-calcium release, thus occupying larger areas. Release events that membrane (20 nm), ring (1 mm), and core cylinder. Each submembrane don’t share any intensity level with other events are calcium sparks that do RyR cluster is approximated as a Calcium-Release Unit (CRU) residing not live for a long time and do not propagate. Collisions and splits of LCRs within the respective junctional SR linked to free SR which pumps/collects are handled as follows. When an LCR separates into different parts, all parts cytosolic calcium. Instead of assuming a fixed restitution period like prior are still considered part of the LCR. On the other hand, when an LCR collides 2D-models, the release activation and termination are controlled in each with another, the one with the weaker signal mass is considered dead and the CRU by local calcium-dependent mechanisms (based on RyR interactions one with the larger signal mass takes its signal mass as its own. An LCR via Calium-Induced-Calcium-Release, reported recently). The new model may also die by stochastic attrition when all its components fade out. simulations are substantially faster (vs. the original 3D-model), but predict Under voltage clamp, LCR areas and signal masses were paradoxically all essential features of LCRs crucial for pacemaker rate autonomic modula- smaller at larger Pup, likely reflecting uptake of cytosolic calcium before it tion. Thus, super-resolution SIM allowed fine localization of calcium- can propagate. Under spontaneous beating conditions, however, higher Pup dynamics and validated the new model of integrated cardiac pacemaker cell greatly increased diastolic LCR signal mass and beating rate as predicted function at the level of individual CRUs, filling an important niche between by the coupled-clock theory. Interestingly, the total integral of all LCRs individual-molecule-level detail and common pool models (lacking LCRs). during diastolic depolarization in both cases remained almost the same as This new faster 3D-model allows parametric sensitivity analyses and provides longer integration time with smaller events is comparable to shorter time a new mechanistic formulation of CRU function that is important for multi- with larger events. scale modeling of heart function. 2880-Pos Board B310 2882-Pos Board B312 Basal Activity of Epidermal Growth Factor Receptor (EGFR) and Glycoside-Induced Collapse of the Sodium and Calcium Gradients Leads Phospholipase C (PLC) are Required to Sustain Spontaneous Beating of to a Biphasic Effect on Cardiac Cell Pacemaker Function Cardiac Pacemaker Cells Rostislav Bychkov1,2, Syevda Sirenko2, Yael Yaniv3, Victor A. Maltsev2, Tatiana M. Vinogradova, Kirill V. Tarasov, Edward G. Lakatta. Edward G. Lakatta2. BRC, NIA, NIH, Baltimore, MD, USA. 1Pharmacology, Universidad Central del Caribe, Bayamon, PR, USA, Spontaneous firing of sinoatrial node cells (SANC) is controlled by sarco- 2Laboratory of Cardiovascular Science, National Institute on Aging, plasmic reticulum (SR) generated local subsarcolemmal Ca2þ releases Baltimore, MD, USA, 3Biomedical Engineering Faculty, Technion-IIT, (LCRs), which appear during diastolic depolarization (DD) and activate an in- Haifa, Israel. ward Naþ-Ca2þexchange current to regulate DD and SANC beating rate. We A major postulate of a modern coupled-clock pacemaker-cell theory is based have previously demonstrated that constitutive PLC activation is critical for upon electrochemical driving force for sodium and calcium (ENa and ECa). regulation of Ca2þ cycling and spontaneous beating of rabbit SANC. PLC However, [sodium] gradients and their changes induced by glycosides (inhib- could be activated by multiple pathways, including the cAMP mediator, iting sodium/potassium-ATPase) have never been measured. We measured Epac, or activation of EGFR. Activation of Epac signaling by cpTOME, time-dependent changes in intracellular [sodium](Nai), [calcium](Cai), however, was without effect on the spontaneous SANC beating rate, ruling and action-potentials (AP) [using Sodium-Binding-Benzofuran-Isophthalate this pathway out. Whether EGFR is present in rabbit SANC or involved in (SBFI), Indo-1, and perforated patch-clamp, respectively] induced by cardiac the regulation of cardiac pacemaker function is unknown. Here we report glycosides (digoxegenin, 10mM) in single, isolated rabbit sinoatrial node that both EGFR and PLC (assessed by RNA-sequencing) are expressed in cells. During 5 minutes of drug application spontaneous AP firing rate under- rabbit SANC. Specific EGFR inhibitor AG1478 decreased the DD rate and went biphasic changes, i.e. an increase followed by a decrease. During this spontaneous SANC beating rate (perforated patch-clamp recordings). Further- time Nai monotonically increased from 8.550.6 mM to 13.151.1 mM more, EGFR inhibition by AG1478, in a time-dependent manner, suppressed (n¼7 cells). The diastolic Cai also monotonically increased 12456nMto LCRs (confocal microscopy, Ca2þ indicator Fluo-3), i.e. decreased average 149511 nM (n¼5). This Cai increase and biphasic drug effect on AP firing LCR’s size, number per each spontaneous cycle and prolonged the LCR period rate were closely reproduced by a coupled-clock Maltsev-Lakatta model sim- (the interval between AP-induced Ca2þ transient and subsequent LCR); and ulations when the experimentally measured Nai time course was included subsequently eliminated LCRs and abolished SANC firing. The time- depen- in the model as an independent parameter. Model simulations also repro- dent increase in the LCR period in response to AG1478 predicted the concom- duced the initial AP rate increase via a moderate Cai increase (predicted itant increase in the spontaneous cycle length, suggesting that Ca2þ cycling by the model and validated by our Cai measurements). Specifically, when could be a major target of EGFR-PLC-dependent regulation of SANC firing. more cell calcium is available for pumping and release, it boosts the calcium All effects of AG1478 were reversible upon washout. We conclude that clock, driving membrane clocks and the entire coupled-clock system. basal EGFR-PLC-dependent regulation of SR Ca2þ cycling and LCR charac- According to model predictions, subsequent marked increases in Nai and teristics represent novel mechanism to control normal automaticity of cardiac Cai collapse ENa and ECa and lead to AP rate decrease and dysrhythmic pacemaker cells. beating as sodium/calcium-exchanger current decreases, resulting in clock uncoupling. Thus, our results not only explain the complex glycoside 2881-Pos Board B311 effects on pacemaker cell AP-firing rate, but also provide new evidence A New Simplified 3D Model of Cardiac Pacemaker Cell Based on Super- supporting the idea that the normal automaticity of SANC emerges from resolution Structured Illumination Microscopy (SIM) coupled-clock functions, i.e. Cai and membrane ionic currents, that are 1 1,2 3 3 Victor A. Maltsev , Oliver Monfredi , Hari Shroff , Andrew G. York , controlled by sodium and calcium gradients to regulate sodium/calcium- 4 1 1 Anna V. Maltsev , Edward G. Lakatta , Michael D. Stern . exchanger current. 1Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Baltimore, MD, USA, 2Institute of Cardiovascular Sciences, University of 2883-Pos Board B313 Manchester, UK, Manchester, United Kingdom, 3Section on High Resolution Autonomic Stimulation Modulates Action Potential Firing Rate in Cardiac Optical Imaging, National Institute of Biomedical Imaging and Pacemaker Cells via Synchronization of Local Calcium Pumping and Bioengineering, NIH, Bethesda, MD, USA, 4Dept of Mathematics, Release University of Bristol, UK, Bristol, United Kingdom. Oliver J. Monfredi1, Edward G. Lakatta2, Victor A. Maltsev2. The contribution of diastolic Local Calcium-Releases (LCRs) to sinoatrial- 1Institute of Cardiovascular Sciences, University of Manchester, Manchester, node-cell pacemaker function is presently approximated by three numerical United Kingdom, 2Laboratory of Cardiovascular Sciences NIA IRP, NIH, models: 1)‘‘common-pool’’ (Maltsev-Lakatta,2009); 2)‘‘2D’’ (Anna Maltsev Baltimore, MD, USA. et al.,2011-2013); 3)‘‘3D’’ (Stern et al.,2014). While the most advanced A modern view on regulation of cardiac pacemaker cell function postulates 3D-model describes stochastic states of each ryanodine receptor (RyR) and that local calcium releases (LCRs) contribute to diastolic depolarization via L-type calcium-channel, its high computational demand prevents parametric electrogenic sodium/calcium exchanger. An unresolved problem, however, sensitivity analyses. Here we developed a new 3D-model having a lower remains: how intrinsically stochastic and heterogeneously distributed release computational demand, but reproducing all essential features of calcium channels (RyR) generate a strong, synchronized ensemble LCR signal and dynamics measured in isolated rabbit SA node cells by SIM (achieving how this signal is effectively regulated by autonomic system to insure pace- approximately double the resolution of conventional microscopy). Our cell- maker rate flexibility. cross-section SIM measurements revealed that LCRs occur mainly within We measured calcium dynamics by a high-speed camera in isolated, single ~1 mm under the plasma membrane, in line with immunofluorescence rabbit sinoatrial node cells (SANC) and assessed the kinetics of local data on RyR cluster localization. Therefore, the model cell interior is approx- calcium-pumping synchronization by examining the distributions of time 570a Wednesday, February 11, 2015 constants (t) of calcium-transient decay among cell neighborhoods at baseline provide the first direct evidence for gating of TPC1 by NAADP and indicate and during stimulation of either beta-adrenergic receptors or cholinergic that endosomal, rather than lysosomal, Ca2þ release is the key trigger Ca2þ 2þ receptors. pool that sensitizes the IP3-mediated Ca release from the ER. The interaction Beta-adrenergic receptor stimulation (isoproterenol) not only decreased cycle between TPC1-expressing endosomes and the ER are being examined. length (CL) and average t vs. baseline, but also decreased the standard devia- tion (SD) of all t’s across all neighborhoods, suggesting a shift into a less 2886-Pos Board B316 heterogeneous, i.e. more synchronized calcium pumping throughout SANC. Mitochondrial Calcium and Bioenergetics Controlled by Tight Coordina- Conversely, cholinergic receptor stimulation (carbachol) not only increased tion of MCU and NCLX CL and average t vs. baseline, but also increased the local SD(t), suggesting Ming-Feng Tsai, Chuck Phillips, Christopher Miller. Brandeis University, waltham, MA, USA. a shift into a more heterogeneous, i.e. less synchronized calcium pumping 2þ 2þ within SANC. Furthermore, on a beat-to-beat basis, the relationship between Mitochondrial Ca is regulated by Ca transport proteins in the mitochon- either t or SD(t) and CL was linear under both baseline conditions and auto- drial inner membrane. Among these are the mitochondrial calcium uniporter (MCU), a Ca2þ channel responsible for most Ca2þ uptake in mitochondria, nomic stimulation. þ 2þ 2þ Conclusions: The degree of heterogeneity of local calcium pumping is a new and NCLX, a Na /Ca exchanger, mediating Ca release to the cytosol. universal factor that affects the CL and insures effective rate and rhythm After more than five decades of intensive study, the MCU gene was finally regulation of the coupled-clock pacemaker system via autonomic modulation. cloned in 2010. Strikingly, MCU-KO mouse, whose mitochondria lose >90% Ca2þ uptake capacity, was born normally with very mild phenotype More synchronized and faster calcium pumping is presumably achieved via 2þ phospholamban phosphorylation and allows cell neighborhoods to reach and only slight reduction of mitochondrial Ca . How does the animal maintain the calcium release threshold quicker and more synchronously, thereby syn- normal physiology without MCU, which is highly conserved and tightly regu- chronizing LCRs and amplifying their ensemble diastolic signal, accelerating lated in most eukaryotes? pacemaker rate. To address this issue, we use an MCU-KO HEK293T line as our model system. Consistent with animal experiments, these cells have normal mitochondrial 2884-Pos Board B314 morphology, matrix [Ca2þ], and ATP production via oxidative phosphoryla- Excitation-Metabolism Coupling in Mouse Heart tion. Interestingly, we observed a slow Ca2þ uptake in MCU-KO mitochondria, Andrew P. Wescott, W.J. Lederer, George S.B. Williams. operating at 5-10% of the MCU capacity, resistant to a potent MCU inhibitor BioMET, University of Maryland Baltimore, Baltimore, MD, USA. Ru360. Furthermore, these mitochondria lose Naþ-dependent Ca2þ release, A beating heart must balance energy consumption with production. This due to decreased expression of the NCLX protein. We therefore propose that balance requires cellular ATP production to respond dynamically to changes mitochondria compensate the loss of MCU by invoking a backup Ca2þ uptake in cardiac output and energy demand. While it is well understood that mito- mechanism, and by suppressing Ca2þ release. Supporting this hypothesis, we chondrial oxidative phosphorylation serves as the primary pathway for ATP show that expressing NCLX in MCU-free mitochondria drastically reduces production in heart, the mechanisms that govern the regulation of mitochon- matrix [Ca2þ], and abolishes >80% ATP output. Moreover, overexpression drial metabolism remain unclear. Calcium (Ca2þ), specifically Ca2þ in the of WT-NCLX, but not a loss-of-function mutant S468A-NCLX, induces rapid 2þ mitochondrial matrix ([Ca ]m), has been implicated as a likely signaling death of MCU-KO cells. 2þ pathway for this regulation. [Ca ]m has been suggested to regulate nearly We’re now investigating how mitochondria invoke these compensatory every stage of mitochondrial metabolism including the activity of the tricarbox- mechanisms. Naþ/Ca2þ exchange can be restored by introducing human ylic acid cycle (TCA) via Ca2þ-sensitive dehydrogenases (DHOs), critical MCU gene into MCU-KO cells, but not by expressing transport-capable proteins in the electron transport chain (ETC), and even the F1/F0 ATP synthase MCU homologues from A. thaliana or D. Discoideum, implicating that itself. Here we expand our local control mathematical model of excitation- mitochondria might balance Ca2þ uptake and release through an MCU- contraction coupling in mouse heart to investigate excitation-metabolism NCLX interaction that stabilizes inner-membrane expression of NCLX. coupling. The model features mechanistic initiation and termination of Ca2þ 2þ 2þ sparks and cytosolic Ca ([Ca ]i) transients in a system that maintains Nucleo-Cytoplasmic Transport sarcoplasmic reticulum (SR) Ca2þ pump/leak balance. Mitochondrial Ca2þ up- take and export is simulated using experimentally constrained formulations 2887-Pos Board B317 of the mitochondrial Ca2þ uniporter (MCU) and the mitochondrial sodium/ Observing Signal Transduction Directly at the Single-Molecule Level in Ca2þ exchanger (NCLX). The model employs key mechanisms of ATP con- Live Eukaryotic Cells sumption (i.e. SR Ca2þ ATPase and myosin ATPase) and buffering (phospho- Adam J.M. Wollman1, Sviatlana Shashkova2, Erik Hedlund1, creatine) as well as a Ca2þ dependent model for mitochondrial ATP generation. Stefan Hohmann2, Mark C. Leake1. 1 We investigated mitochondrial Ca2þ dynamics at physiological pacing fre- Departments of Physics and Biology, University of York, York, United 2 quencies for mouse heart and show that changes in ATP consumption can be Kingdom, Department of Chemistry & Molecular Biology, University of 2þ translated to increased energy production through [Ca ]m signals. Our model Gothenburg, Gothenburg, Sweden. provides new insights into mitochondrial Ca2þ dynamics and how these All cells dynamically sense their environment through signal transduction 2þ [Ca ]m signals may function to preserve energy homeostasis in the face of mechanisms, allowing them to respond to environmental changes at the genetic increased demand. level. The glucose sensory mechanism in Sacchoromyces cerevisiae is a model system of signal transduction. It uses the multi-copy inhibitor of the GAL gene 2885-Pos Board B315 expression Mig1 protein to repress unwanted genes in the presence of elevated Similarities and Differences in Gating of the Two-Pore Channels TPC1 level of extracellular glucose. In wild-type cell strains, and in genetically and TPC2 and biochemically impaired signal transduction phenotypes, we have fluores- Archana Jha, Malini Ahuja, Shmuel Muallem. cently labelled the Mig1 protein with the green fluorescent protein GFP via nidcr,nih, rockville, MD, USA. chromosomal integration at native levels of expression, in addition to the Endolysosomal Ca2þ homeostasis is implicated in several diseases and controls þ RNAP nuclear reporter protein Nrd1 with the red fluorescent protein mCherry. many endolysosomal functions. A key to understanding endolysosomal Ca2 [1] Using millisecond dual-color fluorescence excitation with Slimfield signaling to understand the function of the newly discovered endolysosomal microscopy, [2] we track the fate of single molecules of Mig1 in live yeast cells PI(3,5)P2-regulated two-pore channels (TPCs) and their potential activation at millisecond timescales. We present data showing a consistent difference be- by NAADP. Our recent work concluded that the lysosomal TPC2 function as tween confined, high stoichiometry assemblages of bound Mig1 in the nucleus 2þ a NAADP and PI(3,5)P2-activated channel regulated by cytoplasmic Mg . and freely diffusing single Mig1 molecules across the entire cell as well as more The properties and gating of the mostly endosomal TPC1 are not known. complex behavior in Mig1 traversing the nuclear membrane. The mobility and Recording whole-organelle currents of enlarged endosomes expressing GFP- stoichiometry distribution of Mig1 assemblages we observe is functionally TPC1, we discovered that TPC1 is potently activated by PI(3,5)P2 and dependent on the signal transduction pathway and the concentration of extra- NAADP. Moreover, PI(3,5)P2 facilitates TPC1 activation by NAADP. Unlike, þ cellular glucose, allowing us to directly observe gene regulatory events. TPC2, the activity of TPC1 showed poor inhibition by Mg2 . Most notably, the 1. Bendrioua, L. et al. Yeast AMP-activated Protein Kinase Monitors Glucose concentration dependence of activation of TPC1 and TPC2 by NAADP are Concentration Changes and Absolute Glucose Levels. J. Biol. Chem. 289, remarkably different. While activation of TPC2 by NAADP followed normal 12863-75 (2014). saturation dependence with no sign of inhibition by high NAADP concentra- 2. Reyes-Lamothe, R., Sherratt, D. J. & Leake, M. C. Stoichiometry and archi- tion, activation of TPC1 by NAADP followed a bell shaped dependency, þ tecture of active DNA replication machinery in Escherichia coli. Science 328, resembling the NAADP-mediated Ca2 release in intact cells. These findings 498-501 (2010). Wednesday, February 11, 2015 571a

2888-Pos Board B318 Phe to surrounding water molecules. iii) The addition of importin b to the In Vivo Analysis of Protein Crowding in the Nuclear Pore Complex during crowded FG-peptide environment resulted in a conformational change of Interphase and Mitosis importin b mediated mainly by the Phe residue in the peptide. Comparison Hide A Konishi1, Suguru Asai1, Tomonobu M Watanabe2, of two different peptides revealed the important role of Ser/Thr residues in Shige H Yoshimura1. the conformational change of importin b. These results suggested that the 1Kyoto university, Kyoto, Japan, 2RIKEN Quantitative Biology Center hydrogen bond network among water and hydroxyl groups of nucleoporins (QBiC), Suita, Osaka, Japan. plays an important role in the formation of the barrier within the NPC and The nuclear pore complex (NPC) of eukaryotic cells regulates macromolecular also in the interaction with transport receptors. traffic between the cytoplasm and the nucleoplasm. The central channel of the pore is thought to form a crowded hydrophobic environment, due to the high 2891-Pos Board B321 content of Phe-Gly (FG) motifs in pore-forming subunits (Nups), which deter- Monitoring and Modeling Effects of IGF1, Insulin and Green Tea mines the selectivity of the pore. Here, we analyzed the spatiotemporal forma- Compound EGCG on Nuclear-Cytoplasmic Distribution of Foxo1-GFP tion of crowded environments within the NPC by utilizing a crowding-sensitive in Skeletal Muscle Fibers fluorescent protein probe (GimRET), which was constructed by fusing ECFP Robert Wimmer1, Sarah Russell1, Bradford Peercy2, Martin Schneider1. 1Department of Biochemistry and Molecular Biology, University of with YFP carrying a single amino acid insertion (YFP-G1). The fluorescent 2 properties of GimRET exhibited sensitivity to high concentrations of protein Maryland, Baltimore, MD, USA, Department of Mathematics and Statistics, (> ~100 mg/mL). When GimRET-fused Nups were expressed in HeLa cells. University of Maryland Baltimore County, Baltimore, MD, USA. The FRET signal in the nuclear envelop revealed that the extent of crowding The transcription factor Foxo1 promotes transcription of the ubiquitin ligases is different among Nups. Nups located in the outer rim of the pore (Nups50, MuRF1 and MAFbx/atrogin1, which contribute to muscle fiber protein break- 153, 214 and 358) exist in a highly crowded environment, whereas Nups down, leading to muscle atrophy and wasting. Foxo1 phosphorylated at its located in the middle channel (Nups54, 58 and 62) exhibited a minimal crowd- protein kinase B (Akt) sites is transported out of nuclei, and Foxo1 dephos- ing, suggesting that a large entropic barrier exits at both ends of the pore. We phorylated at these sites is transported into nuclei. Foxo1 net nuclear efflux also investigated the formation of a crowded environment during post-mitotic decreases Foxo1 transcriptional activity, thus opposing muscle protein break- reassembly of the NPC. Quantitative analysis of the probe signals from meta- down and atrophy. Here we use quantitative fluorescence confocal imaging phase to G1 phase indicated that some Nups are in a less crowded environment of the time course of Foxo1-GFP net nuclear fluxes together with mathematical when dispersed in the cytoplasm at metaphase than when assembled in the NPC modeling of Foxo1-GFP nuclear movements to examine the effects of upstream during interphase. However, some Nups exhibit a similar amount of crowding signaling on nuclear Foxo1. IGF1 and insulin , which activate Akt via the in both cases, suggesting that some Nups remain in subcomplexes, but others PI3k/PDK1/Akt signaling pathway, caused concentration dependent decreases dissociate during mitosis. In addition, a lag period between the localization of nuclear Foxo1-GFP. High IGF1 or insulin caused similar rapid and of Nups around the chromatin surface and the detection of crowding suggests marked Foxo1-GFP net nuclear efflux. ECGC caused a more delayed net that there is a dynamic rearrangement of Nups after assembly on the chromatin loss of nuclear Foxo1-GFP, possibly indicative of need for EGCG to enter surface. the fiber to initiate its effect, whereas IGF1 and insulin work at cell surface receptors. Excluding the delay, the final rate and extent of decay of nuclear 2889-Pos Board B319 Foxo1-GFP was similar for EGCG, IGF1 and insulin. Analysis using our Microinjection of fl-tRNA for the Study of tRNA Subcellular Dynamics recently presented reduced (2 state)model for Foxo nuclear fluxes (Wimmer Sean E. Anderson1, Anna Kashina2, Haim H. Bau1, Barry S. Cooperman3. et al, 2014) showed that IGF1, insulin and EGCG all markedly and similarly 1Mechanical Engineering and Applied Mechanics, University of increased the apparent unidirectional rate constant for Foxo1-GFP nuclear Pennsylvania, Philadelphia, PA, USA, 2Animal Biology/Biochemistry, efflux, as well as markedly decreasing the apparent unidirectional rate constant University of Pennsylvania, Philadelphia, PA, USA, 3Chemistry, University for nuclear influx, indicative of increased Foxo1 phosphorylation in both of Pennsylvania, Philadelphia, PA, USA. cytoplasm and nuclei, both of which contribute to the observed decrease in Transfer-RNA (tRNA) is the key adaptor molecule in protein synthesis, nuclear Foxo1-GFP. Thus, IGF1, insulin and EGCG all activate Akt, both in but recent studies demonstrate behavior and interactions that suggest a far the cytoplasm and within the nuclei. Supported by NIH R01AR056477 and greater role in cellular function. This includes priming viral protein synthesis, by UMB-UMBC Seed Grant retrograde nuclear trafficking, binding to cytochrome C, and post-translational arginylation of proteins. Defects in tRNA modifications are also linked 2892-Pos Board B322 to a number of human diseases with relatively unknown mechanisms. The Autophagosome Marker LC3 Undergoes Regulated Targeting to the Here we describe the use of live-cell imaging techniques to monitor Nucleus and Nucleolus tRNA nuclear-cytoplasmic trafficking and localization in real time via micro- Lewis J. Kraft1, Jacob Dowler2, Anne K. Kenworthy2. 1Chemistry and Chemical Biology, Harvard University, Cambridge, MA, injection of fluorescently-labeled tRNA (fl-tRNA) into adherent mammalian 2 cells. These results represent an important step toward our eventual goal of USA, Molecular Physiology and Biophysics, Vanderbilt University Medical developing assays for the full characterization of the subcellular dynamics Center, Nashville, TN, USA. of specific isoacceptor tRNAs. Autophagy is a lysosomal degradation pathway that is important for the main- tenance of cellular homeostasis. Although the formation of autophagosomes 2890-Pos Board B320 occurs in the cytoplasm, it is increasingly evident that several key proteins in Involvement of Water Molecules in the Formation of Hydrophobic Barrier the autophagy pathway shuttle in and out of the nucleus. We previously in the Nuclear Pore Complex reported that LC3, a protein that participates in autophagosome formation Suguru Asai, Hide A. Konishi, Shige H. Yoshimura. and cargo selection, is enriched in the nucleus in a slowly diffusing form. How- Graduate School of Biostudies, Kyoto University, Kyoto, Japan. ever, the mechanisms that retain LC3 in the nucleus and control its nuclear The Nuclear pore complex (NPC) is a protein complex existing in the nuclear dynamics remain poorly understood. To address this, we used a combination envelope which functions as a selective channel between the cytoplasm and the of fluorescence microscopy and FRAP to study Venus-LC3 in HeLa cells. nucleoplasm. The central channel of the pore, which is composed of Phe/Gly We find that mutating residues involved in binding of LC3 to other proteins motif-rich subunits (FG-nucleoporins), is thought to form a hydrophobic and RNA moderately decreases its nucleocytoplasmic ratio, but disrupting barrier and regulate the passage of soluble proteins and nucleotides. In this LC3 lipid modification does not change its nucleocytoplasmic ratio. This study, we combined biochemical approaches and molecular simulations to suggests that the majority of LC3 in the nucleus is soluble, and it is retained reveal the structural dynamics of nucleoporin interactions with transport recep- in the nucleus by interactions with either proteins or RNA. Consistent with tor proteins. i) Structural analyses of purified transport receptor proteins, such this, soluble nuclear LC3 diffuses more slowly than predicted for a monomer. as importin b, revealed that amphiphilic helices undergo conformational Perturbing the autophagy pathway with rapamycin or chloroquine has little changes in their tertiary structure upon exposure to hydrophobic environments, effect on LC3’s nuclear localization, but does change the apparent size of indicating that the structural flexibility of importin b plays a critical role LC3-associated complexes. This implies that soluble nuclear LC3 associates in passage through the crowded hydrophobic environment of the NPC. ii) with complexes with functions possibly related to autophagy. Unexpectedly, Molecular dynamics simulations of FG-containing peptides (STFGST and we also find that nuclear LC3 is enriched within the nucleolus. We show that AVFGAV) at a high concentration (200 mg/mL) were performed to reveal the triple arginine motif of LC3, a region previously shown to bind to both pro- the dynamics of water molecules around the peptides. The result suggested teins and RNA, shares similarities with a consensus nucleolar detention that Ser/Thr residues around Phe are involved in the formation of hydrogen sequence. Furthermore, perturbation of LC3’s triple arginine motif completely bond networks between the peptides and water and contribute to exposing abolishes its localization to the nucleolus. Together, these findings reveal a 572a Wednesday, February 11, 2015 potential role for both protein-protein and protein-RNA interactions in target- response to sub-threshold depolarization. This work was supported by NIH ing LC3 to the nucleus and nucleolus. 2P20GM103408 to ISU.

Voltage-gated Na Channels 2896-Pos Board B326 Gating Pore Currents are Common Defects of Two Nav1.5 Mutations in 2893-Pos Board B323 Patients with Mixed Arrhythmias and Dilated Cardiomyopathy Resting State of S4 Identified for Each Domain of Nav1.2 using Omega Adrien Moreau1, Pascal Gosselin-Badaroudine1, Lucie Delemotte2, Current Technique Michael L. Klein2, Mohamed Chahine1,3. Claudia Lehmann, Hansjakob Heldstab, Nikolaus G. Greeff. 1Electrophysiology, CRIUSMQ, Quebec, QC, Canada, 2Institute of Biophysics Institute Greeff, Uetikon am See, Switzerland. Computational Molecular Science, Philadelphia, PA, USA, 3Department of During gating transitions the voltage sensor S4 slides through the narrow Medicine, Universite´ Laval, Quebec, QC, Canada. so-called gating pore. The positively charged amino acids of S4, arginine The gating pore current, also called omega current, consists of a cation leak (R) or lysine (K) sense the transmembranal electric field and promote S4 through the typically non-conductive voltage sensor domain (VSD) of voltage in or out. If one or more long R or K is replaced by the short neutral gluta- gated ion channels (VGIC). While the study of gating pore current refined the mine (Q), a leak (omega-) pore is created through which a leak current, the knowledge of the structure and the function of VGIC, their implication in omega current will flow when S4 is at the appropriate position. The occu- cardiac disorders has not been established. Two Nav1.5 mutations (R222Q pancy of this leaky position can then be electrically monitored. Rat brain and R225W) localized in the VSD are associated with complex arrhythmias sodium channels Nav1.2 were studied at high expression in X. laevis oocytes and dilated cardiomyopathy. Using the patch clamp technique, in-silico with two-electrode voltage clamping at strong hyperpolarization to force mutagenesis and molecular dynamic simulations, we tested the hypothesis S4 into the resting state. Mutant channels with single gaps R1Q, R2Q or that these two mutations may generate gating pore currents potentially account- R3Q as well as with double gaps RRn,nþ1QQ were tested for the presence ing for their atypical clinical phenotypes. Our findings suggest that the gating of omega leaks. We found unambiguous omega currents for double gaps pore current generated by the R222Q and R225W mutations could constitute in domain DI (RR12QQ), DII (RR12QQ), DIII (RR23QQ) and DIV the yet unrevealed pathological mechanism linking Nav1.5 VSD mutations (RR12QQ), indicating the resting position of S4 in each domain. These find- with cardiac arrhythmias and dilatation of cardiac chambers in humans. ings are in contrast to skeletal muscle sodium channels Nav1.4 where single gap omega leaks are reported for DII and DIII. However, our single gap 2897-Pos Board B327 mutants in Nav1.2 produced only very small leak currents similar to artifacts Interaction of the Cardiac Sodium Channel Alpha-Subunits Leads to sometimes also occurring at wild-type channels at very strong hyperpolariz- Coupled Gating Properties ing pulses and at least ten times smaller than those with double gaps. Based Je´roˆme Clatot, Haiyan Liu, Eckard Ficker, Isabelle Descheˆnes. on this study, we currently use our double gap channel constructs as a tool to Case Western Reserve University, Case Western Reserve University, OH, selectively investigate which S4 of the four domains I-IV are immobilized by USA. inactivation. Objective: The cardiac sodium channel has been linked to cardiac arrhythmias. We have shown the existence of dominant-negative mutations in Brugada 2894-Pos Board B324 Syndrome due to interactions between alpha-subunits. Here we investigated Selective Immobilization of S4 in Domain III and IV of Rat Brain Nav1.2 the stoichiometry of the interaction and whether the interaction leads to coupled Shown by Omega Currents gating properties. Nikolaus Greeff, Hansjakob Heldstab, Claudia Lehmann. Methods: Single-molecule pull-down experiments and blue native gels have Biophysics Institute Greeff, Uetikon am See, Switzerland. been performed to study the stoichiometry of the interaction, while FRET/ Recently, we have identified the minimally necessary number of mutations of TIRF experiments were performed to investigate the interaction at the cell the outer positively charged arginine (R) or lysine (K) to glutamine (Q) in the surface. Biophysical properties were studied by patch-clamp analysis in the voltage sensor S4 of each domain, producing an inward omega leak current in whole-cell configuration. the resting state. In all four domains a double gap (RRn,nþ1QQ) gave distinct Results: Biochemistry results support the dimerization of alpha-subunits. omega currents. In this study we use these double gap channel constructs as FRET/TIRF experiments showed interacting channels at the plasma mem- a tool to investigate which S4 of the four domains I-IV is immobilized by brane. Also, we investigated if the dimerization of the channel leads to inactivation. The recovery time constant of sodium current after inactivation biophysical consequences. To do so, we used different mutants leading to spe- was measured with a classical double pulse protocol for a wide range of re- cific biophysical. R1860X and R1629Q mutants were used for inactivation covery potentials from 100 to 240 mV. In addition, the onset of the omega and R878C for gating deficiency. When cells expressed WT and R1629Q current at the same recovery potentials was measured twofold: without and mutant channels, inactivation properties behaved more closely to the WT with an inactivating prepulse. We found that the onset of omega current contrarily to what would be expected of 2 channels working independently. was fast and not affected by inactivation in domain I and II; however, in In addition, when R1629Q was coepxressed with the gating deficient mutant domain III and IV the onset was fast without prepulse but was slowed after R878C we showed a significant improvement of the R1629Q inactivation the inactivating prepulse. The return to the resting state seems to be hindered properties, even though R878C is not conducting. We then used a truncated due to immobilization. The time constant of the recovery of omega current channel R1860X and once again the coexpression with R878C significantly matches well the recovery of sodium current over the wide potential range improved the inactivation defect, which was not the case with the expression studied. We corroborated our results by using the mutation R4H in S4DIV, of a C-terminus fragment alone. This, strongly suggest that the presence of which slows the sodium current recovery about twentyfold (Ku¨hn and Greeff, the full length channel could rescue the inactivation defect of the delta-Cter 1999). Adding the mutation R4H in S4DIV to our double gap constructs, we channel. found that the omega current was also slowed by the same factor. This sug- Conclusions: Our data indicate that the alpha-subunits of the cardiac sodium gests that the same mechanism which keeps the alpha pore closed for ionic channel present coupled gating properties due to the formation of dimers. current in inactivated channels would also hinder the return of S4III and S4IV to the resting position. 2898-Pos Board B328 Superresolution Microscopy Reveals Sodium Channel Localization within 2895-Pos Board B325 Intercalated Disk Microdomains: Implications for Ephaptic Coupling Voltage Sensor Domains and Closed-State Inactivation in Sodium Rengasayee Veeraraghavan1, Joyce Lin2, James P. Keener3, Channels Steven Poelzing1, Robert G. Gourdie1. James R. Groome. 1Virginia Tech Carilion Research Institute, Virginia Polytechnic University, Biological Sciences, Idaho State University, Pocatello, ID, USA. Roanoke, VA, USA, 2California Polytechnic State University, San Luis Sodium channels enter into a state of fast inactivation after opening, or directly Obispo, CA, USA, 3Dept. of Mathematics, University of Utah, Salt Lake from closed states. We examined the roles of the four voltage sensor domains City, UT, USA. in hNaV1.4 in closed-state fast inactivation using a mutagenesis approach. Pore-forming (Nav1.5) and auxiliary (b1; SCN1b) subunits of cardiac sodium Charge reversing mutations of outer arginine residues in domains I, III and channels are enriched at the cardiomyocyte intercalated disk (ID). Mathemat- IV depolarized the steady-state fast inactivation curve and accelerated entry. ical models suggest that this may facilitate conduction via ephaptic mecha- Similar effects on closed-state fast inactivation were observed for charge- nisms. We previously demonstrated anisotropic conduction slowing during reversing mutations of inner negative charges in domains I and IV, suggesting acute interstitial edema (AIE), possibly due to weakened ephaptic coupling. that electrostatic interaction of these residues limits S4 translocation in Here we assessed Nav1.5 and b1 localization to ID microdomains using Wednesday, February 11, 2015 573a electron microscopy (EM) and super-resolution microscopy (gSTED, STORM) observed that for the two mutations tested with increased sustained current and used optical mapping and computer modeling to investigate the implica- but without increase in current availability (window current), D1784K and tions for ephaptic conduction in the heart. gSTED and STORM revealed D1790G, the patients had significant lower risk of cardiac events, suggesting Nav1.5 and b1 enrichment within ID regions not containing dense clusters of an increase in current availability may be associated with increased risk for Cx43 and N-Cadherin. Notably, both were identified within the perinexus, a mi- these patients. In addition, we measured mutation specific effects of ranolazine crodomain surrounding Cx43 gap junctions. Overall, 22% of Nav1.5 was for these mutants. For all mutants tested, ranolazine preferentially blocked late located within perinexal regions while only 2% was within Cx43 clusters. sodium currents. Ranolazine shifts steady-state availability of the inactivation EM revealed closer membrane apposition at perinexal (<10nm) vs. non- was dependent on the specific mutant tested. Our results suggest that ranolazine perinexal intercalated disk sites (>10nm) under control conditions. AIE may have a mutation dependent effect. Mutations dysfunction may affect drug increased intermembrane distance at perinexal, but not at non-perinexal sites. binding and drug actions in the channel and may alter treatment effectiveness in Functionally, this correlated with decreased transverse conduction velocity patients. (CV-T; 15.250.3 vs. 19.650.1cm/s) and increased anisotropic ratio (AR; 3.050.2 vs. 2.850.1) relative to control, in perfused guinea pig ventricles. 2901-Pos Board B331 Next, we investigated AIE effects on Nav1.5 function in conduction. Nav1.5 Rotational Symmetry of Two Pyrethroid Receptor Sites in the Mosquito blockade (0.5 mM flecainide) by itself decreased CV (18%) without changing Sodium Channel 1 1 1 2,3 AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T Yuzhe Du , Yoshiko Nomura , Ke Dong , Boris S. Zhorov . 5 5 1Michigan State University, East Lansing, MI, USA, 2McMaster University, (13.0 0.6cm/s), increased AR (3.3 0.2) and increased spontaneous arrhyth- 3 mias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model Hamilton, ON, Canada, Sechenov Institute, RAS, St. Petersburg, Russian including ephaptic coupling and the ID localization of Nav1.5 could recapitu- Federation. late these results. In summary, sodium channel complexes localized to ID mi- Pyrethroid insecticides target voltage-gated sodium channels. Emerging crodomains such as the perinexus may enable ephaptic conduction in the heart. mosquito resistance to widely used pyrethroids demands development of Further, Nav1.5 functional availability and perinexal membrane spacing new insecticides. Earlier the X-ray structure of the open Kv1.2 channel and emerge as novel determinants of anisotropic conduction. mutagenesis data were used to build two homology models of insect sodium channels with pyrethroid receptors PyR1 (O’Reilly et al., 2006) and PyR2 2899-Pos Board B329 (Du et al., 2013) located, respectively, in the II/III and I/II domain interfaces. Loss of Calmodulin-Mediated Regulation of NaD Channel Causes Remod- The models differ in the number of contributing transmembrane helices, eling of Electrical and Junctional Proteins; and Induces Dilated Cardiomy- orientation of the bound pyrethroid molecules, and the depth of their penetra- opathy in IQ/AAD/ Mice tion in respective domain interfaces. Here we employed our PyR2 model to Rosy Joshi-Mukherjee1, Hana Cho2, Takeshi Aiba3, Deborah DiSilvestre1, elaborate an analogous PyR1 model. Computational docking yielded a revised Gordon F. Tomaselli1. PyR1 model with deltamethrin bound between the linker helix IIS4-S5 and 1Medicine, Johns Hopkins Medical University, Baltimore, MD, USA, transmembrane helices IIS5, IIS6 and IIIS6 with its dibromoethenyl and 2Sungkyunkwan University School of Medicine, Suwon, Korea, Democratic diphenylether moieties oriented, respectively, in the intra- and extracellular People’s Republic of, 3National Cerebral & Cardiovascular Center, Osaka, directions. Comparison of the PyR2 and revised PyR1 models predicted Japan. new deltamethrin-channel contacts. Model-driven mutagenesis followed by Sodium channel mutations near the IQ and EFL motifs in the carboxyterminal electrophysiological measurements unveiled two new pyrethroid-sensing res- (CT) domain have been linked to long QT (LQTS) and Brugada syndromes idues in PyR1 and four such residues in PyR2. Taken together, the new and (BrS). IQ-calmodulin (CaM) interaction is important for regulation of cardiac previously published data support the following conclusions. (i) PyR1 is Na channels. The aim of this study was to assess the role of Naþ–Ca2þ/CaM formed by helices IIS4-S5, IIS5, IIS6, and IIIS6. PyR2 is formed by helices signaling via IQ motif of the Naþ channels in development and maturation IS4-S5, IS5, IS6, and IIS6. (ii) Helix IIS6 contains four residues that of intercalated disc (ID). We studied transgenic mice with alanines knocked contribute to PyR1 and four residues that contribute to PyR2. (iii) Seven pairs into IQ positions in the Nav1.5 CT. The homozygous mice are embryonic lethal of pyrethroid-sensing residues are located in analogous positions of domain and heterozygous mice (IQ/AAþ/ mice), develop cardiomyopathy (DCM). interfaces I/II and II/III indicating rotational symmetry of the two pyrethroid We measured the signal and distribution of Nav1.5, syntrophin, Cx43 and rya- receptor sites. (iv) Pyrethroids bind to both sites in similar orientations, nodine in 3 and 9 month old IQ/AAþ/ mice. Results were compared to those deeply penetrating in the respective domain interfaces. Our study elaborates obtained from age matched wild type mice. By immunohistochemistry we the dual pyrethroid-receptor sites model and provides a structural background þ/ show that Nav1.5 protein in 9 month-old IQ/AA mice is significantly for rational development of new pyrethroid insecticides. Supported by NIH reduced at the ID. Syntrophin that traffics Na channels to the membrane, is and NSERC. not altered. Cx43 which is co-located with Nav1.5 at the ID, is significantly reduced. The expression of these proteins were not altered in 3 month-old 2902-Pos Board B332 IQ/AAþ/ mice. We also assessed the implication of IQ domain on the local- Nav1.7 Inhibitor, PF-05089771, Inhibits Fast- and Slow-Inactivated ization of Ca2þ handling protein such as ryanodine receptor and found that it Channels with Similar Affinities was significantly altered in 9 month-old IQ/AAþ/ mice. The data suggest Jonathan Theile, Matthew Fuller, Mark Chapman. þ/ Pfizer - Neusentis, Durham, NC, USA. that enhanced late INa,L in IQ/AA mice contributes to DCM via remodeling of electrical and junctional proteins and demonstrate a dynamic interplay of Voltage-gated sodium channel (Nav) inhibitors are used clinically as analgesics Naþ–Ca2þ/CaM signaling via IQ motif of the Naþ channels in ID development and local anesthetics. However, the absence of Nav channel isoform selectivity and maturation. Our study highlights the importance of Ca2þ/CaM-mediated of current treatment options can result in adverse cardiac and CNS side effects, regulation of Naþ channels in DCM and arrhythmia. limiting their therapeutic utility. Human hereditary gain- or loss-of-pain disorders have demonstrated an essential role of Nav1.7 sodium channels in 2900-Pos Board B330 the sensation of pain, thus making this channel an attractive target for new Mutation Specific Drug Response and Cardiac Risk in Long QT Type 3 pain therapies. We have identified a novel, human Nav1.7 selective inhibitor Elsa Ronzier, Yitschak Biton, Alessandra Matavel, Arthur Moss, (PF-05089771, IC50 ¼ 11 nM) that preferentially interacts with, and stabilizes, Wojciech Zareba, Coeli Lopes. inactivated conformation(s) of the channel via an interaction with the voltage- University of Rochester, Rochester, NY, USA. sensor domain (VSD) of Domain 4. The current study demonstrates that Long QT type 3 (LQT3) is caused by mutations that cause an increase in the PF-05089771 exhibits concentration-dependent slowly developing inhibition cardiac sodium current during late phases of the cardiac action potential. For (tau ¼ 209 sec and 33 sec, at 100 nM and 1 mM, respectively), and a similarly a number of LQT3 mutants this is caused by a failure to inactivate fully, and slow recovery from block upon washout (tau ~7 min). PF-05089771 exhibits a consequent increase in late sodium current. For several mutations, shifts in minimal use-dependent inhibition until concentrations exceed 10-fold the voltage dependence of activation and inactivation cause increased sodium IC50, which is consistent with the observed slow onset of block and/or a low channel contribution to more depolarized voltages, an effect referred as in- affinity for resting or fast-inactivated channel conformations. To evaluate crease in window current. The goal of this study was to perform a study in a this further, we employed whole cell patch clamp protocols to separate large number of LQT3 associated mutations and compare the biophysical effect channels into predominantly fast- or slow-inactivated Nav populations. Inhi- of the mutation to cardiac risk in LQT3 patients and response to treatment. We bition by PF-05089771 develops with similar rates using protocols that biases measured the function of eight common mutations associated with LQT3 with for either fast- or slow-inactivated states, suggesting that preference for a different mechanism underlying channel dysfunction. We compared the func- particular inactivated state (fast, intermediate or slow) appears less critical tional effects of the channel with the clinical course of these patients and than the relative time that the channel is in an inactivated state during 574a Wednesday, February 11, 2015 compound exposure. The inhibition profile of PF-05089771 suggests that a (LCA) and through site-direct mutagenesis identified ‘‘hot-spots’’ at the FGF14 conformational change in the Domain 4 VSD couples to multiple downstream surface critical for binding to Nav1.6. Based on the FGF14 monomer structure inactivated states and immobilizing the voltage-sensor via a small molecule generated in silico, we have designed short peptide fragments that align with interaction with this site may lock the channel into long term inactivation pockets defined by the FGF14 b12-strand and b8-b9 loop and validated their from which recovery is slow. in-cell activity as inhibitors of the FGF14:Nav1.6 complex. We then applied patch-clamp electrophysiology and show exciting preliminary data indicating 2903-Pos Board B333 that two peptides, Fpep1 and Epep1, exhibit either a negative allosteric modu- Structural Modeling of Local Anesthetic Binding to the Pore-Domain of lators (NAM)-like or a positive allosteric modulators (PAM)-like activity Human Nav1.5 in Open and Closed States using Rosetta against Nav1.6-encoded currents. For one peptide, Fpep1, we have begun me- Kevin DeMarco. dicinal chemistry efforts to generate novel peptidomimetics that are currently Biophysics, UC Davis, Davis, CA, USA. being evaluated. These breakthrough results identify the FGF14 b8-b9 and Mutations in voltage-gated sodium (Nav) channel isoforms are correlated with b12 as part of potential druggable pockets against the FGF14-Nav1.6 complex a wide range of cardiovascular and neurological diseases in humans, and are and indicate that small molecule inhibitors (SMI) and/or peptidomimetics therefore are important targets for the rational design of novel drugs. The targeting these pockets might give rise to a new class of unconventional PPI- cardiac isoform of the Nav channel, Nav1.5, presents a unique target for the based allosteric modulators of Nav channels that could restore dysfunction of development of antiarrhythmic drugs. In this work, we identify key structural neuronal excitability and plasticity in brain disorders. These results provide motifs for local anesthetic binding in the pore-domain of the open and closed fundamental new knowledge for the design of new leads targeting the Nav chan- states of the human Nav1.5 channel. The Rosetta structural modeling method nel macromolecular complex. We expect our studies to have a broad impact in was used to construct models of human Nav1.5 isoform based on the 3D crystal the drug design against a wide range of still untreatable brain disorders structures of bacterial Nav channels: NavRh (closed state) and NavMs (open state). The resulting lowest free-energy models were selected for local anes- 2906-Pos Board B336 thetic docking simulations. Rosetta loop modeling and global relaxation of Sodium Selective Conduction, Inactivation and Inhibition Mechanisms 10,000 models yielded a convergent motif in the selectivity filter region of a using the Bacterial NavAb Channel stabilizing hydrogen bond network between Tryptophan and Threonine pairs. Ce´line Boiteux1, Igor Vorobyov2, Robert J. French3, Christopher French4, A membrane-facing fenestration near the S6 helix of domain IV and the S5 he- Vladimir Yarov-Yarovoy5, Toby W. Allen1,2. 1 lix of domain III was also structurally conserved, and is a proposed site of School of Applied Sciences and Health Innovations Research Institute, 2 neutral drug entry. Docking simulations of the channel blocker, lidocaine, RMIT University, Melbourne, Australia, Chemistry, University of 3 reveal key protein-ligand binding configurations within the pore. Our prelimi- California, Davis, Davis, CA, USA, Hotchkiss Brain Institute, University of 4 nary models of the human Nav1.5 channel in the open and closed states reveal Calgary, Calgary, AB, Canada, School of Medicine, University of 5 highly conserved structural motifs important for both stabilization of the pore Melbourne, Melbourne, Australia, Physiology, University of California, domain, as well as for drug entry and binding. Future work will use structural Davis, Davis, CA, USA. models of drug interaction with human Nav1.5 as a dynamic testing platform Voltage-gated sodium channels play essential roles in electrical signaling in the for the calculation of the kinetics of drug binding and unbinding. nervous system and are key pharmacological targets. We have carried out multi-microsecond Anton simulations of the bacterial NavAb channel to reveal 2904-Pos Board B334 ion conduction intimately connected with conformational fluctuations of the Understanding the State Dependence of Voltage Sensor Toxin Action on protein pore (C Boiteux, I Vorobyov & TW Allen, 2014 PNAS 111:3454), Voltage Gated Sodium Channels and a multiple-ion mechanism underlying Naþ versus Kþ selectivity. We Phuong T. Nguyen1,2, Ian H. Kimball1, Kenneth S. Eum1,2, Bruce E. Cohen2, observe collapse of the pore domain, involving residues identified in pore- Jon T. Sack1,2, Vladimir Yarov-Yarovoy1. based slow inactivation and drug binding in prokaryotic and eukaryotic chan- 1University of California, Davis, CA, USA, 2Lawrence Berkeley National nels. We demonstrate high affinity binding of anti-epileptic and local anesthetic Laboratory, Berkeley, CA, USA. drugs to F203, that is a surrogate for the highly conserved FS6 in mammalian Voltage gated sodium (Nav) channels are responsible for initiation and propa- sodium channels, as well as low affinity sites with potential roles in channel gation of action potentials in nerve and muscle. Due to their physiological roles, inhibition. We observe two drug access pathways, including a previously Nav channels are prime targets of natural toxins from a variety of organisms suggested lipophilic route via membrane-bound fenestrations, and an aqueous such as spiders, scorpions, snakes and cone snails. ProTx-II, from the tarantula pathway through the channel pore, despite being closed (C Boiteux, et al. 2014 Thrixopelma prurient, is a 30-residue peptide toxin that is a potent inhibitor of PNAS 111:13057). These studies provide new insight into Nav function and Nav channels. It binds to voltage sensor domains (VSDs) II and IV of human modulation, and predictions to assist future drug development. Nav1.7 channels. It is more than 100-fold selective for Nav1.7 versus all other human Nav channel isoforms. Magi-5, from Macrothele gigas, is a 29-residue 2907-Pos Board B337 peptide toxin that stabilizes an activated state of the domain II VSD of Nav Molecular Dynamics Simulations Describe the Mechanism of K Block in channels. Both spider toxins share a structural fold stabilized by the same disul- Bacterial Nav Channels fide bridge network, yet they have opposite effects on Nav function: ProTx-II Van Ngo1, Yibo Wang1, Sergei Noskov1, Stephan Haas2, Robert A. Farley3. 1 stabilizes a resting state of VSDII, while Magi-5 stabilizes an activated state. Department of Biological Sciences Institute for Biocomplexity and 2 We use solid phase peptide synthesis to generate ProTx-II - Magi-5 chimeras Informatics, University of Calgary, Calgary, AB, Canada, Physics & by inserting loop regions between conserved cystines of Magi-5 into ProTx- Astronomy, University of Southern California, Los Angeles, CA, USA, 3 II. Molecular modeling, protein-protein docking and electrophysiology tech- Physiology & Biophysics, University of Southern California, Los Angeles, niques are used to identify critical residues responsible for opposite effects CA, USA. Although extensive electrophysiological characterization is available for eu- of ProTx-II and Magi-5 on Nav channel function. Our findings may be useful þ in the design of novel modulators of human Nav1.7 channel and may elucidate karyotic voltage-gated Na channels (Nav), no high-resolution structures of important structural determinants of VSD toxin activity. these channels are available. Crystal structures of several bacterial Nav chan- nels have been published and molecular dynamics simulations of ion perme- 2905-Pos Board B335 ation through these channels are consistent with many electrophysiological Targeting Protein:Protein Interaction Sites for Drug Development against properties of the eukaryotic channels. Unlike eukaryotic Nav channels, howev- Voltage-Gated Sodium Channels er, the bacterial Nav channels are strongly outwardly rectifying, and the mech- Syed R. Ali1, Zhiqing Liu1, Miroslav N. Nenov1, Neli I. Panova-Elektro1, anism of this rectification has not previously been described. We used step-wise Jia Zhou1, Svetla Stoilova-McPhie2, Fernanda Laezza1. pulling protocols to implement Jarzynski’s Equality in non-equilibrium molec- 1Pharmacology & Toxicology, University of Texas Medical Branch, ular dynamics simulations of ion permeation through the bacterial NavAb Galvesston, TX, USA, 2Neuroscience and Cell Biology, University of Texas channel to obtain a mechanistic description of this outward rectification. Medical Branch, Galvesston, TX, USA. Results of the simulations indicate that two or three extracellular Kþ ions Fibroblast growth factor 14 (FGF14) is a functionally relevant accessory protein bind tightly at the same z-coordinate along the selectivity filter of NavAb of the neuronal Nav channel. Through a monomeric interaction with the intra- and can effectively block the channel in the presence of modest voltages or con- þ cellular C-terminus of neuronal Nav channels, FGF14 modulates Na currents centration driving forces. The configuration with two potassium ions located at in a Nav isoform-specific manner serving as a fine-tuning regulator of excit- the same z-coordinate is also found in the two-dimensional potential of mean ability. In previous studies we have reconstituted the PPI interaction of forces generated from umbrella sampling and weighted histogram analysis. FGF14 and Nav1.6 in live cells using the split-luciferase complementation assay In contrast to Kþ, three Naþ ions move through the selectivity filter together Wednesday, February 11, 2015 575a as a unit in a ‘‘knock-on’’ mechanism of permeation. Differences in the amount 2910-Pos Board B340 of work required to move three Naþ ions through the selectivity filter of NavAb Expression, Purification, and Preliminary Characterization of a Human compared to three Kþ ions predict the large negative reversal potentials Cardiac Sodium Channel Voltage Sensing Domain observed for bacterial Nav channels in instantaneous current-voltage plots. Mohammed H. Bhuiyan1,2, Sebastien F. Poget1,2. The results of the simulations suggest that the block of bacterial voltage- 1Chemistry, College of Staten Island CUNY, Staten Island, NY, USA, gated Naþ channels by extracellular Kþ does not occur in eukaryotic 2Biochemistry, The Graduate Center CUNY, Manhattan, NY, USA. voltage-gated Naþ channels because of differences in the amino acids present Voltage-sensing domains (VSDs) of voltage-gated ion channels sense changes in the selectivity filters of the different channels. in the membrane potential and as a result alter the conduction state of the channel. The human voltage-gated sodium channel NaV1.5 is primarily ex- 2908-Pos Board B338 pressed in cardiac muscle and is responsible for the rising phase of the cardiac Molecular Dynamics Study of Ion Conduction and Selectivity in a action potential. Mutations within NaV1.5 can lead to fatal cardiac arrhyth- Prokaryotic Ion Channel mias. Such mutations have been found throughout the gene, including Karen M. Callahan1, Benoıˆt Roux2. 1 2 missense mutations within the VSD of repeat IV that have been shown to Physiologie, Universite´ de Montre´al, Montre´al, QC, Canada, Biochemistry lead to Brugada Syndrome and LQT3. The VSDs also play an important and Molecular Biology, University of Chicago, Chicago, IL, USA. role as binding sites for gating modifier peptide toxins from tarantula spider Since the publication of crystal structure of NavAb, the first of a (prokaryotic) venoms. Such toxins could serve as good lead compounds for drug develop- voltage gated sodium channel, several computational studies have been aimed ment due to their high specificity and more subtle mode of action compared at determining the mechanisms of ion selectivity and ion conduction in voltage to pore blockers. Therefore, it would be important to know the structures of gated sodium channels. We provide a two-part study, well-converged free en- the human sodium channels VSDs. While no structures of whole eukaryotic ergy surfaces involving one, two and three ions provide results consistent with sodium channel proteins exist, isolated VSDs of other ion channels have microsecond timescale simulations of ion conduction over concentration and been shown to fold into their native conformation in the absence of the voltages. The position of ions in the pore and cavity correlate to coordination pore forming domain. Therefore, we are pursuing the expression and purifica- number and side chain orientation, showing, as suggested by Chakrabarti et tion of the isolated VSDs of human sodium channels in order to investigate al. (PNAS (2013) 110, 11331). More surprising, presence of an ion in the the structural changes within the VSD caused by pathogenic mutations and aqueous cavity beneath the selectivity filter was sufficient to influence gluta- by the binding of gating-modifier toxins. Here, we present the expression mate conformation. and purification of the human NaV1.5 VSD of repeat IV in a bacterial expres- Because the crystal structure of NavAb was in a closed pore conformation, we sion system in isotopically labeled form and preliminary characterization truncated the S5 and S6 helices and restrained these outer helices harmonically of the truncated protein. NaV1.5 VSD IV is expressed in Escherichia coli in a membrane represented by supporting lattice of neon. The turrets, pore heli- in minimal media, and extracted from membranes by solubilization into n-do- ces and selectivity filter were free to move. Conductances were in the range of the decylphosphocholine micelles. Purified NaV1.5 VSD IV was characterized experimental values; however, our studies show no preference for sodium con- by mass spectrometry and gel filtration chromatography and used for prelim- duction over potassium conduction at physiological conditions ( 200mV, 0.15 inary NMR structural studies. M salt). While sodium conductance varied little with respect to concentration and presence of potassium in solution, stronger potassium selectivity is observed 2911-Pos Board B341 at 1 M and in mixed solutions. Our results are consistent with the observation of A Thermodynamic Analysis of Disease-Causing Mutations in the Nav1.5 anomalous mole fraction effect in NaChBac by Finol-Urdaneta et al. (JGP C-Terminus (2014) 143, 157-171), though not sufficient to confirm such an effect. Addition- Ching-Chieh Tung, Ricardo E. Rivera-Acevedo, Bernd R. Gardill, ally, we provide mechanisms for ion conduction showing a greater variety of Filip Van Petegem. states and transitions occupied during potassium conduction, and note that at Biochemistry and Molecular Biology, University of British Columbia, higher concentrations of salt the mechanism of conduction is not as clearly cut. Vancouver, BC, Canada. The opening of voltage-gated sodium channels (Nav) is responsible for the 2909-Pos Board B339 rapid upstroke of action potentials. A key player during myocardial excitation Coupling of Channel Fluctuations in Ion Permeation and Selectivity in is the cardiac channel isoform Nav1.5. The general architecture of mammalian Bacterial Sodium Channel NavAb Navs is comprised of four homologous domains, containing six transmembrane Christopher Ing1,2, Nilmadhab Chakrabarti1, Ning Zheng3,4, segments each, and a C-terminal intracellular domain (CTD) carrying an IQ- William A. Catterall5,Re´gis Pome`s1,2. 1 motif. The individual domains are connected by large intracellular linkers. Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, The linker connecting domain three and four as well as the CTD seem to Canada, 2Department of Biochemistry, University of Toronto, Toronto, ON, 3 play a role in channel inactivation which is different from regular channel clos- Canada, Department of Pharmacology, University of Washington, Seattle, ing but poses an important function to modulate ion conductance. The rapid WA, USA, 4Howard Hughes Medical Institute, Seattle, WA, USA, 5 inactivation of channels limits the influx of ions and therefore depolarization Molecular Structure and Function, University of Washington, Seattle, of the cell per opening signal. In this context the CTD is of particular interest WA, USA. as an interaction partner for regulatory proteins as calmodulin (CaM) as well as Even though crystallographic structures of several cation channels are known at hotspot for disease-causing mutations that have a profound influence on chan- atomistic resolution, the molecular basis for selective ion permeation, and in nel inactivation. To elucidate the functional effects of disease-causing muta- particular, the role of structural fluctuations of the channel in that process, re- tions we expressed mutant channels in Xenopuslaevis oocytes and studied mains unclear. The determination of structures of voltage-gated sodium chan- them by two-electrode voltage clamp. To complement the data we analyzed nels opens the way to elucidating the mechanism of sodium permeation and the thermostability of isolated mutant CTDs and performed isothermal titration selectivity. Recent molecular simulation studies of bacterial sodium channel þ calorimetry experiments. Isothermal titration calorimetry experiments in the NavAb (Chakrabarti et al., PNAS 110, 11331-11336, 2013) suggest that Na absence and presence of Ca2þ were used to determine binding profiles of indi- binding and permeation through the selectivity filter are coupled to the confor- vidual CaM lobes to WT and mutant CTDs. Our data shows that mutations have mational isomerization of Glu177 side chains from an outfacing conformation þ distinct effects on the folding stability and ability to bind CaM. Whereas some to a lumen-facing conformation, resulting in a high rate of Na diffusion mutations cause misfolding of the CTD, others selectively affect binding of through the selectivity filter. apoCaM or both apoCaM and Ca2þ/CaM, and these changes correlate with To clarify the role of channel dynamics on ion permeation and selectivity, we the disease phenotype. examine the effect of structural constraints systematically. Specifically, we characterize the mechanism of cation permeation in the absence of conforma- 2912-Pos Board B342 tional ‘‘dunking’’ of Glu177 side chains. In addition, we investigate the effect Functional Consequences of a Novel Nav1.9 Mutation (L1302F) causing of structural restraints imposed on the pore helices to prevent channel closure, Congenital Insensitivity to Pain as well as of applied voltage, on channel fluctuations and transport properties. Carlos G. Vanoye1, Tatiana V. Abranova1, Chris C. Ramdoski2, Results of simulations totaling over 100 microseconds indicate that restricting Paul Goldberg3, Charles J. Cohen2, Alfred L. George1. Glu177 conformations, either directly or through global structural restraints on 1Pharmacology, Northwestern University Feinberg School of Medicine, the helices of the pore domain, modulates cation binding and permeation. Chicago, IL, USA, 2Xenon Pharmaceuticals, Burnaby, BC, Canada, 3Xenon Further, applying strong external voltage gradients significantly displaces the Phamaceuticals, Burnaby, BC, Canada. conformational equilibrium of the Glu177 side chains, thereby also modulating The contribution of the peripheral nerve voltage-gated sodium (NaV) channel the mechanism of ion permeation in NavAb. NaV1.9 to nociception has been demonstrated in NaV1.9 knockout mice that 576a Wednesday, February 11, 2015 have greatly reduced inflammatory pain sensation. More recently, mutations in Methods: Commercially available hiPSC-CMs were plated at high density to SCN11A encoding human NaV1.9 have been associated with either loss of pain form monolayers or low density to yield single cells. AP recordings from perception, or familial episodic pain and painful peripheral neuropathy. Here monolayers were made using high resistance electrodes at 36C. Whole cell we elucidate the functional consequences of a novel heterozygous NaV1.9 mu- patch clamp was used to record INa in single hiPSC-CMs at room temperature. tation (L1302F) discovered in a female diagnosed with congenital insensitivity Results: AP recordings showed spontaneous activity with a maximum diastolic to pain (CIP). The mutation was stably expressed in ND7/23 cells and whole- potential (MDP)¼69.251.4 mV and upstroke velocity¼41.956.7 V/s. cell currents were elicited with 50 ms pulses from 120 to þ40 mV from a Application of tetrodotoxin resulted in a slowing of the AP rate but had little holding potential of 140 mV in the continuous presence of 150 nM TTX to effect on AP upstroke or duration. In single hiPSC-CMs, a large INa was þ block endogenous TTX-sensitive sodium currents. Cells expressing NaV1.9- recorded when external Na was reduced to 40 mM (73.656.18 pA/pF). WT exhibited whole-cell current that peaked at 40 mV (31.2 5 3.5 pA/pF, Recovery of INa (hp¼ 120 mV) was very fast; at hp¼ 80 mV, recovery n¼10) with a voltage-dependence of activation defined by V½ ¼61.8 5 of INa was slower and the size of peak INa was greatly reduced (27.053.38 þ 1.5 mV and slope factor (k) of 6.1 5 0.4 (n¼10). By contrast, NaV1.9- pA/pF). Molecular analysis showed that SCN5A was the predominate Na L1302F whole-cell current peaked at 70 mV (39.5 5 8.7 pA/pF, n¼12) channel subtype in both adult and iPSC-CMs. In adddition, we found that and exhibited a significantly hyperpolarized voltage-dependence of activation iPSC-CMs express both the fetal (exon 6A) and adult (exon 6) isoforms of (V½ ¼86.6 5 1.1 mV; k ¼ 6.5 5 0.4; n¼12). These results initially ap- SCN5A. Action potential clamp experiments showed that application of a peared to indicate that the mutation potentiates channel function by enabling ventricular or Purkinje cell waveform to the same hiPSC-CM elicited a large activation at more negative membrane potentials. However, when currents INa while application of a SA node waveform elicited no INa. were recorded using a holding potential of 90 mV, mutant channel activity Conclusion: A large INa is present in hiPSC-CM but its contribution to the was reduced substantially by ~95% (1.5 5 0.3 pA/pF @ 70 mV, n¼7), AP upstroke is minimal. The depolarized MDP coupled with the presence of whereas the WT channel had more preserved activity ~50% (15.6 5 3.1 pA/ phase 4 depolarization results in a take-off potential of 60.651.7 mV which pF @ 40 mV, n¼6). These results suggest that the effect of this mutation inactivates the majority of Naþ channels. is more likely a loss-of-function under physiological conditions, and this will reduce neuronal excitability leading to impaired pain sensation. 2915-Pos Board B345 Nav1.5 C-Terminal Domains Influence Calcium Regulation of Fast Inacti- 2913-Pos Board B343 vation Separately from Calmodulin Interaction Infant Sudden Death: Novel Mutations Responsible for Impaired Nav1.5 Franck Potet1,2, Svetlana Stepanovic2, Sabina Kupershmidt2, Channel Function Alfred L. George, Jr.1. Jace Morganstein1, Kundan Jana1, Monique N. Foster1, 1Pharmacology, Northwestern University, Chicago, IL, USA, Tomoe Y. Nakamura2, Thomas V. McDonald3, Yingying Tang4, 2Anesthesiology, Vanderbilt University, Nashville, TN, USA. þ William A. Coetzee1. The cardiac sodium channel (Nav1.5) has a complex ‘intracellular Ca2 1NYU School of Medicine, New York, NY, USA, 2National Cerebral and sensing apparatus’ within its C-terminal domain (CTD) consisting of a partial Cardiovascular Center Research Institute, Suita, Osaka, Japan, 3Albert EF-hand domain (CTD-EF) and a calmodulin (CaM) binding IQ motif (CTD- Einstein College of Medicine, New York, NY, USA, 4Office of Chief IQ). There are additional CaM binding motifs within the DIII-DIV linker. Vari- Medical Examiner, New York, NY, USA. ation in intracellular Ca2þ concentration influences the voltage-dependence of Sudden infant death syndrome (SIDS) is the leading cause of mortality in steady-state fast inactivation (SSI) by an unclear mechanism. Here, we mutated apparently normal infants. During 2008 to 2012, the New York City Office 16 key residues implicated in the interactions between CTD-EF and CTD-IQ, of Chief Medical Examiner (OCME) examined 274 cases of sudden unex- CTD-IQ and CaM, or DIII-DIV and CaM and explored their impact on NaV1.5 plained death (SUD) of which 141 were infants below 1 year of age, with function and biochemistry. We used quantitative yeast-two-hybrid assays to ~93% of these less than 6 months of age at the time of death. Several measure effects of mutations on the interaction between the full length CTD ion channelopathies were found during genetic screening. An African- on CaM, and evaluated SSI in high (1 mM free Ca2þ) and low [Ca2þ]i condi- American/Hispanic girl who died suddenly in her sleep at the age of 5 weeks tions. Using either BAPTA or HEDTA as chelator, 1 mM free [Ca2þ]i was suf- carried two SCN5A mutations: c.5494 C>G and c.5830 C>T, which respec- ficient to shift SSI (elicited by 50 ms prepulses) towards depolarized potentials. tively introduces a missense mutation Q1832E and an early stop codon We observed that 3 of 5 CTD-IQ mutations (F1912A, A1924T and IQ/AA) R1944X in the distal C-terminus of the cardiac Naþ channel -subunit, strongly reduced the interaction between the CTD and CaM. However, these Nav1.5. HEK-293 cells were transfected with cDNAs of wild-type Nav1.5, mutations did not affect the [Ca2þ]i effect on SSI suggesting that SSI [Ca2þ] Nav1.5-Q1832E, Nav1.5D1944 (the C-terminal truncation) or a cDNA with i sensitivity does not depend on CaM binding to the CTD-IQ. Unexpectedly, both mutations (Nav1.5-Q1832E-D1944) and were subjected to whole-cell single and combination mutations of the CTD-EF (L1786A, F1791A, patch clamping. The peak Nav1.5-Q1832E current was reduced by almost Q1807A, L1862A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) 10-fold (e.g. at 20mV the wild-type Nav1.5 was 283549.1 pA/pF, n¼8 diminished the CTD-CaM interaction, and some of these mutations (L1786A, cf. 31511.8 pA/pF, n¼4, for Nav1.5-Q1832E, p<0.001), whereas E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) also suppressed the Nav1.5D1944 and Nav1.5-Q1832E-D1944 currents were not significantly [Ca2þ]i effect on SSI. Indeed, the only mutations we studied that blunted different from wild-type. The inactivation time constants were unaffected the [Ca2þ]i effect on SSI were within the CTD-EF. These results suggest by any of the mutations (e.g. at 10mV, 1 and 2 respectively were that the CTD-EF influences NaV1.5 [Ca2þ]i sensitivity and mutations in this 1.350.15 and 7.750.87 ms, n¼8 for wild-type cf. 1.150.21 and domain can also alter the interaction of the CTD with CaM, but CaM interac- 7.551.61 ms, n¼4, for Nav1.5-Q1832E). No significant differences were tion with the CTD-IQ is not required for the effect of intracellular Ca2þ on observed for the time course of the recovery from inactivation or the voltage inactivation. dependence of the activation and inactivation kinetic variables. Preliminary biotinylation experiments suggest that the Nav1.5-Q1832E surface expression 2916-Pos Board B346 was unaltered compared to wild-type, suggesting a defect independent of CaMKII-Dependent Regulation of Cardiac Sodium Channel trafficking. In conclusion, the Q1832E mutation was sufficient to produce a Federica Farinelli, Deborah DiSilvestre, Peihong Dong, Yanli Tian, severely dysfunctional Nav1.5 channel, which may have been contributing Gordon Tomaselli. to the victim’s sudden death. Cardiology, Johns Hopkins School of Medicine, Baltimore, MD, USA. Voltage-gated Naþ channels are key determinants of conduction, action po- 2914-Pos Board B344 tential profile and refractoriness in mammalian myocardium. Naþ channels Biophysical and Molecular Analysis of the Sodium Current in Human are regulated by a number of protein kinases and alterations in phosphoryla- Induced Pluripotent Stem Cell-Derived Cardiomyocytes tion are associated with the phenotypic expression of inherited and acquired Brian K. Panama, Robert J. Goodrow, Serge Sicouri, Charles Antzelevitch, heart diseases. Ca2þ/calmodulin-dependent protein kinase II (CaMKII) phos- Jacqueline A. Treat, Jonathan M. Cordeiro. phorylates NaV1.5 at multiple sites in the domain I-II linker, with effects Experimental Cardiology, Masonic Medical Research Laboratory, Utica, on channel gating. The CaMKII phosphorylated sites we identified by mass NY, USA. spectrometry (MS) include serines 459, 460, 484, 539, 571, 664, 667 and Background: Human induced pluripotent stem cell-derived cardiomyocytes threonine 486 in the I-II linker and serines 1925, 1937 and 1969 in the (hiPSC-CMs) have been used for safety pharmacology and to investigate carboxyl-Terminus (CT). In addition we evaluated the effect of CAMKII genetic diseases affecting cardiac ion channels. We examined INa in hiPSC- phosphorylation on NaV1.5-S528A and NaV1.5-R526H channels in which CMs and determined its contribution to action potentials (APs) recorded direct phosphorylation by PKA is abolished (Aiba, 2014). In Wild-type from monolayers of hiPSC-CMs. NaV1.5 channels, acute exposure to CaMKII increased the peak current, Wednesday, February 11, 2015 577a produced a-10 mV shift in the availability curve, slowed recovery from inac- with few states cannot account for all of the kinetics observed with multiple tivation and increased the late Na current (INa-L). These changes were pre- protocols. Increasingly complex models become computationally infeasible vented by inclusion of the CaMKII inhibitor peptide, AIP in the pipette or for multi-scale simulations and their results are difficult to interpret. þ partially prevented by exposure to the PKA inhibitor PKI. NaV1.5-S571A, Methods: Following previously published work for the neuronal Na channel, NaV1.5-S526H and NaV1.5-S528A channels eliminated the CaMKII induced we have implemented a genetic algorithm to optimize model topology and rate shift in inactivation, slowing of recovery and increase in INa-L S571D or parameters for the cardiac sodium channel. The advantage of this approach S528D mimicked the effects of phosphorylation. In NaV1.5-S1969A channels is a flexible topology, with unnecessary states and edges removed in favor exposure to CAMKII increases INa-L compared to NaV1.5-WT with a further of model plausibility and computational speed. We improved this model by increase in INa-L in the presence of CaMKII; however, the steady state inac- parallelizing the computation and including subpopulations with random tivation curve is not shifted. These data suggest the presence of functional mutation rates. Patch-clamp data for model parameterization was collected phosphorylation sites in the CT and the interaction of CaMKII and PKA in by recording transiently transfected HEK293T cells expressing Nav1.5 chan- the I-II linker. The cross talk between CaMKII and PKA modulation of the nels 24 hours post-transfection. The temperature of the recordings was channel may have important implications for electrophysiological properties controlled at various temperatures ranging from room temperature to physio- of the heart. logical temperatures. Models were successfully fit to patch-clamp protocols, consisting of activation curves, inactivation, recovery from inactivation, and 2917-Pos Board B347 activation current traces. Recruitment of Calmodulin to the Tail of the Voltage-Gated Sodium Results: The final optimization employed 32 subpopulations each with Channel Nav1.2 50 members and was run for 2000 generations. After 2 x 106 models were Liam Hovey, Corinne Andresen, Dagan Marx, Madeline Shea. evaluated, the algorithm yielded a model composed of 8 states and 10 intercon- Biochemistry, University of Iowa, Iowa City, IA, USA. necting edges. The model was able to reproduce 20ms, 100ms, and 1000ms Voltage-gated sodium channels (NaV) found in excitable cells are responsible inactivation holds, activation, and recovery from inactivation protocols with for the rising phase of action potentials. These multi-domain transmembrane high fidelity. Fast kinetic data was reproduced by fitting traces for 30mV, proteins are regulated by calmodulin (CaM), a highly conserved eukaryotic 10mV, and 10 mV directly. This novel model fits a wide range of experimen- protein that mediates many calcium-triggered signaling events. Inactivation tally collected data and contains significantly fewer parameters than current, of sodium channels depends on CaM-mediated feedback during repolari- widely used models. zation. In the neuronal sodium channel NaV1.2, CaM binds at least two well-separated sites: an intracellular ‘‘inactivation’’ loop between domains 2920-Pos Board B350 DIII and DIV, and an IQ motif [IQRAYRRYLLK] in the cytosolic C-termi- Rate Constant Models cannot Describe Movement of Charged Atoms nal tail. The IQ motif is hypothesized to recruit calcium-free (apo) CaM, or Molecules making it available to move to the III-IV linker after an influx of calcium. Bob Eisenberg. Despite a high degree of sequence identity, the equilibrium constants for Molecular Biophysics and Physiology, Rush University Medical Center, CaM binding to nine human NaV IQ motifs span more than 3 orders of Chicago, IL, USA. magnitude. Apo CaM binds to the NaV1.2 IQ motif with a dissociation Rate constant models built on laws of mass action are used widely to describe constant (Kd) of ~6 nM, while the Kd for binding the NaV1.9 IQ motif movement of ions and electric current through channels, and chemical reactions is ~ 4 mM. Mutational analysis within the IQ motif has not been sufficient of charged substrates. But mass action laws are derived from conservation of to explain the full range of CaM-binding affinities observed for human mass and say nothing about electric charge. Laws of electricity (Maxwell’s NaV sequences. Thus, we hypothesized that isoform-specific differences in equations: a generalization of Kirchoff’s current law) are about conservation upstream sequences were making energetically significant contributions to of charge, not mass. In classical rate models, flows of ions are not correlated the free energy of binding CaM to NaV1.2. The roles of these residues are by laws of electricity. But Maxwell’s equations strongly correlate flows of being investigated by monitoring CaM binding to biosensors containing charge (including displacement current), without known exception, within 18 mutant sequences of sodium channels bracketed by auto-fluorescent proteins one part in 10 or so. Correlation coefficients (describing correlated flows of YFP and CFP. Residue-specific information obtained by NMR will provide charges in rate models) should then be nearly one, something like 0.999 999 structural insight into the contributions of residues in the binding interface 999 999 999 999. Correlations of charge movement are ignored in classical formed by NaV IQ motif sequences binding to calmodulin from multiple rate models, so classical rate models cannot describe movements of charged eukaryotes. NIH R01 GM57001. atoms or molecules with one set of rate constants over a range of conditions. 2918-Pos Board B348 A proof goes like this: Coupling Compartmental Models to Live Neurons to Investigate Action Consider the spatial series of reactions Potential Mechanisms X <¼¼>Y <¼¼>Z Marco A. Navarro1, Sarah L. Debs2, Lorin S. Milescu1. Currents in a series of reactions analyzed by mass action are not (in general) 1Biological Sciences, University of Missouri, Columbia, MO, USA, equal: 2 Whitman College, Walla Walla, WA, USA. IXY/F ¼ zX kXY [X]-zYkYX [Y]; IYZ/F ¼ zYkYZ[Y]-zZkZY[Z] In mammalian central neurons, action potentials are initiated in the axonal Kirchoff’s current law requires IXY ¼ IYZ under all circumstances and initial segment (AIS) by Nav1.2 and Nav1.6 channels, and shaped and termi- conditions. nated by other voltage-gated ion channels. From the AIS, the AP travels Details can be found at http://arxiv.org/abs/1409.0243 on the physics archive. down the axon towards the presynaptic site, but also back-propagates towards The artifactual difference IXY -IYZ can have large effects. It can produce net the soma. The role of axonal sodium channels in AP initiation and propagation charge and electric fields strong enough to break down membranes, proteins, is still incompletely understood, mostly because it is difficult to record from chemical bonds, and even ionize atoms, because of the enormous strength these channels at the AIS. Instead, most experimental evidence of axonal of the electric field, as described unforgettably in p.1-1, of ‘‘Feynman’s activity is obtained indirectly, from electrical recordings at the soma. To better Lectures on Physics, Vol. 2, Mainly Electromagnetism.’’ http://www.feyn- understand these mechanisms, we developed a real-time computational proce- manlectures.caltech.edu/II_toc.html. dure where a compartmental model of the axon is coupled to a live neuron using dynamic clamp. The properties of this computational model (e.g., spatial distribution and kinetics of ion channels) are varied until the firing activity of Voltage-gated Ca Channels the hybrid construct (neuron þ axon compartmental model) best matches the normal activity of the neuron. 2921-Pos Board B351 Targeting T-Type Channels with Protxii-Like Toxins 2919-Pos Board B349 Autoosa Salari, Mirela Milescu. Optimizing a Nav1.5 Markov-Model with a Genetic Algorithm University of Missouri, Columbia, Columbia, MO, USA. Zach R. Teed, Arie Krumholtz, Jonathon R. Silva. Few gating-modifier toxins have been reported to specifically target T-type Department of Biomedical Engineering, Washington University in St. Louis, calcium channels, and the structural basis of toxin sensitivity remains St. Louis, MO, USA. incompletely understood. Unlike the homotetrameric Kv channels, voltage- þ Background: Markov models of cardiac voltage-gated Na (NaV) channels gated calcium channels are comprised of four different domains, presenting have been widely used for cell and tissue simulations of cardiac electro- the possibility of multiple toxin binding sites. Studies of Kv channels iden- physiology. These NaV1.5 models are of varying complexity. Simple models tified a S3b-S4 helix-turn-helix motif, termed paddle motif, which moves at 578a Wednesday, February 11, 2015 the protein-lipid interface to drive activation of the voltage-sensors. This wildtype: 8.950.5 pA/pF). Summary and discussion: Our data support motif is an important pharmacological target for amphipathic neurotoxins the idea of Rad being involved in regulation of ICaL in diabetes. Regarding and it has been suggested that it is conserved in other voltage-gated ion ICaL Rad seems to act either causal (ICaL decrease in IRS2-k.o. at 16 weeks) channels. Here we show that the four S3b-S4 paddle motifs within the or compensatory (increased Cav1.2 expression but unchanged ICaL in ob/ob T-type calcium channel could be transplanted into four-fold symmetric Kv at 28 weeks). Differences observed between the two investigated diabetic channel to individually examine their contributions to the kinetics of voltage mouse models might be explained by differences in the underlying pathome- sensor activation and pharmacology. Using these chimeric constructs, chanisms (lack of IRS2 vs. leptin deficiency, respectively). we screened existing gating-modifier toxins against the putative paddle motif from each domain of T-type calcium channel, Cav3.1. We found 2924-Pos Board B354 that the four individual paddle motifs of Cav3.1 channels display unique Inhibition of Human CaV2.3 Channels via m-, d- and k-Opioid Receptor toxin binding capabilities, suggesting that gating-modifier toxins can bind Activation to T-type calcium channels in a domain-specific fashion. Comparing Geza Berecki, Leonid Motin, David J. Adams. ProTx-II-like toxins effect on T-type calcium channels and chimeras Health Innovations Research Institute, RMIT University, Melbourne, suggests potential amino acids involved in the direct interaction between Australia. toxin and channels. Neuronal voltage-gated Cav2.3 channels are widely expressed in the central and peripheral nervous system where they contribute to neurotransmission and pain 2922-Pos Board B352 sensation. However, modulation of the Cav2.3 channel through G protein- Engineering Selectivity in RGK Protein Inhibition of Cav1/Cav2 Channels coupled (GPC) m- and d-opioid receptors is poorly defined and has not previ- Akil Puckerin. ously been reported for k-opioid receptors. We hypothesized that activation Columbia University, New York, NY, USA. of human m-, d-ork-opioid receptors modulates Cav2.3 channels via G protein 2þ High-voltage activated calcium channels (CaV1.1–CaV1.4; CaV2.1–CaV2.3) signaling. Whole-cell Ba currents were recorded in HEK293T cells co- link electrical signals to vital physiological responses in excitable cells. expressing human Cav2.2 or Cav2.3 channels and m-, d-ork-opioid receptors. Molecules that block CaV1/CaV2 channels are important therapeutics. Rad/ Selective opioid receptor agonists and antagonists were used to study receptor Rem/Rem2/Gem (RGK) proteins are small Ras-like G-proteins that potently modulation. The involvement of intracellular signaling pathways was investi- and indiscriminately inhibit all CaV1/CaV2 channels. The practical utility gated using specific inhibitors of GPC receptor-G protein coupling. Activation of RGKs as genetically-encoded CaV channel blockers would be vastly of m-, d-ork-opioid receptors inhibited Cav2.3 and Cav2.2 channel current improved if it were possible to engineer versions that display selective inhi- amplitude by ~45% and ~60%, respectively. Inhibition of Cav2.3 was not bition of distinct CaV1/CaV2 isoforms. CaV1/CaV2 pore-forming a1 subunits dependent on the type of subunit co-expressed. Inhibition of the Cav2.3 channel require binding to auxiliary CaVbs to generate functional channels. All RGKs was primarily voltage independent, as depolarizing prepulses could not relieve bind wild-type (wt) CaVb and this interaction is disrupted in a mutated the inhibited current. This was in marked contrast with the primarily voltage- CaVb (CaVbTM). We compared the ability of Rem and Gem to inhibit four dependent modulation of Cav2.2 channels that showed nearly complete recov- distinct CaV1s (CaV1.2, CaV1.3, CaV2.1 and CaV2.2) reconstituted with either ery of the inhibited current with depolarizing prepulses. For all three types of wtCaVb or CaVbTM in HEK293 cells. While both Rem and Gem blocked opioid receptors, the pathway leading to Cav2.3 channel inhibition was sensi- all channels reconstituted with wtCaVb, Rem uniquely suppressed CaV1.2þ tive to pertussis toxin and intracellular application of GDP-b-S. Similarly, b2aTM channels, a signature of CaVb-binding-independent inhibition. Using the overexpression of a G protein subunit scavenger, myristoylated- FRET analyses, chimeric Rem/Gem proteins, and electrophysiology we phosducin, significantly reduced the magnitude of Cav2.3 channel inhibition. show that CaVb-binding-independent inhibition of CaV1.2 involves direct Here we demonstrate that Cav2.3 channels are efficiently inhibited by activa- interaction of Rem C-terminus with CaV1.2a1C N-terminus, and additionally tion of m-, d-ork-opioid receptors. Inhibition occurs via voltage-independent requires the Rem nucleotide-binding domain. A mutant Rem that no longer G protein signaling mechanisms. These results suggest opioid receptor controls interacts with CaVb (Remb-null) selectively inhibited CaV1.2þwtCaVb specific members of the Cav2 channel family via differential signaling channels. We further profiled the prevalence of b-binding-dependent pathways. Neuronal Cav2.3 channels are therefore potential targets for opioid and -independent mechanisms of inhibition by Rem2 and Rad across CaV1/ analgesics. CaV2 channel families. While Rem2 relied on CaVb binding to inhibit all four CaVs tested, Rad displayed b-binding-independent inhibition of 2925-Pos Board B355 CaV1.2 and CaV2.2. Consistent with this, Remb-null selectively inhibited Control of Functional Targeting of Cav1.2 Channels by the g6 CaV1.2 and CaV2.2 channels reconstituted with wtCaVb. In summary, our Roman Shirokov, Thomas Comollo, Rose Rendon. results have revealed a latent capability of distinct RGK proteins to block Pharmacology and Physiology, NJMS, Newark, NJ, USA. particular CaV1/CaV2 channels in an a1-subunit-specific manner. We have g6 subunit of voltage-gated calcium channels is expressed in the heart and exploited this feature to generate genetically-encoded CaV-isoform-selective in the brain. It modulates gating of T-type channels (Hansen et al., 2004; inhibitors. Lin et al., 2008). It also associates with CaV1.2 channels and enhances their inactivation in the presence of the b1b, but not b2b, subunit (Yang et al., 2010). 2923-Pos Board B353 We found that g6 subunit dramatically reduces the number of functional a1/ Ventricular L-Type Ca2D Channels and Expression of RGK Proteins in b2a channels expressed in tsA-201 cells. Channels with the g6 are trapped in Mouse Models Associated with Diabetes the Golgi complex. Deletions of N-termini of a1 and g6 subunits restore func- Jessica Ko¨th, Christian Fabisch, Stefan Herzig, Jan Matthes. tional targeting to the plasma membrane. Department of Pharmacology, University of Cologne, Cologne, Germany. We propose that the g6 regulates functional expression of CaV1.2 channels by interacting with the pore-forming a1 subunit or with another protein in the Background: In a diabetic mouse model (db/db) we have shown reduced ICaL density with unchanged single-channel activity and reduced expression of the complex. LTCC pore Cav1.2 (Pereira et al., Diabetes 2006;55:608-15). Of note, LTCC expression and function can be decreased by RGK proteins, including the 2926-Pos Board B356 D diabetes-associated protein Rad. Aim of the study: In the present study, we L-Type Ca2 Channel Cavb Subunits Associate with and Differentially D investigate the association between cardiac Rad expression with the expression Regulate the Cardiac Cav3.2 T-Type Ca2 Channel Currents and function of ventricular LTCC in two mouse models with diabetes-related Marites T. Woon1, Ravi C. Balijepalli2. metabolic disturbances (leptin-deficient obese ob/ob mice and insulin receptor 1University of Wisconsin, Madison, Madison, WI, USA, 2Medicine, substrate 2 deficient IRS2-k.o. mice). Methods: We obtained expression of Rad University of Wisconsin, Madison, Madison, WI, USA. and Cav1.2 protein (Western-blot) and mRNA (qRT-PCR) in murine ventricles Low voltage activated T-type calcium channels (TTCC) play a pivotal role in and recorded whole-cell ICaL in freshly isolated ventricular myocytes. Results: the developing heart. Although the TTCC isoforms, Cav3.1 and Cav3.2, under- The only significant change at the mRNA level was an increased Rad expres- lie cardiac TTCC current (ICa,T) and are expressed in atrial and ventricular sion in IRS2-k.o. mice at 16 weeks of age (206517%). In line with this finding myocytes during development, their expression and roles recede in the adult ICaL density was significantly decreased (IRS2-k.o.: 7.850.8 pA/pF; wild- heart. However, previous studies have demonstrated the re-expression of type: 10.950.9 pA/pF). At an age of 28 weeks, we found expression of ICa,T in pathological cardiac hypertrophy, suggesting that TTCCs contribute 2þ both Rad and Cav1.2 protein to be significantly increased in ventricles from to the altered Ca cycling and signaling in these pathological conditions. ob/ob mice compared to age-matched widtypes (273523% and 159514%, In addition, the reported altered expression of some Cavb subunits (specif- respectively) while ICaL density was unchanged (ob/ob: 8.450.4 pA/pF; ically b1 and b2 subunits) of the high voltage activated L-type calcium Wednesday, February 11, 2015 579a channels (LTCC) has been reported in hypertrophy and heart failure. We hy- was sensitive to the inhibitors of intracellular protein trafficking. The NE- pothesize that the altered expression of the Cavb subunits in cardiac hypertro- induced T-type channel mediated an increased calcium entry during short de- phy and heart failure results in the altered coupling and regulation of ICa,T.We polarizations and supported modest transient electrical responses to depolariz- found an increase in the Cavb1 and Cavb3 mRNA and Cavb2 and Cavb4 sub- ing stimuli. Such experiments reveal a potential for circadian regulation of unit isoform at the protein level in mouse ventricles in a transthoracic aortic pinealocyte electrical excitability and calcium signaling. This work is sup- constriction (TAC) induced pathological cardiac hypertrophy model. Whole- ported by National Institutes of Health grants GM-83913, NS-08174 and cell patch clamp electrophysiology using transiently transfected HEK293 cells DK-080840. revealed that co-expression of Cavb1 or Cavb2 with Cav3.2 channel isoform resulted in a significant increase in peak ICav3.2 and a rightward shift in the 2929-Pos Board B359 V1/2 of activation and significantly slower inactivation of ICav3.2. On the other Single-Channel Analysis of the Inhibition of the Calcium Dependent Inac- hand, co-expression of Cavb3 or Cavb4 significantly reduced the peak ICav3.2. tivation by the C-Terminal Modulator Domain of Cav1.3 Channels In contrast, co-expression of Cavb isoforms did not alter ICav3.1. Furthermore, Elza Kuzmenkina, Elena Novikova, Wanchana Jangsangthong, co-immunoprecipitation studies in transiently transfected HEK293 cells also Stefan Herzig. demonstrated that the Cav3.2 channel separately co-immunoprecitated with Pharmacology, University of Cologne, Cologne, Germany. anti-Cavb1 or anti-Cavb2 antibody. In conclusion, our data suggest that the Cav1.3 channels belong to the family of the voltage-gated L-type calcium chan- Cavb1 and Cavb2 subunits of the LTCC may regulate ICav3.2 in cardiomyocytes nels. Because of activation at low voltage thresholds, Cav1.3 channels are during pathological cardiac hypertrophy. involved in regulation of the cell firing but they are also linked to the generation of the oxidative stress. Calcium-dependent inactivation (CDI) is a negative 2927-Pos Board B357 feedback process terminating the potentially toxic calcium influx (Johny et Homologous Serine/Threonine in the CaV 2.2a1 and 2.3a1 Subunits Behave al., Nat Commun 2013;4:1717). Multiple Cav1.3 splice isoforms, which can Similarly, as Stimulatory and Inhibitory PKC Sites be simultaneously expressed in the same tissue, differ in their channel gating Ganesan L. Kamatchi. and CDI (Bock et al., J Biol Cem 2011;286:42736). Biology, Norfolk State University, Norfolk, VA, USA. We applied single-channel patch-clamp measurements to compare two native High voltage-gated calcium (Cav) channels are regulated by PKC isozymes. isoforms with alternatively spliced C-terminus. Cav1.3 42 isoform has a long These isozymes target selected serine/threonine (Ser/Thr) PKC phosphoryla- C-terminus containing C-terminal modulator domain (CTM), whereas in the tion sites in the intracellular regions of Cava1 subunits of these channels. It short C-terminus of Cav1.3 42A isoform CTM is truncated. CTM shifts channel has been found earlier using Cav2.2a1 subunits that stimulatory (Thr-422, activation to higher voltages and inhibits CDI. Ser-2108 and Ser-2132) and inhibitory (Ser-425) PKC sites exist and their Here, we observed that calcium influx from a few single-channel openings lead activation with PKC isozymes led to potentiation and depression of calcium to CDI, visible as a decay of the average single-channel current. However, the currents (ICa) respectively. Based on the above report, it was planned to extent of CDI was significantly reduced in Cav1.3 42 isoform as compared with examine if the homologous sites in the Cav2.3a1 subunits behave similarly. 42A isoform. Furthermore, we observed the shortening of the open times as a In this regard the WT Cav2.3a1 or Ser/Thr Ala mutants of stimulatory result of CDI (Imredy and Yue, Neuron 1994;12:301). The degree of the open- (Thr-365, Ser-1995 and Ser-2011) or inhibitory (Ser-369) sites were expressed time shortening was dependent on the prior calcium influx (Josephson et al., along with b1b and 2/d cDNA subunits in Xenopus oocytes and the barium cur- J Physiol 2010;588:213) with a steeper dependence for Cav1.3 42A isoform. rents (IBa) were studied. Intracellular injection of PKC isozymes bII or ε Additionally, we performed experiments with a channel agonist S(-)BayK potentiated WT Cav2.3 currents. While both PKC bII and ε potentiated IBa 8644. The boosting of the channel activity by BayK 8644 eliminated the differ- through Thr-365 (T365/S369A/S1995A/S2011A), only PKC ε increased IBa ence between the isoforms, resulted in a high extent of CDI for both splice through Ser-1995 (T365A/S369A/S1995/S2011A) channels. Ser-2011 failed variants. to act as a stimulatory site contrary to its homologous site, Ser-2132 in the Our findings suggest the ability of CTM to inhibit CDI depends on the channel Cav2.2a1 subunits. However, Ser-369 acted as inhibitory site as its homolog activity, which can be in turn modified by CTM. Ser-425 in the Cav2.2a1 subunits. Both PKC bII and ε inhibited IBa through Ser-369 (T365A/S369/S1995A/S2011A) channels. When both Thr-365 and 2930-Pos Board B360 Ser-369 were present (T365/S369/S1995A/S2011A), IBa was neither stimu- Serum Factor Alters T-Type Cav3.2 Gating Kinetics and Current Density lated nor inhibited. However, stimulation was dominant when two stimulatory Gray Evans, Slobodan M. Todorovic. sites (Thr-365 & Ser-1995) were present along with Ser-369. Experiments Department of Anesthesiology, University of Virginia, Charlottesville, VA, with other mutants, including Ser/Thr Asp constructs are being studied and USA. will be discussed. T-type Calcium channels play a critical role in regulating neuronal excit- ability and modulating sensory transmission. The Cav3.2 channel isoform 2928-Pos Board B358 is highly expressed in peripheral nociceptors as well as in the pain- Norepinephrine Upregulates T-Type Calcium Channels in Rat Pinealo- processing regions of the dorsal horn of the spinal cord. Augmenting cytes Cav3.2 currents has been shown to induce hyperexcitability in nociceptive Haijie Yu1, Jong Bae Seo1, Seung-Ryoung Jung1, Duk-Su Koh1,2, neurons in vitro and hyperalgesia in vivo. These studies strongly suggest Bertil Hille1. that potentiation of the Cav3.2 channel results in abnormal nociceptive trans- 1Physiology and Biophysics, University of Washington, Seattle, WA, USA, mission, which could contribute to a variety of clinical pain syndromes. 2Physics, Pohang University of Science and Technology, Pohang, Korea, Therefore, it is important to identify endogenously produced molecular spe- Republic of. cies that modulate Cav3.2 currents. Using the patch-clamp technique and sta- The mammalian pineal has a circadian rhythm of melatonin secretion at night bly transfected human embryonic kidney cells (HEK-293) expressing the triggered by norepinephrine (NE) released from sympathetic nerve terminals. Cav3.2 channel, we have begun to characterize a factor found in fetal bovine We asked whether functional expression of voltage-gated calcium channels in serum (FBS) that profoundly affects Cav3.2 channel gating kinetics. Specif- rat pinealocytes is changed by culturing them in NE as a surrogate for the ically, when compared to baseline recombinant currents, 1% serum produces night signal. Channel activity was assayed as ionic currents under patch maximal increases in current magnitude (350%; p<.001), conductance clamp. Cultured without NE, pinealocytes showed only non-inactivating L- (150%, p<.001), rate of macroscopic inactivation (47.1%; p<.001) and deac- type dihydropyridine-sensitive calcium current. After 24 h in NE, an addi- tivation (74.3%; p<.001). Furthermore, 1% serum induces a hyperpolarizing tional low-voltage activated transient calcium current developed whose phar- shift in voltage-dependence of activation (V50) (4.77mV; p <.001) with macology and kinetics corresponded to a T-type channel. This change was minimal effect on voltage-dependence of inactivation. In contrast, we found initiated by b-adrenergic receptors, cyclic AMP, and protein kinase A as re- that recombinant CaV3.1 currents were completely insensitive. Similar to re- vealed by pharmacological experiments. Quantitative PCR experiments combinant Cav3.2 currents, T-currents from rat dorsal root ganglia (DRG) showed mRNA for Cav1.4 (L-type), Cav3.1 and Cav3.2 (both T-type). cells exhibited comparable changes, with 1% serum producing maximal in- Cav3.1 T-type channel mRNA was significantly elevated by culture in NE, creases in current magnitude (210%; p<.05), conductance (183%; p<.05), but those for Cav1.4 and Cav3.2 were not. After only 8 h of NE treatment, and rate of inactivation (71.4%; p<.001), in addition to inducing a hyperpo- Cav3.1 mRNA was already elevated, but the transient calcium current was larizing shift in V50 (9.402mV; p<.01). Future studies will focus on iden- not. Even a 16 h wait without NE following the 8 h NE treatment induced lit- tifying this serum factor in order to evaluate its potential role in nociceptive tle additional transient current. However, these cells were primed to make signal modulation and cellular excitability. Supported in part by NIH grant transient current after a second NE exposure. Induction of transient current R21DA034448 (SMT). 580a Wednesday, February 11, 2015

2931-Pos Board B361 (Lancet (2013) 381:1371). Our group has recently found three missense Convergent Modulations by Carboxyl-Termini across L-Type Calcium mutations in the CaVb2 gene in ASD patients; while two mutants (G167S, Channel Subtypes S197F) resulted in a retardation of inactivation behavior, one mutant Yaxiong Yang1, Min Liu1, Nan Liu1, Xiaodong Liu1,2. (F240L) accelerated the inactivation of whole-cell Ba2þ currents (PLOS 1Department of Biomedical Engineering, Tsinghua University, School of One (2014) 9(4): e95579). In the present study, we performed single channel 2 Medicine, Beijing, China, School of Life Sciences, Tsinghua University, patch clamp of HEK cells co-transfected with CaVb2 mutants and CaVa1C. Beijing, China. The gating parameters revealed a pronounced biophysical phenotype for all L-type calcium channels (LTCCs), also known as CaV1 family, are subject mutations: G167S and S197F persisted longer in an open state by elongating to diverse perturbations while acting as signaling hubs in cells, including its mean open time (p ¼ 0.005). F240L showed a trend for an increased open autonomous modulations by the distal carboxyl terminus (DCT) of its own. probability. Further, the transition rate constants obtained from Markov Previous studies show that DCT of CaV1.3 and CaV1.4 (i.e., DCTD and modelling of the single-channel data were consistent with the observed DCTF respectively) competes with calmodulin (CaM) for IQ and IQ vicinity gating parameters. Here, the Markov model revealed significantly deceler- (IQ-IQV) of the channel, which results in attenuation of calcium dependent ated transition from open to closed state for G167S (p ¼ 0.008) and inactivation (CDI) (Liu et al. 2010). In parallel, DCTC (i.e., DCT of S197F (p ¼ 0.03). Both G167S and S197F showed slower transition rates CaV1.2) could autonomously inhibit voltage-gated activation (VGA) of suggesting a preference for deeper closed states (for S197F p ¼ 0.04). the channel (Hulme et al. 2006). Here, our data demonstrate that distal That would explain why their open probabilities are not increased while carboxyl-terminus regulatory domain (DCRD) across CaV1 family could mean open times are elongated. We conclude that the three mutations, mediate both inhibition of CDI (I-CDI) and inhibition of VGA (I-VGA), sug- each exhibiting different biophysical mechanisms, lead to the same outcome: gesting a unified scheme of competitive autoregulation by DCRD across more channel activity. CaV1 family. Thus, the apparent discrepancy in phenotypes of I-CDI and/ or I-VGA among CaV1 subtypes can be largely attributed to the different 2934-Pos Board B364 configurations of DCT/IQ-IQV complex. Under such scheme, in addition to Divergent Regulation of Cardiomyocyte Cav1.2 Currents by Calmodulin well-documented I-VGA, the proteolyzed carboxyl-terminus (CCT) of Mutants Associated with Human Sudden Death Syndromes LTCC in neurons could also induce I-CDI. Both I-VGA and I-CDI play Dmytro O. Kryshtal1, Hyun S. Hwang1, Christopher N. Johnson2, crucial roles in CaV1 signaling in neurons. Our results here provide Walter J. Chazin2, Alfred L. George, Jr.3, Bjorn C. Knollmann1. converged mechanisms across CaV1 channel subtypes in their DCT modula- 1Department of Medicine, Vanderbilt University, Nashville, TN, USA, tions, highlighting the importance of comparative studies in the context of 2Center for Structural Biology, Vanderbilt University, Nashville, TN, USA, whole-family of CaV1 channels. Moreover, this study also reveals innovative 3Department of Pharmacology, Northwestern University, Chicago, IL, USA. features from CaV1.1 and CaV1.2, inviting further investigations into mecha- Recent reports identified six calmodulin (CaM) missense mutations associated nism and physiology of DCTs. with three distinct human arrhythmia syndromes: N54I and N98S are associ- ated with catecholaminergic polymorphic ventricular tachycardia (CPVT)- 2932-Pos Board B362 like clinical presentations, D96V, D130G and F142L with long QT syndrome Modal Bifurcation of Cav1.3 Signaling in Cortical Neurons (LQTS) and F90L with idiopathic ventricular fibrillation (IVF). We previously Min Liu1,2, Yaxiong Yang1, Nan Liu1, Ji Tang1, Xuyang Sun1, reported that all LQTS-CaMs exhibit reduced Ca affinity and normal or Xiaodong Liu1,3. decreased (compared to WT CaM) ryanodine receptor (RyR2)-binding 1School of Medicine, Tsinghua Univ, Beijing, China, 2Tsinghua-Peking affinity, whereas CPVT-CaMs have normal or modestly lower Ca affinity Center for Life Sciences, Beijing, China, 3School of Life Sciences, Beijing, but increase RyR2 single-channel open probability and show enhanced China. binding affinity to RyR2, causing significant increase in spontaneous þ L-type Ca2 channels, emerging as one of the central signaling hubs in Ca wave and spark activity in cardiac myocytes. Here, we investigate the healthy and diseased neurons, play prominent roles in diverse essential pro- effect of these mutant CaMs on another target potentially involved in cesses. However, it still remains largely as a puzzle how the same channel arrhythmogenesis - Cav1.2 channels. could simultaneously fulfill opposed tasks of signaling in neurons. Here we Using whole-cell patch clamp, Cav1.2 current (ICa) was measured in freshly report that CaV1.3 channels are bifurcated into subpopulations with two isolated murine ventricular myocytes pre-treated with ryanodine (50 mM) and distinct modes of signaling: n-mode signaling and p-mode signaling. This thapsigargin (10 mM) to prevent SR Ca release. Cells were dialyzed with either þ modal bifurcation is first demonstrated by genetically-encoded Ca2 channel wild-type or mutant CaM (6 mM) via patch pipette. All LQTS-CaMs and IVF- tuners (GECTs) of iCaMp and eCaMp, bioengineered to perturb autonomous CaM caused significant impairment of ICa inactivation: t¼254534 ms competition between calmodulin and distal carboxyl tail of CaV1.3 channel. (D96V), 251510 ms (F142L), 187513 ms (D130G) and 19058 ms (F90L) Channels driven into p-mode by eCaMp tend to produce ‘‘sharp and large’’ vs. 11055 ms for WT (p<0.001, n¼4-7 myocytes/group), whereas CPVT- þ Ca2 influx, which is kinetically distinguished from ‘‘blunt and small’’ CaMs had no effect on ICa inactivation(t¼10853 ms (N54I), t¼132514 þ Ca2 influx via n-mode channels by iCaMp. Moreover, in cultured cortical ms (N98S) (NS, n¼4-6 myocytes/group)). The effect of LQTS-CaMs and neurons, modal bifurcation of CaV1.3 channels provides a pair of homeostatic IVF-CaM was dominant, with a mixture of 25% mutant/75% WT CaM slowing opponents leading to constructive and destructive signaling events, eventually ICa inactivation to a similar degree as 100% mutant CaM. Importantly, average to morphological changes. In these neurons, kinase and phosphatase are pref- peak current amplitude was unchanged in all groups. These results support the erentially activated by respective p-mode or n-mode channels and signals. hypothesis that different targets are affected in arrhythmogenic disorders asso- Correspondingly, CaV1.3 signaling complex switches between its configura- ciated with LQTS-CaMs (impaired regulation of Cav1.2) vs. CPVT-CaMs tions to facilitate such signaling preference. Finally, the capabilities of (altered regulation of RyR2). The F90L CaM mutation shares characteristics GECTs, especially those of eCaMp in promoting p-mode channels and sig- with both CPVT and LQTS CaMs. nals, highlight a new strategy to modulate L-type Ca2þ channels, as potential therapeutics for disorders with certain malfunctioned channels and/or dys- 2935-Pos Board B365 regulated homeostasis. A PQ-Channel Mutation Associated with Epilepsy Alters the Voltage Dependence of Channel Inactivation 2933-Pos Board B363 Ellie Dubrovina, Gabrielle Suppa, Keith Thomas, Zafir Buraei. The Effect of Autism Candidate-Gene Mutations in the Voltage-Gated Pace University, New York, NY, USA. Calcium Channel b2 Subunit on Single Channel Kinetics Epilepsy affects 1 in 140 people, or nearly 50 million people worldwide. It is Alexandra F. Breitenkamp, Ajay K. Singh, Vincent Mortier, characterized by seizures that often result from neuronal hyper-excitability. Patrick Despang, Marion Brill, Elza Kuzmenkina, Stefan Herzig. A recent genome-wide study uncovered many mutations associated with epi- Institute of Pharmacology, Cologne, Germany. lepsy in voltage-gated calcium channels (VGCC). However, the molecular The electrophysiological properties of voltage-gated calcium channel and cellular consequences of these mutations, and hence, their epileptogenic (VGCC) complexes are determined by the specific combination of subunit mechanisms remain unknown. Here, we investigated two mutations that isoforms, i. e. of one pore-forming CaVa1 subunit and the auxiliary subunits occur in PQ-type voltage-gated calcium channels, which are responsible for b, a2-d (and g). Therefore, VGCCs differentially modulate the cellular neurotransmitter release: R477H (in the I-II loop) and Q1957X (a truncation response to stimuli, which is relevant for neuronal function. Interestingly, mutation). The mutations were introduced into a human PQ channel in the CaVa1C and CaVb2-subunit genes have been shown to be risk loci for the pGEMHE vector. The DNA for WT or mutant channels, together with five major psychiatric disorders including autism spectrum disease (ASD) the DNA for other VGCC subunits required for proper channel expression, Wednesday, February 11, 2015 581a was subjected to in vitro cRNA synthesis. The resulting cRNA was subse- the pore (ionic currents) and from the individual VSDs (fluorescence), we quently injected into Xenopus oocytes, and channel currents were examined us- estimated the energetic contribution of each VSD to pore opening in the ing Two-Electrode Voltage Clamp four days later. We thus compared the presence and in the absence of a2d-1. The model predicts that, in channels biophysical properties of WT and mutant channels. We found that the not associated with a2d-1, all VSDs were poorly energetically-coupled to R477H mutation shifts the voltage dependence of inactivation to more depolar- pore opening. The association of a2d-1 with a1cþb3 specifically increased ized voltages, rendering the mutant channels more difficult to inactivate the coupling energy of VSDs II and III to the pore. In agreement with the model compared to the WT channel. In addition, the time constant of inactivation prediction, we found that without a2d-1 subunits the ionic current deactivation was slower for the mutant. At the same time, the voltage dependence of activa- is largely voltage-independent. tion was unchanged. These results suggest a potential mechanism for epilepto- A low-resolution structure of CaV1.2 channel complexes shows that the a2d-1 3 genesis: reduced channel inactivation would contribute to increased cellular subunit forms a cap that embraces ~ =4 of the extracellular surface of the a1c excitability and excitotoxicity - two hallmarks of epilepsy. The results also subunit (Walsh et al., JBC 2009). Based on our results, we propose that the highlight the role of the I-II loop of VGCC in channel inactivation. As for exposed ¼ of the a1c subunit is VSD IV, which does not show evidence of the Q1957X mutation, our current hypothesis is that this mutant generates no functional interaction with a2d-1 subunits. currents. In addition, we hypothesize that the Q1957X mutant will act as a dominant negative regulator of coexpressed WT channels via the unfolded pro- tein response. 2938-Pos Board B368 Genetic Ablation of KLHL1 Alters CaV3.2 Expression in DRG Neurons and Mechanical Pain Transmission 2936-Pos Board B366 Elizabeth Martinez-Hernandez1, Yungui He2, Paula P. Perissinotti1, b2A and b3 Differentially Modulate Time- and Voltage-Dependent Erik Almazan1, Michael D. Koobb2, Erika S. Piedras-Renteria1. 1 Properties of Individual Voltage Sensors in the Human Cav1.2 Channel Cell and Mollecular Physiology, Loyola University of Chicago, Maywood, Nicoletta Savalli1, Daniel Sigg2, Alan Neely3,4, Riccardo Olcese1,5. IL, USA, 2Institute for Translational Neuroscience and Dept. of Lab 1Anesthesiology, UCLA, Los Angeles, CA, USA, 2dPET, Spokane, WA, Medicine & Pathology, University of Minnesota, Minneapolis, MN, USA. USA, 3Universidad de Valparaı´so, Valparaı´so, Chile, 4Centro Pain signaling is initiated by the detection of noxious stimuli through Interdisciplinario de Neurociencias de Valparaı´so, Valparaı´so, Chile, specialized primary nociceptors located in peripheral endings in dorsal 5Physiology, UCLA, Los Angeles, CA, USA. root ganglion (DRG) neurons. Low voltage activated CaV3.2 T-type calcium The human L-type calcium channel (CaV1.2) is a multi-protein complex channels play a major role in sensory perception in these neurons; silencing consisting of the pore-forming a1c subunit and at least two auxiliary subunits, their activity with antisense RNA or genetic ablation results in anti- b and a2d, which assemble in a 1:1:1 stoichiometry. b subunits regulate mul- nociceptive, anti-hyperalgesic and anti-allodynic effects1-3. These channels tiple aspects of CaV1.2 channel physiology, including surface expression, are regulated by a plethora of proteins, including KLHL1, a neuronal actin degradation and gating. Investigating the effect of b2a and b3 subunits on binding protein that stabilizes channel activity by increasing its recycling human CaV1.2 channels (a1cþa2d) expressed in Xenopus oocytes, we found back to the plasma membrane via recycling endosome activity. We explored that the voltage dependences of total charge displacement (QV) and conduc- the role of KLHL1 and its effects on CaV3.2 and mechanical peripheral pain tance (GV) were ~10mV more negative in the presence of b2a. We tested transmission using the KLHL1 KO mouse model. Total T-type calcium cur- the hypothesis that b2a and b3 subunits differently modulate the activation rent amplitude was smaller in KO DRG neurons (12 5 2.03 pA/pF, n¼18) of the four voltage-sensor domains (VSDs) in the a1c subunit. Using the compared to controls (18 5 1.89 pA/pF, n¼13; p<0.05), without significant Voltage-Clamp Fluorometry technique, we fluorescently tracked the move- changes in voltage dependence as expected in the absence of its modulator. ment of individual VSD in conducting human CaV1.2 channels. We found Western blot analysis confirmed CaV3.2 but not CaV3.1 or CaV3.3 protein that, in the presence of b2a, the voltage-dependent activation of VSDs II was significantly decreased. Interestingly, von Frey hair tests show paw and III was left-shifted as compared to b3-associated CaV1.2 (VSD II withdrawal threshold in KO mice was increased. Our data establishes 0 with b2a:Vhalf¼31.151.0 mV, z¼ 4.550.8 e ; VSD II with b3:Vhalf¼ KLHL1 is a calcium channel regulator in DRG neurons, providing a novel 0 26.551.8 mV, z¼ 2.450.1 e ; VSD III with b2a:Vhalf¼36.653.9 mV, target to control peripheral pain sensation. Downregulation of CaV3.2 acti- 0 0 z¼ 1.850.1 e ; VSD III with b3:Vhalf¼16.951.4 mV, z¼ 1.250.1 e ). vity by as little as ~30% was sufficient to alter pain perception in the KO Moreover, activation and deactivation kinetics of VSD II in channels asso- model. ciated with b2a were dramatically slower than in the presence of b3, while voltage- and time-dependent properties of VSD IV remained the same in the presence of either Cavb subunit. These results suggest that, in spite of 2939-Pos Board B369 their highly-conserved , different b subunit isoforms Morpholino Oligomer Peptide Therapy Improves Mitochondrial Function distinctly modulate the biophysical properties of Ca 1.2 voltage sensors to in mdx Cardiomyopathy V 1 2 3 3 mediate their effect on Ca conductance. Victoria P. Johnstone , Abbie M. Adams , Steve D. Wilton , Sue Fletcher , Livia C. Hool1,4. 1Anatomy, Physiology and Human Biology, The University of Western 2937-Pos Board B367 Australia, Perth, Australia, 2Centre for Neuromuscular and Neurological The a2d Subunit Efficiently Couples VSDs Activation to Pore Opening in Disorders, The University of Western Australia, Perth, Australia, 3Centre for 4 Human Cav1.2 Channels Comparative Genomics, Murdoch University, Perth, Australia, Victor Nicoletta Savalli1, Antonios Pantazis1, Daniel Sigg2, Alan Neely3,4, Chang Cardiac Research Institute, Sydney, Australia. Riccardo Olcese1,5. Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disorder. It is 1Anesthesiology, UCLA, Los Angeles, CA, USA, 2dPET, Spokane, WA, commonly associated with cardiomyopathy that is characterised by disrupted USA, 3Anesthesiology, Universidad de Valparaı´so, Valparaı´so, Chile, cytoskeletal architecture and mitochondrial dysfunction. Activation of the 4Centro Interdisciplinario de Neurociencias de Valparaı´so, Valparaı´so, Chile, L-type Ca2þ channel (LTCC) provides the Ca2þ influx required for contraction, 5 Physiology, UCLA, Los Angeles, CA, USA. but also causes an increase in mitochondrial membrane potential (Jm)ina The a2d-1 auxiliary subunit binds to the pore-forming a1c subunit of voltage- calcium-independent manner. This involves the movement of cytoskeletal gated L-type calcium channels (CaV1.2) and facilitates both membrane proteins and regulation of mitochondrial VDAC. The increase in Jm (JC-1 trafficking and voltage-dependent activation. Using voltage-clamp fluorometry fluorescence) after activation of LTCC is absent in cardiomyocytes isolated to study human CaV1.2 channels expressed in Xenopus oocytes, we have from hearts of mdx mice, a murine model of DMD (1.551.0%, n¼4 vs. recently found that a2d-1 association, in the presence of b3 subunits, causes 12.151.4%, n¼10 in C57BL/10ScSnArc controls) but can be restored voltage sensor domains (VSDs) I, II and III, but not IV, to activate at more hy- using antisense morpholino oligomers to induce exon skipping of dystrophin perpolarized potentials (DVhalf¼39mV, 32mV, 18mV for VSDs I-III exon 23 (M23D). Here we investigate the effects of M23D peptides on respectively) and with steeper voltage-dependence (z fold increase with a2d- changes in Jm and mitochondrial oxygen consumption (flavoprotein auto- 1: 1.9, 2.2 and 1.6 for VSDs I-III respectively). Thus, the a2d-1-induced fluorescence) induced by LTCC activation. Male (M) and female (F) neonatal facilitation of CaV1.2 voltage-dependent activation seems to be mediated mdx mice were injected (i.p.) for 3 weeks and divided into two treatment through the remodeling of three VSDs. groups: (i) 120mg/kg once per week; (ii) 30mg/kg four times per week. We analyzed our voltage-clamp fluorometry data with a 32-state allosteric Activation of LTCC with the dihydropyridine agonist BayK(-) induced an model for CaV1.2 activation, consisting of five gating particles (one pore, increase in Jm in cardiomyocytes from animals treated with a single weekly four VSDs). By simultaneously fitting kinetic and steady-state data from dose (M: 1752%, n¼5; F: 1653%, n¼12) and with multiple weekly doses 582a Wednesday, February 11, 2015

(M: 1652%, n¼6; F: 1352%, n¼15). BayK(-) increased flavoprotein the revertants that restored ion transport, rapidly lost weight and died. In oxidation in mdx cardiomyocytes from animals treated with a single weekly contrast, mice infected with mutants lacking IC activity, which did not incor- dose (M:1353%, n¼13; F: 1053%, n¼9) and with multiple doses per porate mutations within E gene during the experiment, recovered from disease week (M: 1752%, n¼16; F: 1452, n¼24). These data indicate that either and most survived. single or multiple i.p. injections per week of morpholino oligo peptides are We have shown that SARS-CoV E protein IC activity is a virulence effective at restoring cardiac mitochondrial function in both male and female determinant. mdx mice. [1] DeDiego, M.L., et al 2008. Virology 376, 379-389. [2] Verdia´-Ba´guena C., et al. 2012. Virology. 432: 485-494. 2940-Pos Board B370 High Susceptibility to Non-Alcoholic Fatty Liver Disease in Two-Pore 2942-Pos Board B372 Channel 2-Deficient Mice Divalent Copper Compound as Inhibitory Agent of Influenza A Christian Grimm, Cheng-Chang Chen, Elisabeth Butz, Martin Biel, Kelly L. McGuire, Nathan A. Gordon, Roger G. Harrison, David D. Busath. Christian Wahl-Schott. Physiology and Developmental Biology, BYU, Provo, UT, USA. Pharmacy, Pharmacology, LMU University of Munich, Muenchen, Germany. Influenza A virus (IAV) exhibits a high mutation frequency. Mutations in the Endolysosomal organelles play a key role in trafficking, breakdown and proton channel M2 have created substantial IAV drug resistance to previously receptor-mediated recycling of different macromolecules such as low- effective drugs, such as amantadine (AMT), along with its analogs. The main density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or drug-resistant variation in the M2 proton channel, which has become ubiqui- transferrin. In the liver, receptor-mediated uptake of low-density lipoproteins tous in humans, is the mutation S31N. Divalent copper has previously been (LDLs) and subsequent intracellular transport is essential for hepatic choles- shown, using in vitro assays involving SSNMR and Xenopus oocytes, to bind terol homoeostasis and plasma lipoprotein metabolism. Dysfunction within and block wild type M2 at the His37 selectivity filter. Here we report initial this pathway results in liver disease such as non-alcoholic fatty liver disease tests of the hypothesis that, given the essential, conserved nature of the selec- (NAFLD), which is associated with increased cardiovascular and liver- tivity filter, a complex consisting of copper bound to an amantadine derivative related mortality. It has been estimated that as many as 30% of adults might serve as a broad-spectrum anti-IAV drug. The EC50 of AMT, two AMT 2þ in these countries have NAFLD. This liver disease has thus emerged as a analogs, CuCl22H2O, and four previously published Cu complexes were substantial public health concern. Here, we examine the role of two-pore tested for antiviral activity against the California/07/2009 (H1N1) IAV strain channel (TPC) 2, an endolysosomal cation channel, in these processes. containing the S31N M2 proton channel in viral mini-plaque assays and for Embryonic mouse fibroblasts were generated from wild type and TPC2 defi- M2(22-62, S31N)-mediated proton transport block in liposomes. A novel, cient mice and characterized by single lysosome patch clamp employing AMT-based divalent copper compound, NAG 107, emerged as a plausible conventional patch clamp as well as planar patch clamp. In addition, traf- lead with an EC50 of 2.91 5 0.29 mM in the viral mini-plaque assay and ficking assays were performed. Embryonic mouse fibroblasts and hepato- 4.5 5 0.6 mM in the liposome assay, 21-fold and 11-fold better than amanta- cytes lacking TPC2 display a profound impairment of LDL-cholesterol dine in these assays, respectively. and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway 2943-Pos Board B373 most likely on the level of late endosome to lysosome fusion. Importantly, Insights on Molecular Determinants of hERG KD Channel Inhibition: endolysosomal acidification or lysosomal enzyme function are normal in Design, Synthesis, and Biological Evaluation of Lubeluzole Derivatives TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic Roberta Gualdani1, Maria Maddalena Cavalluzzi2, cholesterol overload and liver damage consistent with non-alcoholic Francesco Tadini-Buoninsegni1, Maria Rosa Moncelli1, Giovanni Lentini2. fatty liver hepatitis. These findings indicate reduced metabolic reserve of 1Department of Chemistry, University of Florence, Sesto F.no (FI), Italy, hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial 2Department of Medicinal Chemistry, University of Bari, Bari, Italy. role in trafficking in the endolysosomal degradation pathway and, thus, is Drug-induced block of ‘Human ether-a-go-go-related Gene’ (hERG) Kþ chan- potentially involved in the homoeostatic control of many macromolecules nels is the main reason of long QT syndrome, a disorder of cardiac repolariza- and cell metabolites. tion which may lead to sudden death due to ventricular fibrillation. This side effect has been the most frequent cause for drug withdrawal from the pharma- Ion Channels, Pharmacology, and Disease ceutical market. For this reason, understanding the molecular determinants of hERG channel inhibition is an interesting strategy to avoid the cardiotoxicity 2941-Pos Board B371 of drugs. Relevance of SARS-CoV E Protein Ion Channel Activity in Virus Lubeluzole, a neuroprotective compound, has been associated with the acquired long QT syndrome and ventricular arrhythmias; however its effects Pathogenesis þ Carmina Verdia´ Ba´guena1, Jose´ L. Nieto-Torres2, Marta L. De Diego2, on the hERG K channel have not been described to date. Thus, the molecular Jose M. Jimenez-Guarden˜o2, Jose A. Regla-Nava2, determinants for lubeluzole action on hERG channel were studied using the Raul Fernandez-Delgado2, carlos Castan˜o-Rodrı´guez2, Jaume Torres3, patch clamp technique. We found that lubeluzole and its enantiomer are highly Antonio Alcaraz1, Vicente M. Aguilella1, Luis Enjuanes2. potent inhibitors of hERG current with an IC50 value of around 10 nM 1Department of Physics, Universitat Jaume I, Castello´n de la Plana, Spain, (no stereoselectivity observed). Moreover we observed that hERG inhibition 2National Center of Biotechnology (CNB-CSIC), Campus Universidad by lubeluzole is time- and voltage-dependent. In fact lubeluzole causes a nega- Auto´noma de Madrid, Madrid, Spain, 3School of Biological Sciences, tive shift in the voltage dependence of hERG current activation and accelerates Division of Structural and Computational Biology, Nanyang Technological tail current deactivation, suggesting that it preferentially blocks the activated University, Singapore, Singapore. state of the channel. Coronaviruses (CoV) are vertebrate pathogens that cause common colds, bron- To go further in details, we synthesized a series of lubeluzole congeners chiolitis and acute respiratory distress syndrome. In fact, their relevance designed to explore the role of lipophilicity and electronic distribution. increased when the causative agent of the severe acute respiratory syndrome Structural variations were designed on the basis of the indications gained by (SARS) was identified as a CoV. CoV E protein is a small transmembrane pro- quantum-mechanical calculations run at several DFT levels. tein of between 76-109 amino acids in length that modulates coronavirus For each compound we determined the relative hERG affinity and the state- morphogenesis, tropism and virulence [1]. dependent mechanism, to evaluate how specific modification of drug structure We sought to elucidate the role of E protein IC activity in virus pathogenesis affected hERG binding interaction. Main SAR results will be presented. by combining our knowledge of residues essential for E protein ion conduc- tivity with the manipulation of SARS-CoV genome. To test the contribution 2944-Pos Board B374 of E protein IC activity in virus pathogenesis, two recombinant mouse- Validation of KCa3.1 Channel Nifedipine Interaction Site Predicted by adapted SARS CoVs, each containing one single amino acid mutation that Rosetta Modeling Method suppressed ion conductivity, were engineered. After serial infections, mutant Hai M. Nguyen1, Heike Wulff1, Vladimir Yarov-Yarovoy2. viruses, in general, incorporated compensatory mutations within E gene 1Pharmacology, University of California, Davis, Davis, CA, USA, that rendered active ion channels. Furthermore, IC activity conferred better 2Physiology and Membrane Biology, University of California, Davis, Davis, fitness in competition assays, suggesting that ion conductivity represents an CA, USA. advantage for the virus. Interestingly, mice infected with viruses displaying Calcium-activated potassium channels, including the intermediate-conduc- E protein IC activity, either with the wild-type E protein sequence or with tance KCa3.1 channel and the related small-conductance KCa2 channels, Wednesday, February 11, 2015 583a monitor and regulate intracellular calcium and are ideal pharmacological 21) folds respectively. Once these two reagents were applied together, how- targets for immunosuppression, fibroproliferative disorders, hypertension and ever, the Po was increased by 28.98 5 7.6 fold (n¼ 10), less than the expected various neurological diseases. However, the development of drugs specifically magnitude (~ 60-fold) if NPPB and VX-770 work independently. Hence, NPPB for these medically relevant channels faces serious challenges because there is only increases the Po of G551D-CFTR by 3.7 5 0.4 fold (n ¼ 9) in the pres- no available crystal structure that could be used for structure-assisted drug ence of VX-770. Similarly, a much reduced enhancement (4.8 5 1.8 fold, n ¼ design. We used the Kv1.2-Kv2.1 channel structure (pdb id: 2R9R) as a tem- 5) of the Po by VX-770 in the presence of NPPB was seen. Interestingly, we plate to generate a homology model of the KCa3.1 channel transmembrane also observed that VX-770 effectively potentiates a CFTR mutant with its sec- region using the Rosetta modeling method. Docking of small molecules that ond nucleotide-binding domain completely removed (i.e., dNBD2), whereas are known to block KCa3.1 channel currents using Rosetta generated structural NPPB has virtually no effect (n ¼ 12). These results can be explained by a models of channel - drug complexes that can be validated using experimental gating model featuring an energetic coupling between opening/closing of the approaches. Here we report the validation of the binding sites for the dihydro- gate in TMDs where VX-770 binds and dimerization/dissociation of NBDs pyridine nifedipine predicted by Rosetta’s lowest energy stimulation of the where NPPB acts on. KCa3.1 channel complex. We used a combination of site-directed mutagenesis and electrophysiological patch-clamp recording to test the identified amino 2947-Pos Board B377 acid residues located between the pore lining S5 and S6 segment. Blocking ‘‘Use Dependence’’ Without a Ball and Chain - Inhibition of Bacterial of the KCa3.1 channel by nifedipine was reduced by altering the side chains Sodium Channels by m-Contoxins at positions L209, T212 and V278 to either alanine or valine, with T212 the Rocio K. Finol-Urdaneta, Denys McMaster, Robert J. French. most affected residue. Bulkier side-chain change to phenylalanine confirmed Physiology & Biophysics, University of Calgary, Calgary, AB, Canada. T212 as the main interacting site for nifedipine without compromising the ‘‘Use-’’ or ‘‘state-’’ dependent block of voltage-gated sodium channels is channel’s affinity for TRAM-34. Thus, by correctly identified the interaction characteristic of therapeutic agents used to moderate electrical activity in path- of the dihydropyridine nifedipine with the KCa3.1 channel complex, Rosetta ologically hyperactive tissues and combat certain forms of epilepsy, cardiac models can be used to understand the molecular mechanism of action of arrhythmia, myotonia, and pain. Externally acting Na channel pore blockers KCa channel blockers and gating modulators and assist with drug design are not commonly used as therapeutic drugs, but intraperitoneal m-conotoxin efforts. (mCTX) KIIIA can act as an effective analgesic against inflammatory pain in mice, at concentrations that do not cause obvious systemic side effects (Zhang 2945-Pos Board B375 et al 2007 J Biol Chem 42:30699). This action appears to result from highly GBR-12909: Potent Blocker of Peak and Late Nav1.5 Currents specific targeting to neuronal Na channels, without essential reliance on the Carlos Obejero-Paz, James Kramer, Andrew Bruening-Wright, use-dependent potency that contributes to the efficacy of clinical local anes- Antonio Lacerda, Arthur Brown. thetics. mCTXs, although highly selective among closely related eukaryotic ChanTest Corporation, Cleveland, OH, USA. Na channels, have recently been found to block bacterial Nav channels with GBR-12909 (vanoxerine) is a potent highly selective dopamine transporter high affinity (Chen & Chung 2012 Biophys J 102:483; Finol-Urdaneta et al antagonist. It was initially developed for treatment of Parkinson’s disease 2013 Biophys J 104:136a). In addition, the block was potentiated at more de- and while safe in man, lacked efficacy. Although a potent hERG blocker, polarized holding potentials. Here, we explore the dependence of mCTX PIIIA the drug exhibits multiple ion channel effects including frequency- block of NaChBac and NavSp1 on varying patterns of conditioning depolari- dependence. The drug effectively terminated induced, sustained atrial fibril- zation. Briefly, in the low picomolar range, PIIIA application was associated lation (AF) and atrial flutter in a canine model of sterile pericarditis. with faster inactivation decay during a test depolarization, and block of the Subsequently the drug terminated AF in a Phase 2B ascending dose trial. Pre- peak current. The speeding of inactivation precedes the block of current vious in vitro measurements used protocols that did not address the issue of the following PIIIA application, and this kinetic effect was obvious at more nega- late Nav1.5 current which is important in understanding safety of a potent tive holding potentials than was the decrease in peak current. In NaChBac, both hERG blocker. activation and inactivation were shifted in the hyperpolarizing direction by Peak and late sodium currents were measured in HEK-293 cells stably ex- ~25-30 mV in the presence of 5pM PIIIA. The data suggest that PIIIA binding pressing the human Nav1.5 channel. Peak currents were measured using a is conformation dependent, with high affinity for non-conducting, depolariza- step-ramp protocol consisting of a 40 ms step to 15 mV (where the peak tion-induced states. At present, the relative importance of ‘‘pre-activated’’ current was measured), a 180 ms step to þ30 mV and a 200 ms ramp to and ‘‘in-activated’’ states in potentiating inhibition by PIIIA is unclear. the holding potential of 80 mV (1 Hz stimulating frequency). The effect of the drug was parameterized with a single binding isotherm having an 2948-Pos Board B378 5 5 IC50 of 29 3 nM (95% CI: 22-37) and a Hill coefficient of 0.91 0.10 W493R Gain of Function Mutation in Atypical Cystic Fibrosis Rewires the (95% CI: 0.68-1.18) (seven cells, three concentrations). We also evaluated Epithelial Sodium Channel Dynamics the effect of GBR-19209 on currents increased using the sea-anemone toxin Mahmoud Shobair1, Yan H. Dang2, Hong He2, Jack M. Stutts2, ATX-II as a surrogate of the late sodium current. The calculated IC50 and Nikolay V. Dokholyan1. Hill coefficients from five cells and five concentrations were 93 5 9nM 1Biochemistry and Biophyiscs, UNC-CH, Chapel Hill, NC, USA, 2Cystic (95% CI: 7-117) and 1.57 5 0.24 (95% CI: 1.05-2.67), respectively. Thus, Fibrosis Center, UNC-CH, Chapel Hill, NC, USA. GBR-12909 block is at least sixty times more potent than ranolazine’s Sodium absorption in epithelial cells is rate-limited by the epithelial sodium ¼ (IC50 6 mM).The combination of potent peak and late sodium block may channel (ENaC) activity in lungs, kidney and the distal colon. Pathophysiolog- explain the antiarrhythmic and safe profile of the drug. ical conditions, such as cystic fibrosis and Liddle syndrome, result from water- electrolyte imbalance partly due to malfunction of ENaC’s molecular regu- 2946-Pos Board B376 lation. The molecular mechanism(s) of pathologically implicated mutations VX-770 and NPPB Modulate CFTR Gating via Different but Dependent in ENaC subunits are largely unknown due to absence of structural models Mechanisms for ENaC’s oligomers. Here, we propose a dynamics-driven mechanism of Wen Ying Lin, Kangyang Jih, Tzyh-chang Hwang. the gain of function mutation aW493R implicated in atypical cystic fibrosis. University of Missouri, Columbia, MO, USA. We utilize a combination of discrete molecular dynamics simulations (DMD) Understanding how small molecules potentiate CFTR gating is critical for of the extracellular region of ENaC’s heterotrimer abg and functional data developing novel therapy for cystic fibrosis. Our previous studies have sug- from whole-cell electrophysiology experiments. Structure-function studies gested that VX-770 (ivacaftor), a CFTR potentiator now used in clinics, suggest that tetramers and trimers are the major characterized functional olig- increases the Po of CFTR by shifting the gating conformational changes in omeric states of ENaC. Using the crystal structure of the acid sensing ion chan- CFTR’s transmembrane domains (TMDs) to favor the open channel configura- nel, ENaC’s structural homolog in the ENaC/degenerin mechanosensitive tion. Lately, another CFTR potentiator NPPB was reported to enhance CFTR ion channel family, we have constructed homology models of ENaC subunits activity through a modus operandi that exploits the ATP hydrolysis-driven, alpha, beta and gamma, in addition to the heterotrimers abg, agb and the non-equilibrium gating mechanism unique to CFTR. It is however puzzling heterotetramer abag. Electrophysiology data show constitutive activity of that NPPB can also increase the activity of non-hydrolytic G551D-CFTR, aW493Rbg in oocytes indicated by higher open probability and elevated basal the third most common pathogenic mutation. We therefore set forth to unravel activity compared to WT. Our DMD simulation provides an allosteric frame- the mechanism of NPPB using VX-770 as a reference. Once the blocking effect work, which agrees with experimental data. W493R rewires the electrostati- of NPPB was corrected, we found that NPPB and VX-770, when applied alone, cally mediated inter-residue interaction network in close proximity to W493, increased the Po of G551D-CFTR by 6.9 5 0.8 (n ¼ 12) and 9.3 5 1.4 (n ¼ resulting in widening of the pore geometry in the outer mouth of the pore in 584a Wednesday, February 11, 2015 the extracellular region. Rewiring effect of inter-residue interactions in 493R cating that PP1-R/KVxF- binding is a prerequisite for dephosphorylation of mutant pocket allosterically propagates across the channel resulting in a pSer68-PLM. more stabilized global conformational ensemble of the channel. These findings Conclusion: R/KVxF motif in NCX1-CBD1 anchors PP1, does not change the predict a novel mechanism of ENaC’s constitutive activity, in which changes activity of the enzyme and facilitates dephosphorylation of pSer68-PLM. in local dynamics can affect the relative population of the channel’s active states and its open probability. 2951-Pos Board B381 The Pore-Domain of TRPA1 Mediates the Inhibitory Effect of the Antag- 2949-Pos Board B379 onist 6-Methyl-5-(2-(Trifluoromethyl)Phenyl)-1H-Indazole Role of Threonine 338 in CFTR Gating Hans Moldenhauer1, Ramon Latorre1, Jorg Grandl2. Hsuan-Ting Kuo1,2, Tzyh-Chang Hwang1. 1Facultad de cencias, Universidad de Valparaiso CINV, vin˜a del mar, Chile, 1Medical Pharmacology and Physiology, University of Missouri, Columbia, 2School of medicine Duke University, Ion channels research unit, Durham, MO, USA, 2Medicine, National Yang-Ming University, Taipei, Taiwan. NC, USA. As a member of the ATP-binding cassette (ABC) protein superfamily, CFTR, a The pore-domain of TRPA1 mediates the inhibitory effect of the antagonist phosphorylation-activated but ATP-gated chloride channel, is comprised of 6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole two transmembrane domains (TMDs) that form a gated anion conducting The ion channel TRPA1 gives to the organisms the ability to detect noxious pore and two nucleotide binding domains (NBDs) whose dimerization/dissoci- chemicals to sensory neurons and as a result of this, mediates chemical nocicep- ation is allosterically coupled to opening/closing of the gate. Recent cysteine tion in vivo. Mouse TRPA1 is activated by electrophilic compounds such as scanning of CFTR’s TMDs not only identified pore-lining residues, but also mustard-oil (MO) and several physical stimuli such as cold temperature. suggested molecular motions of the TMDs involved in opening/closing Both stimuli are mechanistic linked to the N terminus of the channel. Due to of the ‘‘gate’’. Since many of the pore-lining residues exhibit clear state- its sensory function, the inhibition of TRPA1 activity might provide an effec- dependent exposure to the aqueous pore, it is predicted that mutations at these tive treatment against chronic and inflammatory pain, for that reason, TRPA1 positions might affect gating by altering the free energy level of a particular has become an important target for the development of new and better analgesic state. T338 was chosen because this pore-lining residue is mostly concealed drugs. 6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (Compound 31) from the pore in the closed state. We converted T338 to various amino acids has been identified by a chemical screen and lead optimization as an inhibitor and found that the physical properties of the side-chain at this position indeed of chemical activation of TRPA1. However, the structures or domains of affect CFTR gating as well as anion conductance. For hydrophilic residues like TRPA1 that mediate the inhibitory effect of Compound 31 are unknown. threonine itself (0.57 5 0.06, n ¼ 4), serine (0.45 5 0.02, n ¼ 12) and aspar- Here, we screened 12,000 random mutant clones of mouse TRPA1 for their agine (0.88 5 0.02, n ¼ 6), the larger the side-chain, the higher the Po. In sensitivity to mustard-oil and the ability of Compound 31 to inhibit chemical contrast, for hydrophobic ones such as alanine (0.60 5 0.02, n ¼ 6), isoleucine activation by MO. We identified five mutations located within the pore domain (0.19 5 0.03, n ¼ 6) and valine (0.44 5 0.05, n ¼ 7), the larger the side-chain, that cause loss of inhibition by Compound 31, one of them in the residue T877 the lower the Po. Single-channel kinetic analysis revealed that mutations placed in the TM5, important in the binding of menthol, a dose-dependent mainly affect the open time. To exclude possible effects of the mutation on agonist/blocker. This result demonstrates that the pore-domain is a regulator ATP hydrolysis, we introduced some mutations into the E1371S background, of chemical activation and suggests that Compound 31 might be acting directly whose ATP hydrolysis is abolished. Interestingly, mutations that shorten the on the pore-domain. open time under the wild-type background also decrease significantly the Po in E1371S. The implications of our data on the gating and permeation mecha- 2952-Pos Board B382 nisms for CFTR will be discussed. Planar Patch Clamp System Capable of Recording Mechanosensitive Activity of Ion Channels 2950-Pos Board B380 Ken Takahashi, Keiji Naruse. PP1 Anchoring onto NCX1 Facilitates Dephosphorylation of P-SER68-PLM Cardiovascular Physiology, Okayama University Graduate School of Tandekile Lubelwana Hafver1, Pimthanya Wanichawan1, Kjetil Hodne1, Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. Jan Magnus Aronsen1, Bjørn Dalhus2, Marianne Lunde1, Ulla Enger1, Response to mechanical stimuli, which includes proper development of biolog- Marita Mathisen1, William Fuller3, Ivar Sjaastad1, Ole Sejersted1, ical tissues and organs, is essential to the cell. Mechanosensitive ion channels Cathrine Carlson1. are the basic elements among mechanosensing molecules and complexes. 1Institute for Experimental Medical Research (IEMR), University of Oslo, Although the traditional patch clamp technique using glass micropipettes is Oslo, Norway, 2Institute of Clinical Medicine, University of Oslo, Oslo, still widely used for electrophysiology, it is difficult to control the mechanical Norway, 3Division of Cardiovascular and Diabetes Medicine, University of stimulus to ion channels in this system. Here we developed a novel patch clamp Dundee, Dundee, United Kingdom. system with a planar stretchable electrode to record mechanosensitive re- Introduction: The cardiac Naþ/Ca2þ exchanger 1 (NCX1) modulates sponses of ion channels. Planar electrodes of 100 mm thickness were fabricated excitation-contraction coupling and contributes to Ca2þ removal in cardiomyo- using silicone resin. Next, we prepared HEK293 cells transfected with the cytes. Altered expression and activity of NCX1 is linked to dysfunctional Ca2þ stretch-activated KCa (SAKCA) channel, which is mechanosensitive, for handling in chronic heart disease. Consequently, modulation of NCX1 activity planar patch clamp recordings. Using our planar patch clamp system, a giga- is proposed as a therapeutic target. The cytosolic loop of NCX1, ~550 amino ohm seal was achieved with a maximum seal resistance of 10 GU. The success acids, comprises several important docking and regulation sites. In particular, rate of gigaohm seal formation was 37%. Then we recorded single channel phospholemman (PLM) has been shown to interact and inhibit NCX1 activity currents with a slope conductance of 285.4 pS, which is parallel to the known when phosphorylated at serine 68 (pSer68-PLM). Importantly, pSer68-PLM SAKCA current. Using scanning electron microscope, we confirmed elonga- has been shown as a substrate for protein phosphatase 1 (PP1) in the context tion of the aperture by 37.7% when 50% stretch was applied to the planar of PLM regulation of the sodium-potassium pump. PP1 regulation of NCX1 electrode. It is expected that a controllable and sufficient stretch stimulus can is unknown. be applied to the cellular membrane using our newly developed planar patch Hypothesis: PP1 binds to NCX1 and regulates its activity by dephosphorylat- clamp system. ing pSer68-PLM. Methods and Results: Using co-immunoprecipitation in rat heart lysates we 2953-Pos Board B383 have shown that NCX1 exists in a macromolecular complex with PP1 and Single-Channel Analysis of the Molecular Pharmacology of the Long QT PLM. This facilitates specific control of NCX function. Bioinformatic analysis Syndrome Variant 3 revealed three putative PP1 binding sites on NCX1. Co-localization studies, Seth H. Robey, Kevin J. Sampson, Robert S. Kass. co-immunoprecipitations, pull down, mutation- and peptide overlay assays Department of Pharmacology, Columbia University, New York, NY, USA. indicated that PP1 bound directly to the consensus sequence R/KVxF in cal- The Long QT Syndrome (LQTS) is characterized by a prolongation of the QT cium binding domain 1 (CBD1) of NCX1. The reciprocal NCX1 binding site interval on an ECG and occurrences of ventricular fibrillation, polymorphic in PP1 was identified within residues 235-260, a region which harbours ventricular tachycardia, and sudden cardiac death. In patients with LQTS important anchoring sites. A peptide docking model was generated showing variant 3 (LQT3), mutations in the cardiac sodium channel alpha subunit, how the R/KVxF peptide may bind in the hydrophobic pocket of PP1. Surface Nav1.5, disrupt channel inactivation by multiple mechanisms and can cause plasmon resonance analysis indicated that the NCX1-PP1 binding is strong a sustained depolarizing current (INaL) sufficient to prolong the ventricular and stable. This binding does not inhibit PP1 activity. Co-expression of action potential. LQT3 mutant sodium channels are therefore a reliable exper- NCX1 with PLM and PP1 in HEK293 down regulates pSer68-PLM, indi- imental model for the study of the function and pharmacology of dysfunctional Wednesday, February 11, 2015 585a sodium currents in a wide range of more common acquired disorders that are inhibitors and comparison of kinetic of responses were obtained in ‘‘single linked by disease-altered INaL, including ischemic heart disease and heart fail- hole’’ (SH) and PPC modes. Experiment protocols were developed to þ ure. While preferential pharmacological block of INaL by several Na -channel routinely produce signals ~2 nA and ~1 nA for NR1/NR2A and NR1/ blocking drugs has proven to be an important and useful clinical approach to NR2B, respectively, in PPC mode. Under our experimental procedures both manage LQT-3 arrhythmias, we still lack a fundamental understanding of the NR1/N2A and NR1/NR2B showed good uniformity with Z’ factor > 0.6. pharmacological determinants of this selectivity. We have previously identified We found that EC50 concentrations for L-glutamate were 3.8 mM and a novel lethal LQT3 variant, F1473C, which displays slowed inactivation, 2.0 mM for NR1/NR2A and NR1/NR2B, respectively. NMDA appeared to altered channel availability, and a significant INaL. Here we report for the first be a less potent partial agonist at NR1/NR2B (>75% and EC50¼18- time single channel recordings from F1473C channels and find that late reopen- 20 mM), whereas NMDA appeared to be a full agonist at NR1/NR2A with ing events during prolonged depolarizations underlie pathological INaL for this EC50 ¼ 100 mM. These EC50 values were consistent with literature data mutation. To further unravel the mechanism of action of of Ranolazine, a drug obtained with conventional manual patch clamp methods. Additionally, we known to preferentially block INaL, we studied its effects on single F1473C confirmed selectivity of the uncompetitive inhibitor, ifenprodil, towards channels. Ranolazine reduced the number of late reopening events in a manner NR1/NR2B receptors with IC50Ifen¼ 19.3 mM, whereas NR1/NR2A recep- consistent with block of whole cell currents. These experiments represent the tors were unaffected. Potential variability in IC50 values of uncompetitive first exploration of the block of INaL caused by late reopening, and present a inhibitors due to open-pore dependence will be discussed. Our data demon- framework for further studies of the mechanisms of drug block in these strate the potential of a robust HTS patch clamp assay for profiling new channels. chemical entities against NMDA receptor subtypes. 2954-Pos Board B384 2956-Pos Board B386 Automated Patch-Clamp Pharmacology Assays using Human Induced Characterization of Small Molecule Modulators of the Cystic Pluripotent Stem Cell-Derived Cardiomyocytes Fibrosis Transmembrane Conductance Regulator using Backscattering Olaf Scheel1, Stefanie Frech1, Bogdan P. Amuzescu2,1,Jo¨rg Eisfeld1, Interferometry Kun-Han Lin1, Thomas Knott1. Ashley Lockwood1, David Heidary2, Christopher Richards2, 1Cytocentrics Bioscience GmbH, Rostock, Germany, 2Biophysics & Michael Baksh1, M.G. Finn1. Physiology, Faculty of Biology, University of Bucharest, Bucharest, 1Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, Romania. USA, 2Chemistry, University of Kentucky, Lexington, KY, USA. Recent progress in the field of embryonic and induced pluripotent stem cell Cystic Fibrosis (CF) is a genetically-inherited disease characterized by the (iPSC)-derived cardiomyocytes led to flourishing of a variety of pharma- loss of function of the Cystic Fibrosis Transmembrane Conductance Regu- cology and drug safety screening assays, relying on diverse principles, lator (CFTR), a chloride ion channel located in the epithelial lining of such as optical measurements using wide dynamic range Ca2þ-sensitive or the lungs and pancreas. CFTR function is reduced or eliminated as the transmembrane voltage-sensitive dyes, or multi-electrode arrays allowing result of a variety of genetic mutations, leading to harmful fluid buildup, field potential measurements, action potential (AP) conduction velocity and decreased organ function, and eventually death. Our goal is to identify and 2D local activation mapping, and impedance changes in cultured monolayers. characterize the molecular interactions of several disease-relevant mutations In view of new recommendations of the cardiac in vitro pro-arrhythmia of CFTR with a variety of small molecule modulators of the protein. To (CiPA) consortium of multiple ion channel pharmacology assessment this end, we use a highly-sensitive label-free detection method based on (MICE), combined with AP and electrophysiology model-based testing of backscattering interferometry (BSI). By displaying CFTR in a native mem- arrhythmia-prone conditions, we developed a complex automated patch- brane environment, we have observed and characterized the interactions clamp assay, combining current-clamp and voltage-clamp protocols, appli- of several small molecule modulators, obtaining both binding affinities and cable to commercial high-purity human iPSC-derived cardiomyocyte prepa- structural information about CFTR. This information has allowed us to rations, e.g. iCellÒ Cardiomyocytes or Cor.4UÒ cardiomyocytes with the conclusively determine the previously unknown binding site for one of these CytoPatchÔ 2 equipment. To study stimulus-triggered APs we suppressed modulators. spontaneous firing via continuous injection of a faint hyperpolarizing current (10 or 20 pA), and for removal of fast oscillations during rapid AP up- 2957-Pos Board B387 stroke we applied a 60 Hz Gaussian filter implemented in Clampfit10. APs Dantrolene Inhibition of RyR2 requires Calmodulin 1 2 1 1 were analyzed for automated measurement of resting and peak potential, Ye W. Oo , N. Gomez-Hurtado , D.F. vanHelden , M.S. Imtiaz , B.C. Knollmann2, D.R. Laver1. maximum phase 0 slope (dV/dtmax), and various AP durations: APD10, 1School of Biomedical Sciences and Pharmacy, The University of Newcastle, APD50, APD90. Nifedipine elicited pacing frequency-dependent reductions 2 in APD50 (16.1% and 12.0% of control values at 1 and 10 mM, respectively), Newcastle, Australia, Division of Clinical Pharmacology, Vanderbilt cisapride 0.1 mM prolonged APD90 to 176.2%, and TTX 10 mM decreased University, Nashville, TN, USA. Dantrolene is a muscle relaxant that has been used clinically as the treatment dV/dtmax to 33.3% and APD90 to 80.4% of control values. Voltage-clamp for malignant hyperthermia. Dantrolene acts on skeletal and cardiac muscle measurements evidenced a progressive increase from 10 to 15 days in vitro 2þ (d.i.v.) for densities (pA/pF, mean5SD) of peak Naþ current (119.6567.3 to by inhibiting Ca release from the SR. However, single channel studies fail 406.75799.2), L-type Ca2þ current (2.7452.84 to 3.4354.05), plateau cur- to see any effect of dantrolene on RyRs. Calmodulin (CaM) regulates RyR2 rent within a 2-s depolarizing pulse at þ40 mV (1.2851.42 to 3.1654.68), directly by binding to RyR2 and dantrolene influences CaM binding to while transient outward current was more steady (0.7751.14, 10 d.i.v. and RyR2. CaM can dissociate from RyR2 within ~1 min so when RyR2 are iso- 0.6150.75,14 d.i.v.). lated form cells and examined in artificial lipid bilayers, CaM is not usually pre- sent. Thus, we hypothesized that CaM is the missing protein required for 2955-Pos Board B385 dantrolene inhibition of RyR2. Pharmacological Characterization of NR1/NR2A and NR1/NR2B Gluta- Single channel recordings were obtained from RyR2 isolated from sheep mate Receptors using an Electrophysiology Based HTS Assay heart and incorporated into artificial lipid bilayers. Control RyR2 activity Nikolai Fedorov, Yuri Kuryshev, Amy Wright, Zhiqi Liu, Luke Armstrong, was measured for periods of 1 min, and then during 1 min exposure to Glenn E. Kirsch, Arthur M. Brown. dantrolene and then again after washout. This was repeated in the absence ChanTest, Cleveland, OH, USA. and presence of exogenous 100 nM CaM. In diastolic cytoplasmic [Ca2þ] Glutamate NMDA receptors are increasingly employed in drug discovery for (100 nM), dantrolene (50 mM) reduced the mean open probability of RyR2 numerous clinical indications. Multiple receptor isoforms with distinct brain to 45 5 6 % in the presence of CaM (n ¼ 7, p < 0.05, student paired distributions and properties arise by selective splicing of the NR1 transcripts t-test) but had no significant effect when CaM was absent (95 5 9%, and differential expression of NR2 (A-D) subunit subtypes of NMDA recep- n¼20, p ¼ 0.24). Dantrolene exhibited a hyperbolic dose-response in the tors. NR1/NR2A and NR1/NR2B represent the most abundant subtypes. presence of CaM with an IC50 of 0.16 5 0.03 mM and with a saturating rela- 2þ Recent advances in electrophysiology-based HTS assays however, demand tive Po (Emax)of525 4%.Emax increased to one as cytoplasmic Ca was renewed characterization of these receptors on automated patch clamp plat- increased to levels above 1 mM. forms. In this study we used IonWorks Barracuda, an HTS platform that In saponin permeablized mouse cardiomyocytes supplemented with 100 nM can operate in 384-well format, population patch clamp (PPC) mode. Side- CaM, dantrolene reduced the frequency and amplitude of Ca2þ waves by-side comparisons of pharmacological properties of NR1/NR2A and with an IC50 of 0.3 mM. However, when cells were depleted of CaM, NR1/NR2B glutamate receptor responses to L-glutamate and NMDA, phar- dantrolene had no effect. Thus CaM is essential for inhibitory action of macological profiling and comparison of eight competitive and uncompetitive dantrolene. 586a Wednesday, February 11, 2015

2958-Pos Board B388 2960-Pos Board B390 M1 and M2 Microglia Exhibit Significant Differences in their KD Channel New Insight into the Involvement of Large-Conductance Calcium- Expression Activated-Potassium-Channel(BK) in Cell Viability: Pathophysiological Eva Melanie Grossinger1, Hai Minh Nguyen1, Yi-Je Chen1, Implications in Neuromuscular Disorders Izumi Maezawa2, Heike Wulff1. Angela Curci, Antonietta Mele, Giulia Maria Camerino, Diana Conte, 1Pharmacology, University of California Davis, Davis, CA, USA, Domenico Tricarico. 2Department of Pathology and Laboratory Medicine, University of California Dept. of Pharmacy-Drug Science, University of Bari, Bari, Italy. Davis, Sacramento, CA, USA. The large conductance Ca2þ-activated Kþ-channel (BK) is involved in several Microglia effector functions are widely associated with specific phenotypes, pathophysiological conditions including periodic paralysis (PP) and myotonia. which are referred to as M1 and M2. Classically activated M1 microglia Acetazolamide (ACTZ) a carbonic anhydrase inhibitor used in these con- release pro-inflammatory cytokines and neurotoxic molecules and have been ditions, acts targeting BK in PP. Here we investigated the involvement of BK associated with neurological damage in ischemic stroke and Alzheimer’s dis- channel in neuronal viability (SH-SY5Y cell) by combining patch-clamp ease. Alternatively activated M2 microglia exhibit beneficial immunological technique and cell proliferation assays. We performed these measurements effector functions such as phagocytosis of debris and release of anti- in the presence or absence of the selective BK channel blocker Iberiotoxin inflammatory and neurotrophic factors. Similar to B- and T-cells, microglia (IbTX) (10-400x109M), the unselective Kþ-channels blocker Tetraethylam- activity is regulated by calcium (Ca2þ)-signaling, which is maintained by po- monium (TEA) (0.01-1x103M), and the BK channel openers NS1619 (10- tassium (Kþ) channels. We here investigated whether M1 and M2 microglia 100x105M) and ACTZ (0.1-200x106M). Patch-clamp recordings showed differ in their Kþ channel expression by differentiating neonatal mouse that at þ30mV (Vm) IbTX and TEA reduced whole cell Kþ-current in a concen- microglia into M1 and M2 phenotypes using lipopolysaccharide (LPS) tration -dependent manner with an Imax of 46% and 90% respectively. and/or interferon-gamma or IL-4 and studying the cells by whole-cell NS1619 enhanced Kþ-current of þ141% at 10mV (Vm). Acetazolamide, patch-clamp. We identified three types of Kþ channels based on their bio- that in muscle acts as a BK opener, in neurons caused a concentration- physical and pharmacological fingerprints: a use-dependent, outwardly recti- dependent block of Kþ-current at þ30mV(Vm) with an IC50 of 1.73x107M fying current sensitive to the Kv1.3 blockers PAP-1 and ShK-L5, an inwardly an Imax of 40% (slope¼0.37) (Number of patches¼12). These drugs exert rectifying Ba2þ-sensitive Kir2.1 current, and a Ca2þ-activated, TRAM-34- their effects also on neuronal cell viability, enhancing it: IbTX showed a sensitive KCa3.1 current. M1 microglia, obtained by stimulation with LPS maximal proliferative effect (MPE) of þ46% at 108M concentration, reducing or a combination of LPS and interferon-gamma exhibited high Kv1.3 current it at higher concentrations; TEA showed a concentration-dependent increase of densities (~ 30-60 pA/pF at 40 mV) and virtually no KCa3.1 and Kir currents, cell proliferation with a MPE of þ34% at a 104M concentration; NS1619 and while IL-4 stimulated M2 microglia exhibited large Kir currents (~ 10 pA/pF ACTZ showed a MPE of þ181.6% at 5x105M and þ135% at 100x106M at 120 mV). KCa3.1 currents were generally low but moderately increased concentration respectively. Staurosporine (STS) (2x10-6M), a broad spectrum following stimulation with interferon-gamma or ATP. This differential protein kinases inhibitor, prevented the IbTX and TEA proliferative action. Kþ channel expression pattern suggests that Kþ channel modulators could These results suggest that BK channel may play a role in the regulation of be used to selectively inhibit detrimental neuroinflammatory functions of neuronal viability through an intracellular pathway that involves STS-sensitive microglia. protein kinases. These findings may have relevance in the cellular repair mech- This work was supported by RO1 GM076063 and RO1 AG043788A1 from anisms in the neuromuscular disorders. Supported by Telethon GG14096. the NIH. 2961-Pos Board B391 2959-Pos Board B389 Long-Term Modulation of Ion Channels by Aldosterone in Adult Rat Atherogenic Very-Low-Density Lipoprotein Shortens Atrial Action Poten- Atrial Myocytes tial Duration by Increasing Potassium Currents and Calcium Transient Erick B. Rios-Perez, Maricela Garcı´a-Castan˜eda, Guillermo Avila. Hsiang-Chun Lee1,2, Chi Wei3, Liang-Yin Ke3, Pei-Shang Tsai3, Cinvestav, IPN, Mexico, Mexico. Hsin-Ting Lin3, Yi-Lin Shiao3, Bin-Nan Wu4, Chu-Huang Chen3,5, In recent years, both aldosterone and mineralocorticoid receptor (MR) have Sheng-Hsiung Sheu1,2. drawn attention as important factors that promote structural remodeling of 1Division of Cardiology, Department of Internal Medicine, Kaohsiung the atrium. Here, we investigated effects of chronic aldosterone treatment on Medical University Hospital, KMU, Kaohsiung, Taiwan, 2Internal Medicine, both intracellular Ca2þ and ion channels. Atrial myocytes were cultured in College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, either the absence or presence of aldosterone, and then the activity of ion chan- 3Center of Lipid Biosciences, Kaohsiung Medical University Hospital, nels was studied, under whole-cell patch-clamp conditions. Aldosterone Kaohsiung, Taiwan, 4Department of Pharmacology, College of Medicine, increased both the cell membrane capacitance (Cm) and the maximal conduc- Kaohsiung Medical University, Kaohsiung, Taiwan, 5Vascular & Medicinal tance of ion channels that give rise to IKsus, INa, ICaT, and ICaL (30-100%). Research, Texas Heart Institute, Houston, TX, USA. Except for inactivation curves of INa, which were shifted by 10 mV, aldoste- Background: Compared to that of healthy normal subjects, plasma very rone produced no major alterations in the biophysical properties of the chan- low density lipoprotein (VLDL) of patients with the metabolic syndrome nels. Interestingly, at resting membrane potentials the increase in INa was (MetS) has been shown to be more electronegative and atherogenic. cancelled by a greater fraction of inactivation. The onset and recovery of the Given the association between MetS and increased prevalence of atrial changes -in ICaT, ICaL, and Cm- were also assessed. In general, they required fibrillation (AF), we investigated the mechanistic role of VLDL in the 2d to be noticeable, reached their maximal value in 6d, and returned to basal AF pathogenesis. values after 1-3d of aldosterone removal. The effects on both Cm and ICaL Methods: We extracted VLDL via peripheral blood obtained from normal, were further studied -to explore both a potential dose-response relationship healthy volunteers and MetS individuals. The normal-VLDL and MetS- and a possible implication of the MR. In fact, co-incubating with 10 mM of spi- VLDL samples were treated to HL-1 atrial cardiomyocytes respectively for ronolactone (an MR antagonist) abolished both effects. Furthermore, their cor- 12 hours before experiments. Whole-cell patch clamp was used for recording responding magnitudes fitted well with the Hill equation, being the EC50 the action potentials, voltage-gated potassium currents, and L-type calcium cur- values for Cm and ICaL 20 and 130 (nM), respectively. Interestingly, aldoste- rents. Calcium image with Fura-2-AM Ca2þ indicator was applied for the intra- rone did not alter expression levels of Cav1.2, suggesting that the action on cellular calcium measurements. ICaL arises from a stimulus in open probability. The hormone also produced Results: MetS-VLDL treated HL-1 cells exhibited significantly higher den- a 40% increase in the amplitude of Ca2þ transients along with a higher propor- sities of repolarizing potassium currents, IKs and IKr. MetS-VLDL shifted tion of arrhythmic cells (2.5-fold increase). These results contribute to under- the activation curve of ICaL toward more negative membrane potentials. standing the role of the MR and aldosterone in atrial electrophysiology. Intracellular calcium signals were significantly enhanced by MetS-VLDL but not by normal-VLDL. MetS-VLDL significantly shortened action poten- 2962-Pos Board B392 tial durations (MetS-VLDL 178.1 5 32.0 msec vs control 257.2 5 52.7 The Anti-Proliferative Effect of Cation Channel Blockers on T Lympho- msec; P¼0.0017). Addiotnally, frequent occurrences of early after- cytes Stimulated by Anti-CD3 and Anti-CD28 depolarization on action potentials were noted in MetS-VLDL treated HL-1 Zoltan Varga, Zoltan Petho, Andras Balajthy, Adam Bartok, Sandor Somodi, cells. Orsolya Szilagyi, Gyorgy Panyi. Conclusions: The VLDL of MetS individuals augmented repolarizing potas- Dept. of Biophysics and Cell Biology, University of Debrecen, Debrecen, sium currents, increased intracellular calcium release, and significantly short- Hungary. ened action potentials. These changes may contribute in coordination to The ion channels of T lymphocytes form a crucial part of the healthy immune increased AF vulnerability in MetS. system, as they are important for cellular activation and proliferation. In Wednesday, February 11, 2015 587a autoimmune diseases, effector memory T cells have a unique ion channel servation of TOK1 primary structure was greatest in both pore domains and pattern, therefore they are promising therapeutic targets. A number of ion chan- flanking pore lining transmembrane domains, TM6 and TM8. MgTOK1 from nel inhibitors are known as selective inhibitors of T lymphocyte proliferation, Mycosphaerella graminicola (wheat leaf blotch) and FgTOK1 from Fusarium but the data available is contradictory. Our aim was to elucidate this phenom- graminearum (wheat head blight) were cloned by RT-PCR into an expression enon by investigating how the blockage of ion channels affects the activation vector. cRNA was transcribed in-vitro and injected into Xenopus laevis oocytes and proliferation of T cells treated previously with different concentrations and ionic currents measured by two-electrode voltage clamp after 24-48 hours. of mitogens. Both cloned TOK1 channels exhibited Kþ selective, non-inactivating, In our experiments human peripheral blood lymphocytes from volunteers were strongly outwardly-rectifying Kþ currents whose activation threshold was activated via monoclonal antibodies affecting the TCR-CD3 complex on the strictly determined by the transmembrane Kþ gradient, as revealed by isotonic cell surface and the co-stimulator molecule CD28. We applied specific ion replacement of extracellular Naþ with Kþ. Channels varied in their voltage- channel blockers acting on the major cationic channels of the T cell, the dependent activation kinetics and distinct from the canonical TOK1 isolate Kv1.3, the KCa1.1 and the CRAC channel, either alone or in combination from Saccharomyces cerevisae, displayed signs of time-dependent deactiva- with rapamycin, the inhibitor of the mammalian target of rapamycin tion. This appears to be the first reported molecular identification and charac- (mTOR). Five days after the stimulus flow cytometry measurements were per- terisation of TOK1 Kþ channels from plant pathogenic fungi. formed to determine the extent of cellular viability and proliferation. Our measurements indicated that ion channel blockers and rapamycin had a 2965-Pos Board B395 negative dose-dependent effect on the amount of cell division. Simultaneous KCa1.1 (BK) Channels on Fibroblast-Like Synoviocytes: A Novel Thera- application of blockers for each channel along with rapamycin proved to be peutic Target for Rheumatoid Arthritis 1 2 1 1 the most effective, which indicates that they affect independent regulation Mark R. Tanner , Zoltan Petho , Rajeev B. Tajhya , Redwan Huq , 1 3 1 pathways. Upon increasing the rate of stimulation, the anti-proliferative effect Frank T. Horrigan , Percio S. Gulco , Christine Beeton . 1Department of Molecular Physiology and Biophysics, Baylor College of of the blockers diminished. This phenomenon was unknown to date and may 2 prove to be important in understanding the fine-tuning of T cell activation. Medicine, Houston, TX, USA, Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary, 3Division of Rheumatology, Icahn School of 2963-Pos Board B393 Medicine at Mount Sinai, New York, NY, USA. Proton Channels are Present in Cell Membranes of the Breast Cancer Cell Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease attacking Line MDA MB 231 and Affect Recovery from an Acid Load principally freely-moveable joints and affecting approximately 1.3 million in Deri Morgan1, Patrick McIntire1, Vladimir Cherny1, Susan Smith2, the US. Recently, joint-resident fibroblast-like synoviocytes in RA (RA-FLS) Boris Musset3, Thomas DeCoursey1. have been implicated in disease pathogenesis. We have shown that KCa1.1 1Molecular Biophysics, Rush University Medical Center, Chicago, IL, USA, is the predominant potassium channel expressed by RA-FLS and by FLS 2Department of Biology & Physics, Kenshaw State University, Kennesaw, from the pristane-induced arthritis (PIA) rat model of RA. Blocking KCa1.1 GA, USA, 3Institute of Complex Systems, Forschungszentrum Ju¨lich, Ju¨lich, with paxilline or iberiotoxin or reducing its expression with siRNA inhibited Germany. the production of pro-inflammatory cytokines, chemokines, and proteases, Voltage gated proton channels (HV1) have been implicated in late stage breast and the invasiveness of both RA- and PIA-FLS. In contrast, the over- cancer (Wang et al, 2012. J. Biol. Chem. 287:13877), where HV1 expression expression of KCa1.1 increased the invasiveness of PIA-FLS and induced correlated with tumor size and poor prognosis. HV1 knockdown reduced cell the invasiveness of healthy rat FLS. These data demonstrate a crucial role of proliferation and migration as well as matrix metalloprotease release. However, KCa1.1 in regulating the aggressive behavior of FLS during RA. the presence of functional HV1 on cancer cell membranes has not been demon- We induced two models of RA in rats, moderate PIA and severe complete strated and the mechanism by which they affect the function of these cells has Freund’s adjuvant collagen-induced arthritis. Treatment with the small mole- not been elucidated. cule KCa1.1 blocker paxilline, starting after onset of clinical signs, significantly Here we show the definitive presence of functional HV1 on the membranes of reduced disease severity in both models. However, paxilline can cross the MDA MB 231 cells, a highly metastatic triple negative cell line. We performed blood-brain barrier and block all variants of KCa1.1 throughout the body, patch clamp experiments on these cells and were able to detect bona fide thereby inducing side effects that preclude its use as a therapeutic for human voltage- and pH-gated channels that were perfectly selective for protons. The use without significant modification. A strategy to target KCa1.1 on RA-FLS membrane density of the channels in these cells was recorded as 3.5 pA/pF without side effects in other tissues involves the identification of the b and g which is roughly 5-fold lower than the 15 pA/pF displayed in neutrophils. In regulatory subunits of KCa1.1 in RA-FLS through qPCR, western blotting, order to show that HV1 expresses at a level sufficient to impact pH regulation and patch-clamp electrophysiology. This has the potential for the development within these cells, we acid loaded them using the ammonium prepulse tech- of blockers that selectively target KCa1.1 on RA-FLS and do not enter the cen- nique and monitored pHi recovery utilizing SEER with SNARF-1. Inhibiting tral nervous system. HV1 with 1 mM Zn2þ slowed recovery from an acid load by 3-fold, demon- strating that the expression of HV1 on these cells affects pH regulation in 2966-Pos Board B396 these cells. Kidney CLC-K Chloride Channels Inhibitors: Definition of Novel Struc- We conclude that one mechanism by which HV1 may influence the patho- tural Requirements and Efficacy in CLC-K Polymorphism Associated physiology of breast cancer is by improving the ability of breast cancer cells with Hypertension 1 1 1 to regulate their internal pH. Paola Imbrici , Antonella Liantonio , Giuseppe Fracchiolla , 1 1 2 Supported by the Bears Care and the Brian Piccolo fund. Giuseppe Carbonara , Maria Maddalena Dinardo , Michael Pusch , Diana Conte1. 2964-Pos Board B394 1Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy, TOK1 Potassium Channels in Phytopathogenic Fungi 2Institute of Biophysics, CNR, Genova, Italy. William R. Manville1, Andrew Corran2, Anthony Lewis1. The human chloride channels CLC-Ka and CLC-Kb play a pivotal role in kid- 1School of Pharmacy and Biomedical Sciences, University of Portsmouth, ney by controlling chloride and water absorption. Both channels require barttin Portsmouth, United Kingdom, 2Fungicide Biochemistry, Syngenta Limited, as an accessory subunit for full activity. Mutations in CLC-Kb and barttin genes Bracknell, United Kingdom. lead to severe renal salt loss while CLC-K gain of function polymorphisms Fungal plant pathogens are a significant threat to crop yield and global food could predispose to hypertension. Thus, compounds that selectively bind to security and the search for pathogen-specific agricultural fungicide targets is CLC-Ka and/or CLC-Kb channels may have a significant therapeutic potential. of high priority. TOK1 is a structurally and functionally unique plasma mem- Recently, we explored the pharmacological profile of CLC-K/barttin expressed brane potassium (Kþ) channel with no known homologues in plants or animals in mammalian HEK-293 cells and demonstrated that HEK cells represent a and is the only passive Kþ ion efflux pathway in fungi. Activation of TOK1 valid biological system to screen CLC-K high affinity blockers (Imbrici et leads to ion dyshomeostasis and cell death. However, little is known about al., Biochim Biophys Acta, 2014). Here, by using molecular modeling and TOK1 channels in phytopathogenic fungi. Here we describe the distribution, patch-clamp technique, we developed a new series of benzofuran derivatives evolution and molecular conservation of TOK1 homologues across plant fungal and evaluated their efficacy on CLC-K channels expressed in HEK 293 cells. phyla, and the cloning and characterization of TOK1 channels from two phy- Chemical modifications regarding the hydrophobic group at C-5 and C-3 posi- topathogens of significant socio-economic importance. In-silico bioinformatics tion of the benzofuran nucleus of the lead compounds RT-93 and JBL-44 (IC50 identified genes predicted to encode putative TOK1 protein subunits, conform- within 10-30 mM range), allowed us to define the structural requirements to ing to the characteristic eight transmembrane domain two pore domain (8TM/ ensure an efficacious CLC-Ka block, finally identifying SRA-36 the most 2P) structure, in 204/231 sequenced fungal genomes analysed. Molecular con- potent compound so far described, with an IC50 of 2.6 5 1 mM. Interestingly, 588a Wednesday, February 11, 2015 besides capable of inhibiting CLC-Kb and CLC-K1 isoforms, this compound no family history of neurological disorders and generated highly purified was also efficacious in blocking A447T CLC-Ka, a polymorphism associated (>95% TUJ1-positive), terminally differentiated cortical neurons from the with hypertension. Thus, the SRA-36 molecule could represent a useful probe two separate but isogenic iPS cell lines. for exploring CLC-K molecular mechanisms of gating as well as a new poten- Here, we present data on the characterization of these human neurons, tial therapeutic option for hypertensive patients carrying CLC-K gene polymor- including expression of a standard set of neuronal markers at both the genetic phisms (Telethon GGP14096). and protein level. We also highlight functional testing of the cells, with a spe- cific focus on electrophysiological readouts such as multi-electrode array 2967-Pos Board B397 (MEA) analysis. The ability to model neurological disorders through editing Computational Studies on Bladder Smooth Muscle: Modeling Ion Chan- the genome of an iPS cell and subsequently produce previously inaccessible nels and their Role in Generating Electrical Activity human neurons has revolutionized the way we approach studying and treating Chitaranjan Mahapatra, Rohit Manchanda. diseases of the central nervous system. Bio science & Bio engineering, Indian Institute of Technology Bombay, IIT Bombay,Powai,Mumbai,Maharastra, India. 2969-Pos Board B399 Urinary Incontinence (UI) is the involuntary loss of urine that constitutes a so- Novel Mutation of SCN1A in a German Family Presenting with both cial or hygiene problem, which has adverse effects on quality of life. Urinary Hemiplegic Migraine and Epilepsy Bladder Smooth Muscle (UBSM) instability is a major cause of UI. Different Chunxiang Fan, Frank Lehmann-Horn, Karin Jurkat-Rott. ion channels within bladder detrusor smooth muscle (DSM) play a role in Division of Neurophysiology, University ULM, ULM, Germany. generating spontaneous action potentials (sAP) and depolarizations (SD). Familial hemiplegic migraine (FHM) is a rare autosomal dominant migraine The aim is establish a mathematical platform of sufficient biophysical detail subtype with aura associated with reversible hemiparesis. Three causative to quantitatively simulate DSM sAPs and thereby inform future empirical in- genes encoding ion-channels / transporters have been identified: CACNA1A vestigations of physiological and pathophysiological mechanisms governing (FHM1), ATP1A2 (FHM2) and SCN1A (FHM3). SCN1A is a well-known normal and dysfunctional bladder activities. In line with recent experimental epilepsy gene with over 150 known mutations, while until now only few evidence, adapting the Hodgkin- Huxley formulation in the NEURON plat- FHM3 mutations have been described. Here we report a novel SCN1A form, we construct mathematical models for seven ionic currents of UBSM: L1624P mutation in a three-generation family with four patients who present Ca2þ currents (L- and T- type), two voltage gated Kþ currents and three with both FHM and epilepsy. To explore the underlying mechanism, SCN1A Ca2þ - activated Kþ current. The magnitudes and kinetics of each ionic current was transiently transfected in human TSA 201 cells together with the auxiliary system in a cylinder - shaped single cell with a specified surface area are subunits b1 and b2. Whole-cell patch clamp was employed for the electro- described by differential equations, in terms of maximal conductances, electro physiological characterization. L1624P produced similar current density and chemical gradients, voltage-dependent activation/inactivation gating variables similar voltage dependence of activation as those in wild-type channel. It and temporal changes in intracellular Ca2þ computed from known Ca2þ fluxes. displayed a positive shift in the steady-state inactivation, a faster recovery These quantifications are validated by the reconstruction of individual experi- from inactivation, an increase of non-inactivated depolarization-induced mental ionic currents obtained under voltage clamp. Our integrated model has sodium current near threshold potentials, and a decrease of use-dependent been validated by comparing the simulated profile of sAPs with experimental block. Our gain of function findings in L1624P are consistent with consequent recordings and shows good correspondence in terms of amplitude and shape neuronal hyperexcitability, and might be involved in the pathomechanism of in both control and pharmacologically altered conditions. Stimulation is done the phenotype. using an external current clamp, where somatic current injections were used to generate phasic spike. In summary, our advanced mathematical model 2970-Pos Board B400 provides a powerful tool to investigate the physiological ionic mechanisms un- Possible Role of STIM1 Sensor Signal in Memory Loss Connected with derlying the spikes in DSM, which in turn can shed light in genesis of UBSM Familial Alzheimer’s Disease 1 1 2 for a malfunction. Maria Ryazantseva , Ksenia Skobeleva , Anna Goncharova , Nikolai Kamyshev2, Elena Kaznacheyeva1. 2968-Pos Board B398 1Institute of Cytology RAS, St. Petersburg, Russian Federation, 2Pavlov Modeling Neurological Disease with Human iPS Cell-Derived Neurons Institute of Physiology RAS, St. Petersburg, Russian Federation. Containing a KCNT1 Mutation Familial Alzheimer’s disease (FAD) which leads to memory impairments is Kile P. Mangan, Michael McLachlan, Tom Burke, Benjamin Meline, caused by mutations in presenilin-1 (PS1) gene in approximately 40% of cases. Nathan Meyer, Lucas Chase, Brad Swanson, Coby B. Carlson, PS1 is well known as a component of the gamma-secretase enzyme which Susan DeLaura, Eugenia Jones. cleaves APP to A-beta. To become a catalytic part of enzyme PS1 undergoes Cellular Dynamics International, Madison, WI, USA. an endoproteolysis. It was shown that mutations in PS1 gene disrupt endopro- The sodium-activated potassium channel Slack, encoded by the gene KCNT1, teolysis increasing uncleaved protein level in brain tissue of FAD patients. In is expressed in neurons throughout many brain regions, including the frontal our study we found effects of FAD PS1 mutants (PS1DE9, PS1 D247A) on ac- cortex, and mediates a sodium-sensitive potassium current (IKNa). This out- tivity of store-operated calcium (SOC) channels in mice hippocampal neurons ward current regulates neuronal excitability and determines how neurons and Neuro2a cell line. Increased uncleaved PS1 levels led to SOC channels hy- respond to repeated high frequency stimulations, both of which are aspects peractivities detected with direct single-cell electrophysiological measure- of memory and learning. Not surprisingly, mutations in KCNT1 and alterations ments and calcium imaging experiments with fura2-AM. The effects were to the IKNa current have patho-physiological consequences. Recent studies caused by impaired signal transduction from ER to SOC channels in plasmatic have described the emerging role of Slack channels in cognitive deficits, and membrane. The impaired intracellular signal transduction by STIM1 sensor several reports have found KCNT1 mutations in patients with severe early was revealed in live confocal imaging experiments and proved with STIM1 onset ‘‘childhood’’ epilepsies. knock-down. Moreover, a feeding of Drosophila melanogaster transgenes ex- The development of better therapies for neurological disorders has been pressing human mutated PS1 in cholinergic nervous system with pharmacolog- hindered by limited access to clinically-meaningful cell models for research ical inhibitor of STIM sensor signal transduction 2APB led to rescue of the and drug development. The advent of induced pluripotent stem (iPS) cell tech- memory loss detected by courtship based assay with aged animals. Therefore nology provides a platform to facilitate increased understanding of disease hyperactive STIM1 signal transduction leads to increased SOC channels activ- mechanisms in a physiologically-relevant environment. We have leveraged ity which could be the reason for memory loss in FAD. this technology to generate human neurons that express the KCNT1 P924L This work was supported by the program of ‘‘Molecular and Cellular Biology’’ mutation in the Slack channel. To introduce this alteration, we genetically RAS, research grants from the Russian Basic Research Foundation, Russian engineered a ‘‘control’’ iPS cell line from an apparently healthy donor with Scientific Fund and the President of Russia Scholarship. Wednesday, February 11, 2015 589a

Skeletal Muscle Mechanics, Structure, 2973-Pos Board B403 Improved Imaging, 3D Reconstruction and Homology Modeling of and Regulation Tarantula Thick Filaments Shixin Yang1, Fa-Qing Zhao1, Guidenn Sulbara´n2, John L. Woodhead1, 2971-Pos Board B401 Lorenzo Alamo2, Antonio Pinto2, Rau´l Padro´n2, Roger Craig1. Direct Assessment of Skeletal Muscle Contractile Strength in Live 1UMass Medical School, Worcester, MA, USA, 2Instituto Venezolano Wildtype and Rbfox Morphant Zebrafish Larvae de Investigaciones Cientı´ficas (IVIC), Caracas, Venezuela, Bolivarian Brit L. Martin1, Tom L. Gallagher2, Neha Rastogi1, Christine E. Beattie3, Republic of. Sharon L. Amacher2, Paul M.L. Janssen1. Tarantula muscle is an outstanding model system for elucidating thick 1 Department of Physiology and Cell Biology, The Ohio State University, filament molecular structure. In past studies we showed by cryo-EM, single 2 Columbus, OH, USA, Department of Molecular Genetics, The Ohio State particle reconstruction, and atomic fitting with a hybrid myosin head struc- 3 University, Columbus, OH, USA, Department of Neuroscience, The Ohio ture, that relaxed tarantula filaments are characterized by intramolecular State University, Columbus, OH, USA. interaction between heads (J-motif) that is thought to inhibit myosin activity The accessible genetics and abundant skeletal musculature of the zebrafish by blocking actin binding in one head and ATPase activity in the other make it an ideal model for studies of muscle contraction. Current functional (Woodhead et al., 2005). We have been pursuing improvements in technique assays for quantifying the effect of experimental modification of zebrafish to obtain a higher resolution reconstruction. A key problem in reconstructing muscle are indirect and observational, or they underestimate contractile myosin filaments is intrinsic flexibility of the heads, which reduces the res- force. Therefore, we developed an in vivo assay for measuring muscle con- olution we can achieve. We have used staurosporine to minimize RLC phos- tractile force. As proof of concept, we have measured contractile strength of phorylation and thus head disordering, and blebbistatin to stabilize the heads. skeletal muscle in wildtype larvae and in a morphant model from 2-5 days Automated data collection has been used to obtain images on a field emission post fertilization (dpf). Mean maximum tetanic whole-body forces produced cryo-electron microscope (Titan Krios). Preprocessing of images has enabled by wildtype 2, 3, 4, and 5 dpf larvae amounted to 3.03 mN, 7.31 mN, the selection of a data set as homogeneous as possible. Both SPIDER and 8.67 mN, and 10.94 mN, respectively. Mean twitch forces produced by RELION were used for 3D reconstruction, and yielded a similar resolution larvae were 0.90 mN, 5.58 mN, 7.08 mN, and 9.08 mN for 2, 3, 4, and of ~13 A˚ , 2-fold better than our original reconstruction, but presumably still 5 dpf respectively. The morphants we have analyzed are knockdowns of limited by some head flexibility. The J-motif is consistent with the original two zebrafish paralogs of rbfox, rbfox1l and rbfox2, which regulate findings, but the structure of the S2 segment of the myosin tail is much muscle-specific splicing programs. We assessed the contractile force devel- more robust and some details of the paramyosin core are now visible. We oped during contraction and found that our assay is clearly able to differen- have built an homology model of the interacting-heads motif (using the tiate between morphant phenotypes. rbfox2 morphants appear to produce tarantula sequence for both heavy and light chains), that includes all surface maximal tetanic forces similar to wildtype larvae, while rbfox1l morphants loops, and we are using this for molecular dynamics flexible fitting (MDFF). demonstrate significantly impaired function by producing decreased forces Preliminary results suggest the need for repositioning of several loops from at the same developmental time points. This supports the conclusion that their positions in the crystal structure, with possible implications for filament rbfox1l regulates the majority of splicing events in larval skeletal muscle. assembly and stability. rbfox1l/rbfox2 morphants are paralyzed and their lack of significant con- tractile force production in our assay indicated a muscle-specific defect, 2974-Pos Board B404 not just a motoneural defect, causing the characteristic paralysis. We have Spectroscopic Studies of the Super-Relaxed State of Skeletal Muscle also developed an immunohistological assay for empirically determining Leonardo Nogara1, Nariman Naber2, Edward Pate3, Marcella Canton1, the cross-sectional area of larval trunk skeletal muscle in order quantify Carlo Reggiani1, Roger Cooke2. muscle-specific force per cross-sectional area. These functional results quan- 1University of Padua, Padua, Italy, 2UCSF, San Francisco, CA, USA, 3WSU, tify muscle-specific phenotypes sans neural input. Pullman, WA, USA. The super-relaxed state of myosin (SRX) in which the myosin ATPase activity 2972-Pos Board B402 is strongly inhibited has been observed in a variety of muscle types. It has been Zebrafish Myofilaments and their Assemblies are Good Structural Models proposed that myosin heads in this state are inhibited by binding to the core of for Studying Disease Mutations the thick filament in a structure known as the ‘‘interacting heads motif’’ (IHM). Fa-Qing Zhao, John L. Woodhead, Roger Craig. This contrasts with the disordered relaxed state (DRX), in which heads are not UMass Medical School, Worcester, MA, USA. organized around the core of the thick filament and have an ATPase rate that is Zebrafish have become a prominent animal model of human disease because of an order of magnitude greater. In the IHM the N-terminal lobes of the two their genetic tractability and rapid development (Lieschke & Currie, 2007). RLCs bind to each other. We have made a series of five single cysteine mutants Several muscle diseases have been studied using zebrafish, including of the RLC, placed both paramagnetic and fluorescent probes on them, and muscular dystrophy (Johnson et al., 2013), hypertrophic and dilated cardio- exchanged them into rabbit fast skeletal muscle fibers. Probes that were located myopathy, nemaline myopathy (Sehnert et al., 2002; Asnani & Peterson, within the putative RLC-RLC interface tended to disrupt the stability of the 2014; Telfer et al., 2012) and distal arthrogryposis (Ha et al., 2013). Here SRX. Probes far from this interface had no effect on the SRX. Most of the we show that zebrafish myofilaments and their assemblies have molecular spin-labels bound to the RLC were disordered in all states, however one label structures similar to those of higher vertebrates, demonstrating their useful- (C31) showed good orientation in the SRX. Some fluorescent probes adjacent ness in elucidating structural changes due to disease mutations. EM and to the putative interface showed an increased intensity during the transition 3D reconstruction have shown that striated muscle contraction is regulated from the disordered relaxed state to the SRX. Together the results provide 2þ by Ca -induced movement of tropomyosin on thin filaments, which un- further evidence identifying the location of the RLC-RLC interface, and covers myosin-binding sites on actin. Using negative staining EM and single provide a proof of concept that pharmaceuticals that bind to this interface particle reconstruction we find that tropomyosin is well resolved in zebrafish will disorder the SRX leading to a potentially effective treatment for type II 2þ native thin filaments and undergoes the same Ca -induced movement as diabetes and obesity. seen in other species. Zebrafish thin filaments are therefore good models for studying the impact of thin filament mutations (actin, troponin, tropomy- 2975-Pos Board B405 osin, nebulin) on thin filament structure and regulation. Thick filaments are X-Ray Diffraction from Insect Flight Muscle Fibers with Exchanged also readily isolated from zebrafish muscle and their 3D reconstruction is Contractile Proteins similar to that of mammalian filaments (Gonza´lez-Sola´ et al., 2014). We Hiroyuki Iwamoto, Naoto Yagi. have isolated intact filament assemblies (myofibrils and A-segments) from Life & Environ. Div., SPring-8, JASRI, Sayo-gun, Hyogo, Japan. zebrafish and find that these also closely resemble those of other vertebrates, By recording fast X-ray diffraction movies from live bumblebee, we have including mammals, clearly revealing MyBP-C and M-line periodic organi- shown that specific X-ray signals (the reciprocal intensity changes of the 111 zation. These are therefore good models for elucidating the structural impact and 201 reflections) arise in the stretch phase of the flight muscle (IFM), and of mutations in myosin, MyBP-C and M-line proteins. We conclude that the from this observation and model calculations we have suggested that the structures of zebrafish skeletal muscle thick and thin filaments and their stretch-induced distortion of actin-bound low-force myosin head is the trigger native assemblies closely resemble those of higher vertebrates, making for stretch activation (SA). However, there are still some unsolved questions: them excellent models for studying the molecular impact of disease- (1) does IFM myosin have special sensitivity to stretch? (2) Is it the very regular causing mutations on filament structure. spatial arrangement of proteins in IFM sarcomere that causes SA regardless of 590a Wednesday, February 11, 2015 the source of myosin? (3) Does the IFM-specific troponin keep the myosin from Results: The mechanical results from dissociated FDB myofibers match generating force prematurely? To answer these questions we have started ex- findings from dissected EDL myofibers, but SP was up to 14-fold higher in periments to exchange contractile proteins within IFM sarcomere with those the FDB than EDL. SP was 27% lower in mdx myofibers and 50% less in from other sources. It is shown that, when IFM fibers from crane fly are incu- branches of split mdx fibers compared to the trunk. AP amplitude was not bated in a solution containing fluorescent rabbit skeletal muscle myosin, it is altered in between groups, but this work is ongoing. incorporated into IFM sarcomere. This technique utilizes the properties of my- Conclusions: Data indicate a reduction in muscle stiffness, increased sarco- osins that have different extractability in KCl solutions. The IFM fibers with lemmal deformability and instability in mdx muscle. This approach corrobo- rabbit myosin showed a series of strong X-ray meridional reflections with a rates the labor-intensive data obtained from single fiber dissection and allows basic repeat of 14.5 nm. On the other hand, the control IFM fibers incubated a facile high throughput model. Findings suggest mechanical differences due in the same KCl solution without rabbit myosin seem to retain their endogenous to altered morphology, despite comparable excitability. myosin molecules. These results suggest that, even at KCl concentrations too low to extract endogenous myosin, myosin exchange occurs if exogenous sol- 2978-Pos Board B408 ubilized myosin is present in the surrounding medium. We also plan to extract Mir181A Targets the 3’UTR of MG29, a Muscle-Specific the thin filaments in IFM and to replace them with actin filaments from other Family Gene, for Down-Regulation of MG29 Expression in Dystrophic sources. Skeletal Muscle Feng Jin1, Kyoung-Han Choi2, Jae-Kyun Ko2, Ki-Ho Park1, Chao Cheng3, 2976-Pos Board B406 Xiaoli Yao4, Zhenguo Liu3, Jianjie Ma1, Hua Zhu1. The M-Line Protein Obscurin in the Development of Insect Flight Muscle 1Department of Surgery, Davis Heart and Lung Research Institute, Anja Katzemich1, Kevin Leonard2, Sean Sweeney1, John Sparrow1, Columbus, OH, USA, 2Department of Physiology and Biophysics, Rutgers Belinda Bullard1. University-Robert Wood Johnson Medical School, Piscataway, NJ, USA, 1Biology, University of York, York, United Kingdom, 2Wellcome Trust 3Department of Thoracic Surgery, The First Affiliated Hospital of Sun Genome Campus, EMBL-EBI, Cambridge, United Kingdom. Yat-sen University, Guangzhou, China, 4Department of Neurology, The First The Drosophila M-line protein, obscurin (475 kD) has 21 tandem Ig domains, 3 Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. Fn3 domains, a Rho-GEF signalling domain near the N-terminus and two MG29 is a muscle-specific member of the synaptophysin family protein that kinase domains near the C-terminus. Obscurin is needed for the formation of participates in controlling the maturation and development of transverse tubule a symmetrical sarcomere in the indirect flight muscle (IFM). Obscurin forms (TT) structure and the maintenance of intracellular Ca2þ signaling in skeletal periodic striations in the IFM sarcomere 30 h after puparium formation, muscle. Genetic ablation of MG29 leads to defective formation of the TT when kettin and myosin are still in unstructured strands and there are no network in skeletal muscle, which resembles the abnormal TT structure observed Z-discs. Early expression of obscurin acts as a template for the formation of in dystrophic muscles. We found that MG29 protein is drastically reduced in symmetrical thick filaments. Reducing expression by RNAi had no effect on mouse models with muscular dystrophy (e.g. mdx and dysferlin-/-), and such sarcomere length in IFM, but the M-line was missing and the H-zone was reduced MG29 expression and disrupted TT structure were also observed in shifted from the midline of the sarcomere. The length and polarity of thin human patient biopsies with muscular dystrophy. The mg29 gene contains a filaments was determined by the position of the bare zone in adjacent thick fil- unique 3’ untranslated region (UTR) with potential binding sites for microRNA aments. Therefore obscurin is essential for the assembly of correctly overlap- (miRNA) or RNA-stabilizing factors. Mutagenesis studies revealed that miR- ped thick and thin filaments in a symmetrical sarcomere. We have identified 181a could target a region in the 30UTR that is highly conserved between mouse ligands of the two kinase domains in vivo. Kinase constructs with tags were and human mg29 genes to exhibit posttranscriptional control of MG29 ex- injected into embryos and individual kinases with bound proteins were isolated pression in skeletal muscle. Overexpression of anti-sense miR-181a by microin- from IFM. Kinase 1 bound Ball (an active kinase) and kinase 2 bound MASK jection into mouse flexor digitorum brevis fibers significantly increased the (a 400 kD protein with ankyrin repeats). Reducing expression of these proteins expression of endogenous MG29. Studies from other investigators have shown by RNAi produced a phenotype in IFM that was similar to that produced by that miR-181a was elevated in human patients with muscular dystrophy. These reducing obscurin, although obscurin itself was present at wild-type levels. data suggest an intriguing possibility that elevated miR-181a may be a con- Therefore Ball and MASK probably act downstream of obscurin. These pro- tributing factor for the reduced MG29 expression under dystrophic conditions. teins are linked via signalling pathways involved in the development of Further understanding of the post-transcriptional regulation by microRNA of Drosophila muscle. MG29 expression can provide more insights into the therapeutic targets of muscular dystrophy. 2977-Pos Board B407 Sarcolemmal Biomechanics and Excitability in Malformed Muscle Fibers 2979-Pos Board B409 of Dystrophic Mice Calcium Sensitivity after Active Shortening in Rabbit Psoas Fibres Karla P. Garcia-Pelagio1, Erick O. Herna´ndez-Ochoa2, Stephen J.P. Pratt3, Venus Joumaa, Walter Herzog. Kathleen Twomey3, Richard M. Lovering3. University of Calgary, Calgary, AB, Canada. 1Department of Physiology, University of Maryland School of Medicine, Introduction It has been shown that isometric force after active shortening of Baltimore, MD, USA, 2Department of Biochemistry & Molecular Biology, muscles is lower than the purely isometric force performed at the corresponding University of Maryland School of Medicine, Baltimore, MD, USA, lengths. This property is termed force depression. Force depression has been 3Department of Orthopaedics, University of Maryland School of Medicine, observed in isolated muscle preparations and muscles in situ and in vivo (Rassier Baltimore, MD, USA. et al., J Applied Physiol 2004). The origin of force depression is still unknown. It Background: Duchenne muscular dystrophy (DMD), the most common and se- has been suggested that active shortening may affect calcium sensitivity by vere dystrophy, is caused by the absence of dystrophin. Muscle weakness and changing calcium-troponin binding equilibrium leading to decreased force after fragility (i.e. increased susceptibility to damage) are presumably due to struc- active shortening (Ekelund and Edman, Acta Physiol Scand 1982). Our aim in tural weakness of the myofiber cytoskeleton, but recent studies suggest that the present study was to test whether calcium sensitivity was decreased after malformed/split myofibers in dystrophic muscle may also play a role. We active shortening. have previously studied the biomechanical properties of the sarcolemma in sin- Methods Experiments were performed using skinned fibres (n¼7) isolated gle myofibers isolated mechanically from extensor digitorum longus (EDL) from rabbit psoas muscle. Calcium sensitivity was characterized by establish- muscles in wild-type (WT) and dystrophic (mdx, mouse model for DMD) ing the force-pCa curves for isometric reference contractions performed at an mice. PURPOSE: We use similar biomechanical methods on enzymatically- average sarcomere length of 2.4 mm and active shortening contractions from dissociated myofibers (both normal and malformed) from the flexor digitorum an average sarcomere length of 3.0 mm to an average sarcomere length of brevis muscle (FDB) of WT and mdx mice. 2.4 mm. pCa50 and the coefficient of cooperativity (nH) were compared between Methods: FDB muscles were enzymatically-dissociated and plated on special- reference and active shortening contractions. ized coverslips. Suction pressures (P) applied through a pipette to the mem- Results and Discussion pCa50 for the reference contractions was 5.9950.02. brane generated a bleb, which increased in height with increasing P. Larger No change in pCa50 was observed in the force depressed state (6.0050.01). increases in P ruptured the connections between the sarcolemma and myofibrils Furthermore, the coefficient of cooperativity did not change after active short- (speration P, or SP) and eventually caused the sarcolemma to burst. We also ening (3.950.3 versus 4.350.3 for the reference and force depressed states examined excitability using high-speed confocal microscopy and the voltage- respectively). These results suggest that calcium sensitivity does not decrease sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl- after active shortening and that the binding interactions between troponin sulfonate to assess the action potential (AP). and calcium are not altered in the force depressed state. Wednesday, February 11, 2015 591a

2980-Pos Board B410 slack). Force mapping revealed distinct transversal stiffness patterns along Force Transients during Stretch of Myofibrils Activated with MgADP the sarcomere, suggesting relatively higher Z-disk stiffness and somewhat Fabio C. Minozzo, Dilson E. Rassier. lower A-band stiffness. A-band lateral stiffness did not differ between psoas McGill University, Montreal, QC, Canada. and diaphragm sarcomeres at slack. With stretch, this stiffness was increased When muscle fibres are subjected to a fast stretch while activated, the force significantly greater in psoas than in diaphragm. Osmotic compression by increases rapidly to attain a peak, hereby referred to as critical force (Pc). 5% dextran at slack increased the A-band lateral stiffness to a degree matching Although there is still uncertainty on the mechanism responsible for Pc,ithas (psoas) or exceeding (diaphragm) that seen after stretch in 0% dextran. The been commonly associated with the state of crossbridges attached to actin. In stretch-induced and compression-induced contributions to A-band lateral stiff- the present study, we used fast stretches to investigate myofibrils activated ness were additive. Thus, stiff titin contributes more to A-band stiffness than þ with Ca2 or MgADP, which biases crossbridges into a strongly bound state. Iso- compliant titin, confirming a role for titin in lateral force generation during lated myofibrils were attached between an atomic force cantilever and a micro- sarcomere stretching. The electrostatic and the titin-based lateral forces com- needle using micromanipulators. A multi-channel perfusion system connected to pressing the A-band lattice are of the same order of magnitude in stretched þ a double-barreled pipette was used to activate myofibrils with calcium (pCa2 muscle. 4.5) or MgADP (20mM). After force was fully developed, myofibrils were 1 2983-Pos Board B413 stretched by 3% of SLo at 10 SLo.s . MgADP activation resulted in a higher 2þ Residual Force Enhancement in Cardiac Myofibrils Pc (1.37 5 0.07 P/Po) than Ca activation (Pc ¼ 1.23 5 0.03 P/Po)without changing the critical extension required to reach such force (MgADP ¼ 25.01 Kevin R. Boldt, Venus Joumaa, Walter Herzog. 5 1.59 nm/HSL; Ca2þ ¼ 24.99 5 2.68 nm/HSL). This result suggests that myo- Kinesiology, University of Calgary, Calgary, AB, Canada. fibrils activated with MgADP produce less force, but are stiffer than myofibrils Residual force enhancement (RFE) is a property of muscle where an activated activated with Ca2þ. Crossbridges induced to strongly bound state via MgADP muscle is stretched from a short to a long length resulting in greater force than activation may cause further crossbridge formation while generating less force in is produced isometrically at the long length. This history-dependent property comparison with crossbridges that undergo the power-stroke. However they can has been identified across skeletal muscle hierarchy including whole muscle, resist stretching when the myofibrils are stretched. fascicles, fibres, and myofibrils. However, RFE has not been investigated in cardiac muscle. Therefore, the purpose of this study was to determine if RFE 2981-Pos Board B411 was present in cardiac myofibrils. Passive Force Analysis of Single Sarcomeres from Muscles Lacking Rabbit hearts were dissected and strips of left ventricle were skinned overnight Arginyl-tRNA-Protein Transferase (Ate1) with 1% Triton skinning solution and stored at 20C. On the day of experi- Felipe de Souza Leite1, Fabio C. Minozzo1, Neal Trecarten1, Xuemei Han2, ments, the cardiac tissue was blended and a myofibril with a good striation John R. Yates3, Anna Kashina4, Dilson E. Rassier1. pattern was identified and suspended between a glass needle and a nanolever 1Kinesiology and Physical Education, McGill University, Montreal, QC, allowing for length changes and force measurement. The myofibril was set at Canada, 2Department of Animal Biology and Scripps Research Institute, a sarcomere length (SL) of 2.4 mm and activated to establish a reference university of Pennsylvania, Philadelphia, PA, USA, 3department of Animal contraction before being passively stretched to a SL of 3.2 mm. After a rest Biology and Scripps Research Institute, University of Pennsylvania, period, the myofibril was activated at a SL of 2.4 mm, actively stretched to a philadelphia, PA, USA, 4Department of Animal Biology, University of SL of 3.2mm, held for one minute and then relaxed. RFE was calculated as Pennsylvania, Philadelphia, PA, USA. the difference between the steady-state force obtained after active stretch and Introduction: Arginylation is a post-translational process mediated by the the corresponding predicted isometric force at 3.2 mm based on the reference enzyme arginyl-tRNA transferase (Ate1). Recent findings from our laboratory force, calculated according to the force-length relationship scaled to the fila- suggest an important role of arginylation on the architecture and function of skel- ment lengths in rabbit muscles, and accounting for passive force. etal and cardiac muscles. In this study, we investigated the passive force devel- All myofibrils (n¼5) produced more force when stretched actively compared opment of single sarcomeres isolated from soleus muscles of mice lacking Ate1. to the calculated reference isometric force, indicating the presence of RFE in Methods: Soleus muscles from Ate1 KO and wild type (WT) mice were cardiac myofibrils. The average RFE was 54.8510.8%. homogenized to myofibrils. Single sarcomeres in relaxing solution were tested The presence of RFE in cardiac myofibrils supports that RFE is a universal using a pair of micro-needles. The sarcomeres were stretched in steps of 300 nm, muscle property. Investigation in cardiac muscle may shed new light into the starting in a sarcomere length of 2.2mm(50.2mm). The force produced by the mechanisms underlying RFE. sarcomeres was plotted against the sarcomere length. Mass spectrometry was used to evaluate the presence of arginylation sites on titin. Gel electrophoresis 2984-Pos Board B414 was used to test for isoform modifications due to the lack of Ate1. The Effects of Titin Degradation on Passive Stiffness Properties of Skinned Results: Single sarcomeres of Ate KO developed less passive force than sarco- Rabbit Psoas Fibers during Osmotic Compression mere from WT. Mass Spectrometry reviewed 4 sites for arginylation within Ian C. Smith, Walter Herzog. titin’s A-band structure. Electrophoresis gels showed no difference in the titin Kinesiology, University of Calgary, Calgary, AB, Canada. N2A isoform. Muscle is disproportionately stiff over short stretch distances. This effect is Conclusion: Post-translation arginylation of titin A-band does not change titin’s largely calcium-sensitive, and can be eliminated in actively contracting skinned isoform, but is required for normal passive force development in soleus fibers by inhibiting crossbridge formation with pharmacological agents such as muscles. 2,3-butanedione monoxime (BDM). However, elevated short range stiffness can be induced in the absence of calcium in relaxed skinned muscle fibers by 2982-Pos Board B412 osmotic compression, an effect which is not attenuated by BDM. This study Direct Evidence for the Effect of Titin Stiffness on Thick-Filament aimed to determine if titin, the primary source of tension and stiffness in Mechanical Properties in Stretched Muscle Sarcomeres relaxed muscle, could account for the elevated short range stiffness in osmot- Yong Li, Wolfgang A. Linke. ically compressed, relaxed skinned fibers. Accordingly, skinned rabbit psoas fi- Institute of Physiology, Ruhr Univ. Bochum, Bochum, Germany. bers (n¼9) were held at 2.8 mm sarcomere length and osmotically compressed The titin springs of muscle sarcomeres determine tensile muscle stress. How- using 7.5% dextran-T-500 in calcium-free relaxing solution. Muscles were ever, they run not strictly in parallel with the sarcomeric axis, being anchored stretched by 2% (~0.057 mm/sarcomere) over 0.66 s, and returned to initial to actin at Z-disks and myosin at A-bands. On tensile sarcomere strain, which length over another 0.66 s. Dextran treatment resulted in a 5.6 5 0.6 fold in- stretches the titin springs, a force component arises both longitudinally and crease in short range stiffness, with an apparent length limit of 4.550.2 nm laterally. The latter could contribute to increased sarcomeric transversal stiff- per sarcomere (Phase 1). Beyond this limit (Phase 2), stiffness was not different ness, decreased lateral myofilament spacing, and length-dependent activation (100.453.0%) than dextran-free stiffness. To selectively degrade titin, fibers of stretched muscle. We aimed to quantify by AFM force-mapping the titin were held in relaxing solution containing both dextran and trypsin (0.25 mg/ contribution to sarcomeric transversal stiffness in comparison to the contribu- mL) until mechanical failure, with periodic application of the stretch protocol. tion from electrostatic forces modulated by osmotic compression. Single myo- In the stretch immediately preceding failure, Phase 2 stiffness was 8653% fibrils were isolated from skeletal muscles expressing stiff titin-isoform (rabbit lower than initial values, while Phase 1 stiffness only decreased by 3153%. psoas) or compliant titin-isoform (rabbit diaphragm). Myofibrils were placed in Additionally, tension at peak stretch increased 12.552.1 mN (3353%) upon relaxing buffer under the MFP-3D-BIO-AFM (Asylum Research) and force osmotic compression, matching the 12.651.1 mN intercept of the Phase 2 curves (50x50) were performed over a region-of-interest encompassing a whole regression line on the Dtension versus Dlength graph. Thus, short range stiff- sarcomere. Measurements were done in the absence or presence (5%) of ness appears additive to Phase 2, titin-based stiffness, and is best accounted dextran, and in the non-stretched (‘slack’) or stretched state (140-150% of for as a separate entity acting in parallel to titin. 592a Wednesday, February 11, 2015

2985-Pos Board B415 ements within the myosin II catalytic domain (CD). Two complementary EPR Elevated Ionic Strength Diminishes Force/Cross-Bridge and the Number techniques were employed to measure protein orientation (conventional EPR) of Force-Generating Cross-Bridges and intra-protein distances (dipolar electron-electron resonance, DEER). The Li Wang1, Anzel Bahadir2, Masataka Kawai1. use of BSL greatly enhances the resolution of EPR, by virtue of its strongly im- 1Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USA, mobilized and stereoselective bifunctional attachment to the protein backbone 2Department of Biophysics, Duzce University, Konuralp, Duzce, Turkey. at two engineered Cys residues. Crucially, both techniques utilized here permit Ionic strength (IS) is an important parameter to govern the inter- and intra- the elucidation of myosin structure while in complex with actin, generating rele- molecular interactions. In muscle, it has been known that an increase in IS vant constraints for the refinement of actomyosin structural models. In the þ lowers Ca2 activated tension, however, its molecular mechanism is not well current work, Dictyostelium myosin II was used as our model system. We understood. Our aim was to determine whether force/cross-bridge or the num- measured nucleotide-dependent structural transitions of three key helices within ber of force-generating cross-bridges changes with IS. Stiffness during rigor the myosin CD. Three double-Cys sites were engineered, with Cys pairs located was studied on single fibers from rabbit psoas, which showed that there was on the relay helix, helix HK (upper 50kDa domain) and helix HW (lower 50kDa no effect of IS, demonstrating that in-series compliance is not affected by IS. domain), respectively. BSL on a construct with one of these pairs was used to This observation indicates that stiffness of thick filament, thin filament, myosin measure myosin orientation relative to oriented actin. BSL on a construct with head, and actomyosin interface are not affected by IS. Sinusoidal analyses were two pairs was used to measure interprobe distances. The effect of ADP binding þ performed during Ca2 activation, and the effects of ATP, phosphate (Pi), and was clearly detected by EPR, and subsequently modeled using the orientation ADP on three rate constants were studied at IS ranging 150mM-300mM to and distance measurements as constraints. We find that the structural change characterize elementary steps of the cross-bridge cycle. Both ATP binding induced by ADP in the actin-bound myosin CD is clearly different from that (K1) and ADP binding (K0) increased to 2x, and the Pi binding (K5) decreased predicted from actin-free crystal structures. This work was funded by grants to 1/2 when IS was increased from 150mM to 300mM. The effect of Pi can be from NIH (R01 AR32961, T32 AR07612, P30 AR0507220). explained by the electrostatic interaction with the Pi binding site on myosin. The effect on ATP/ADP can be attributed to improved stereoscopic and hydro- 2988-Pos Board B418 phobic interaction with the nucleotide binding site. The increase in IS increased Post-Translational Modification of Tubulin Amplifies X-ROS Signaling cross-bridge detachment steps (k2 and k(-4)), indicating that electrostatic force in Striated Muscle counteracts these steps. However, IS did not affect attachment steps (k(-2) and Jaclyn P. Kerr1, Benjamin L. Prosser2, Guoli Shi1, Patrick Robison2, k4). Consequently, the equilibrium constant of the detachment step (K2) Aaron M. Kempema3, Joseph K. Hexum3, Daniel A. Harki3, Stuart S. Martin4, increased to 2x, and the force generation step (K4) decreased to 0.7x. These ef- Roberto Raiteri5, Christopher W. Ward1. 1University of Maryland, Baltimore, Baltimore, MD, USA, 2Perelman School fects together diminished the number of force-generating cross-bridges by 3 ~10%. Because associated decrease of tension was ~40%, the major effect of of Medicine, University of Pennsylvania, Philadelphia, PA, USA, Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA, 4Physiology, IS is a decrease in force/cross-bridge, but also a decrease in the number of force 5 generating cross-bridge occurs. University of Maryland, Baltimore, Baltimore, MD, USA, Department of Informatics, Bioengineering, Robotics, and System Engineering, University 2986-Pos Board B416 of Genova, Italy, Genova, Italy. Formation of the Motor Protein-Photochromic ADP Analogue- Dysregulated mechano-activated calcium (Ca2þ) and reactive oxygen species Fluorometal Ternary Complex and Photo-Reversible Transition along (ROS) signaling pathways underscore a growing number of diseases; however, the Steps in ATPase Cycle a lack of mechanistic detail has limited the discovery of therapeutic targets. The Akihisa Iwata1, Takeshi Itaba1, Mitsuo Ohmori2, Shinya Mitsuhashi3, cytoskeleton has garnered recent interest as it integrates and focuses mechani- Shinsaku Maruta1,2. cal stress on mechano-activated enzymes, ion channels, and proteins. In striated 1Div. Bioinfo., Grad. sch. Eng, Univ. Soka, Hachioji, Japan, 2Dep. Bioinfo., muscle, we recently discovered the microtubule network as the critical cyto- Fac. Eng., Univ. Soka, Hachioji, Japan, 3Grad. sch. Agri., Univ. Hokkaido, skeletal element which activates X-ROS, a novel pathway in which the me- Sapporo, Japan. chanical stress of stretch/contraction drives NADPH Oxidase 2 (NoX2) ROS In the presence of Mg2þADP, myosin forms stable ternary complexes with production. In healthy muscle, X-ROS sensitizes the activation of ligand or phosphate analogues, fluoroberyllate (BeFn), fluoroaluminate (AlF4-) and or- stretch-activated Ca2þ channels and pharmacologic modulation of MT density thovanadate (Vi), each of which may mimic different transient state along ‘tunes’ X-ROS. In disparate models of muscular dystrophy, we find elevated ATPase cycle. It is known that kiensin also forms similar ternary complexes. MT network density drives excess X-ROS and Ca2þ signaling dysfunction. Our previous studies on the photoaffinity-labelling using photoactive ADP Acute pharmacologic ablation of MT network structure in diseased muscle re- analogue and 19F NMR spectroscopy using 19F-labeled ADP analogue for vealed the MT network as a target with translational potential, however MT the ternary complexes revealed that there is some variation in the myosin- structure is critical to normal cellular function and significant MT ablation nucleotide contacts at the nucleotide base among the ternary complexes. Pre- may limit this approach. We identified elevated levels of a-tubulin de- viously, we have incorporated photochromic molecule, azobenzen derivative tyrosination - a post-translational modification (PTM) associated with into the functional region of ATP driven motor proteins and succeeded to con- increased mechanical properties of the MT network - in muscle exhibiting trol activities and conformation reversibly by photochromic irradiation. Aim of both enhanced MT density and X-ROS. In dystrophic mdx muscle, the pharma- this study is to induce transition between the different steps in ATPase cycle of cologic reduction of a-tubulin de-tyrosination had no effect on the overall den- motor proteins utilizing photoisomerization on the motor protein-phtochromic sity or structure of the MT cytoskeleton, yet significantly reduced X-ROS and ADP analogue-fluorometal ternary complexes. In this study, three types of Ca2þ dysregulation in vitro and limited contraction-induced muscle injury in photochromic ATP analogues composed of azobenznene, spiropyran and vivo. Measures of sarcomere and sub-sarcolemmal cytoskeletal mechanics re- fulgimide derivatives were synthesized. The photo-responsive interaction of vealed that a reduction in a-tubulin de-tyrosination decreased the mechanical the photochromic ATP analogues with myosin and kinesin were examined. stiffness of the MT cytoskeletal network. We conclude that a-tubulin de- Photo-dependent conformational changes of the myosin motor domain were tyrosination is a PTM that regulates the mechanosensitivity of striated muscle monitored by X-ray small angle scattering. And the photo-reversible changes and propose that pharmacological targeting of this PTM will have broad ther- in the velocity of the microtubule glyding for kinesin in the presence of the apeutic potential for the muscular dystrophies. photochromic ATP analogues were also observed. In the presence of fluorome- tal, BeFn, photochromic ADP analogues were trapped within the ATPase site 2989-Pos Board B419 of kinesin and myosin resulted in formation of ternary complexes. The confor- Reloading Injury, Chronic Recovery, and Fiber Type Adaption of Mouse mational changes of the complexes induced by photo irradiation were studied. Soleus Muscle after Four Weeks of Hind Limb Suspension Hanzhong Feng1, Moh H. Malek2, Xue-Qun Chen1, Jian-Ping Jin1. 2987-Pos Board B417 1Physiology Department, Wayne State University, Detroit, MI, USA, 2Wayne Backbone Orientation and Distance Measurements in Myosin II: State University, Detroit, MI, USA. Applications of High-Resolution EPR using a Bifunctional Spin Label Disuse and unloading rapidly result in skeletal muscle atrophy, fiber type switch Benjamin P. Binder1, Andrew R. Thompson1, Sinziana Cornea1, and dysfunction. The recovery from those conditions has broad physiological Rebecca J. Moen2, David D. Thomas1. and medical significances. To understand the recovery process and the molec- 1University of Minnesota, Minneapolis, MN, USA, 2Mankato State ular adaptations in muscle reloading, mouse soleus muscles were studied after University, Mankato, MN, USA. 4 weeks of hind limb-suspension and reloading for 3, 7, 15, 30, 45 and 60 days. We have used electron paramagnetic resonance (EPR) of a bifunctional spin The results showed that 4 weeks of unloading produced significant muscle at- label (BSL) to obtain high-resolution measurements of individual structural el- rophy and reductions of contractile force and fatigue tolerance, accompanied Wednesday, February 11, 2015 593a by switches of myosin isoforms from IIa to IIx and IIb, with no change in type I cross-bridge kinetics. The higher power and shortening velocity of 11a and 11e myosin, and to more low molecular weight fast troponin T splice forms. During fibers enabled increased jumping ability, 76.0% and 48.8% farther, respectively. reloading, soleus muscle mass, fiber size and contractile force gradually recov- Converter homology models, using the new Drosophila myosin S1 crystal ered and reached the control level by 15 days. The levels of type IIa, IIx and IIb structure (pdb 4QBD), revealed minimal tertiary structure differences suggest- myosins recovered at 15 days of reloading. However, fatigue tolerance and post ing residue specific interactions with the relay, N-terminus, or essential light fatigue force recovery showed a trend of worsening during this period with chain are critical. Overall, our data support the hypothesis that the converter significantly inflammatory cell infiltration at 3 and 7 days, indicating reloading helps set muscle shortening velocity under loaded and unloaded conditions injuries. The reloading injury was accompanied by up-regulations of filamin-C and thus contributes to muscle fiber type diversity. and alpha-crystallin-B, which returned to control level after 30 days. During the course of slow recovery, we observed later increases of type I myosin expres- 2992-Pos Board B422 sion and the number of type I fibers to levels significantly higher than that in Actomyosin Regulation by Conserved Sites of Tm5NM1 (Tpm3.1), a normal adult mouse soleus muscle at 30 to 60 days of reloading, which may Nonmuscle Tropomyosin contribute to the recovery of fatigue tolerance. The data demonstrated the value Bipasha Barua, Sarah E. Hitchcock-DeGregori. of increased slow fiber contents as a secondary adaption to compensate for mus- Pathology and Laboratory Medicine, RWJMS, Rutgers University, cle reloading injury, serving as an indicator for monitoring muscle function dur- Piscataway, NJ, USA. ing the chronic recovery from unloading and disuse conditions. Actin filaments carry out diverse cellular functions including cell division, intracellular transport, and muscle contraction. Tropomyosin (Tm) is an a-he- 2990-Pos Board B420 lical coiled-coil protein that regulates actin in most eukaryotic muscle and non- Minimum and Maximum Limit to Number of Myosin II Motors muscle cells. Nonmuscle isoforms of Tm, particularly Tm5NM1 (Tpm3.1) Participating in an Ensemble Motility (short isoform, 247 a.a., TPM3 gene), play an important role in cellular func- Khushboo Rastogi1, Shabeel Hamnaf1, Vikas Pandey2, Sunil Nath1, tions such as cell migration, and cytokinesis as well as in the transformation Ravikrishnan Elangovan1. and metastasis of cancer cells. In previous work, mutation of evolutionarily- 1Department of Biochemical Engg and Biotechnology, Indian Institute of conserved residues in striated muscle aTm (Tpm1.1) (long isoform, 284 a.a., Technology Delhi, New Delhi, India, 2Department of Chemical TPM1 gene) revealed the regions important for actomyosin regulation. In the Enggineering, Indian Institute of Technology Delhi, New Delhi, India. present study, we have mutated evolutionarily-conserved residues in Tpm3.1 Myosin II is an ensemble motor that participates in muscle contraction, actin to determine the molecular basis for isoform-specificity of actomyosin regu- cortex remodeling and cytokinesis ring constriction. It has been an interesting lation by Tms. We mutated surface residues in nonmuscle Tpm3.1 in periods puzzle for scientific community to understand the allosteric rules between P1-P7 at positions homologous to the residues that are important for actomy- motors that regulate/co-ordinate when they work in group. We have performed osin regulation by striated muscle Tpm1.1 (Barua et al., 2012). In vitro motility classical In Vitro motility assay at different heavy meromyosin (HMM) density assays were carried out to determine the effect of mutations on actin filament and ATP concentrations. By changing immobilized HMM density and ATP velocities. Actin-Tm velocities with skeletal myosin are inhibited by Tpm1.1 concentration, we change the number of HMM molecules available for interac- (~60%) but activated by Tpm3.1 (~60%) relative to actin alone. The Tpm3.1 tion/unit length of actin filament. Actin filaments of >10 micron length were mutants had little or no effect on velocity, except for the P3 and P6 mutants added in flow cell and assay was initiated by addition of ATP. Actin filaments that showed a ~50-60% inhibition in filament velocity relative to WT breaks down to smaller pieces within minutes after addition of ATP. Average Tpm3.1. In comparison, amongst the Tpm1.1 mutants, the P3 and P6 mutants length of sliding filaments correlates well with motors density and ATP con- also showed the largest inhibition (~70-80%) in filament velocity relative to centrations in solution. At 633 head/mm2 density and 2 mM ATP concentration WT Tpm1.1. These results indicate that the same regions of Tm (periods 3 the average filament length was 1747.65614 nm and further reduced to and 6) are important for regulation of skeletal myosin by two different isoforms 1290.55385.5 nm at 0.1 mM ATP. At 4000 heads/mm2 density and 2 mM that have contrasting effects (inhibition by Tpm1.1 vs. activation by Tpm3.1) ATP concentration the average filament length was 935.55286 nm and on filament velocity. Supported by NIH. 5 reduced to 599.5 154.2 nm at 0.1 mM ATP. Any filament below this average 2993-Pos Board B423 length does not slide continuously, they detach from surface with time and any The Binding of S1A1-ADP to Skeletal Thin Filaments is Insensitive to the filament above this average length fragments to smaller pieces with time. Presence of Covalently Bound Phosphate in Tropomyosin Assuming Poisson distribution of immobilized heads, we calculated maximum David H. Heeley1, Charitha L. Goonasekara2, White D. Howard3. number of molecules that can interact for a given actin length. Number of mol- 1Memorial University, St. John’s, NL, Canada, 2Medicine, General Sir John ecules required for continuous sliding is independent of motor density and de- Kotelawala Defence University, Colombo, Sri Lanka, 3Physiological 5 pends only on ATP concentration. For 2 mM ATP concentration, 123.6 28.9 Sciences, EVMS, Norfolk, VA, USA. 5 heads and for 0.1 mM ATP concentration 62.3 19.5 heads are required for Striated muscle tropomyosin is phosphorylated at a single site. Effects of this 5 continuous sliding of actin. All values are mean SD. covalent modification include an increase in myosin activation by thin fila- Supported by DST, Government of India. ments (þCa(II)) under steady state conditions (Vmax, 16 vs. 8 sec-1; ionic strength, ~ 30 mM; T, 25 oC). Thin filaments were assembled using skeletal 2991-Pos Board B421 muscle troponin and alpha-tropomyosin (either unphosphorylated or phosphor- Muscle Shortening Velocity is Modulated by Alternative Myosin ylated). Each type of thin filament was mixed with varying concentrations of Converters myosin-S1A1 (4uM F-actin; buffer, 5.5 mM MgCl2, 50 mM KCl, 10mM imid- Christopher S. Newhard, Bernadette M. Glasheen, Debra Sheppard, azole, 3mM MgADP, 1mM dithiothreitol, pH 7 plus 0.5mM EGTA or Ca(II)) Lauren Riley, Douglas M. Swank. and incubated at 4 oC for 1hr with 20uM myokinase inhibitor. The mixtures Biology, Rensselaer Polytechnic Institute, Troy, NY, USA. were then centrifuged for 30min at 150,000 x g at 25 oC. The unbound S1 The myosin converter has been proposed to be critical for setting muscle short- was determined using a NH4/EDTA ATPase versus [myosin-S1A1] standard ening velocity by influencing the amplification of small conformational changes curve. The zero time samples showed no colour development, consistent at the ATPase site into the larger lever arm swing. To test this hypothesis, we with the absence of ATP. At a given pCa the isotherms obtained with each exploited the natural variation of the converter region found in Drosophila mus- type of reconstituted thin filament are virtually superimposable (in terms of cle types. In Drosophila, a single myosin heavy chain gene is alternatively steepness and mid-point). The apparent binding constants range from 0.1 to spliced to generate myosin isoforms. The converter region is encoded by five 0.4 uM. Added Ca(II) strengthens affinity and EGTA weakens it. Pre-steady alternative versions of exon 11 that are expressed in different muscle types. state experiments using double-mixing fluorescent stopped-flow are in progress Through genetic modification we forced the expression of the alternative ver- to investigate further the effect of this modification. sions in the Drosophila jump muscle and found that two of the three versions tested to date, 11a (native to indirect flight muscles) and 11e (embryonic mus- 2994-Pos Board B424 cles), caused faster maximum shortening velocities of skinned jump muscle fi- The Functional Consequences of Hypertrophic Cardiomyopathy Troponin bers relative to the native control 11c. Increases in velocity were primarily C Mutations in the Regulation of Slow Skeletal Muscle Contraction: The responsible for 70.9% and 79.0% higher power outputs for 11a and 11e fibers, Protective Role of Slow Skeletal Troponin I respectively, because maximum force generation was not significantly different Tiago Veltri, Clara A. Michell, Maicon Landim, David Dweck, from control fibers. The fibers expressing 11a and 11e also exhibited a straighter Jose R. Pinto. force-velocity curve, as shown by increased Hill equation parameters a (95.4% Biomedical Sciences, Florida State University, Tallahassee, FL, USA. and 84.5% higher, respectively) and b (72.6% and 65.5% higher, respectively) Mutations in cardiac (cTnC) have been linked to hypertrophic car- relative to control fibers, indicating that the converter modulates load dependent diomyopathy (HCM) in humans. The cTnC subunit of the troponin complex is 594a Wednesday, February 11, 2015 expressed in both slow skeletal and cardiac muscle. The cTnC HCM muta- then implement a stochastic version of the eight step ATPase cycle that in- tions A8V, C84Y and D145E increase the Ca2þ-sensitivity of contraction in cludes calcium regulation of the actin binding steps according to the McKil- cardiac muscle; however, their functional consequences in slow skeletal mus- lop & Geeves Blocked/Closed/Open model of the regulated thin filament. cle regulation are unknown. Here we investigated the Ca2þ-sensitivity of Using this model it is now feasible to model the dependence of the ATPase TnC-extracted rabbit soleus skinned fibers reconstituted with either WT or rate on calcium concentration and predict the occupancy of each state in the HCM mutant cTnC. Surprisingly, the slow skeletal skinned fibers extin- cycle for any defined set of protein and substrate concentrations. This allows 2þ guished the Ca -sensitization typical of cardiac fibers for A8V (pCa50 ¼ us, for example, to explore the interplay of calcium and ATP concentrations 6.01) and D145E (pCa50 ¼ 6.05), while for C84Y (pCa50 ¼ 6.33) the in the cooperative activation of the ATPase cycle and make predictions for 2þ Ca -sensitization was maintained compared to WT (pCa50 ¼ 6.00). To deter- how the system will respond to sudden changes in calcium or ATP concen- mine whether slow skeletal TnI (ssTnI) alone was responsible for the protec- tration. In a further variant of the model the program allows us to predict how tive effect of incorporating the A8V and D145E mutants into the slow skeletal the system will respond if variable ratios of fast and slow isoforms of myofilaments, cardiac skinned fibers were reconstituted with a hybrid cardiac myosin, or variable ratios of proteins carrying cardiomyopathy mutations troponin complex containing cTnT, ssTnI and cTnC-WT or -HCM mutants. are present in the system. This will be a useful tool to explore the behaviour The presence of ssTnI in cardiac fibers partially protected against Ca2þ-sen- of mixed myosin & troponin populations and correlate experimental data sitization by the cTnC mutants in cardiac skinned fibers, with the exception with model predictions. of C84Y. MgATPase activity of slow or cardiac myofibrils replaced with Supported by: R01 AR048776, R01 DC 011528 and W.Trust 085309 exogenous HCM cTnC mutants were also affected. The absence of increased Ca2þ-sensitivity of contraction arising from the A8V and D145E in the soleus 2997-Pos Board B427 muscle suggests that the aberrant effects of cardiomyopathic cTnCs may be Effect of Actin and Nucleotide on the Movement of A1-Type Myosin tissue-specific, and ssTnI is an important partner that partially protects the Essential Light Chain, Detected by Time-Resolved FRET slow muscle from a deleterious effect of the HCM cTnC mutants. Supported Piyali Guhathakurta, Ewa Prochniewicz, David D. Thomas. by NIH HL103840 (JRP). Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. 2995-Pos Board B425 We have used site-directed time-resolved FRET to detect structural changes of Structural and Functional Changes in Skeletal Muscles in an A8V-Troponin the N-terminal extension on the A1 isoform of ELC, during active interaction C Hypertrophic Cardiomyopathy Knock-In Mouse Model with actin. Skeletal muscle myosin subfragment 1 (S1) has two ELC isoforms, Milica Vukmirovic1, Marcos Angel Sanchez-Gonzalez2, Gregory S. Frye3, A1 and A2, which differ by the presence of 40-45 additional residues at the Andrew Koutnik1, David A. Gonzalez1, Edda Ruiz1, Eric Krivensky1, N-terminus of A1. Removal of ELC from myosin results in a loss of movement David Dweck1, Leonardo F. Ferreira3, Jose Renato Pinto1. of actin filaments, and a reduction in isometric force. It has been proposed that 1Biomedical Sciences, Florida State University, Tallahassee, FL, USA, the N-terminal extension of A1 interacts directly with actin to modulate acto- 2Biomedical Sciences, Larkin Health Sciences Institute, South Miami, FL, myosin kinetics. We have used time-resolved FRET to explore the structural USA, 3Applied Physiology and Kinesiology, University of Florida, details of this modulation. We recently showed, using probes on actin and Gainesville, FL, USA. ELC, that the amplitude of the myosin power stroke is much greater for A1 Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal than A2. We have now engineered single cysteine (C16, in the N-terminal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic extension) and double cysteine (C16-C180, in the C-terminal lobe) mutants cardiomyopathy (HCM). The impact of HCM c/ssTnC mutations in skeletal of A1-ELC and used FRET to determine the distance from C16 to actin 374 muscle structure and function is unknown; therefore, we examined the effect or to C180, as affected by the power stroke. Labeled ELCs were exchanged of an HCM-associated c/ssTnC A8V mutation on myosin heavy chain into S1, and the pure isoenzyme (S1A1) was isolated using Talon affinity resin. (MHC) isoform expression, contractile properties of different skeletal muscles, Labeling and exchange preserved the functional properties of S1A1. Intermo- and cardiac autonomic function in a homozygous knock-in mouse (KI-TnC- lecular FRET between C374 of actin and C16 of A1 showed that the distance A8Vþ/þ). The distribution of MHC I/II isoforms in various muscles from increases from 2.9 nm to 3.5 nm upon addition of ATP. Intramolecular FRET 3-month old male WT and KI-TnC-A8Vþ/þ mice were analyzed by glycerol between C16 and C180 does not change in the presence of ATP or actin. We SDS-PAGE and the muscle/body weight ratios (M/BW) were recorded. Me- conclude that (1) the N-terminus of ELC moves away from actin during the chanics of contraction in isolated intact soleus muscle, running/endurance transition between strongly and weakly bound states of acto-S1, and (2) the capacity and heart rate variability (HRV) were also measured. In comparison converter/C-terminal domain of myosin/ELC and the N-terminal extension to the WT mice, MHC isoform switching was observed in diaphragm move as a rigid body during the power stroke. (increased MHCIIb, decreased MHCI) and soleus (decreased MHCIIa, increased MHCI) muscles of KI-TnC-A8Vþ/þ mice; while, the M/BW ratio 2998-Pos Board B428 was unchanged for all tested muscles. The force vs frequency relationship Systematic Variations in Fast-Type Myosin Light Chain 1 Sequence Correlate with Species Body Mass at 15Hz and the t1/2 of relaxation increased in the intact soleus muscle of KI- TnC A8Vþ/þ mice in comparison to the WT; whereas, no changes in maximal Peter J. Reiser, Sabahattin Bicer. force, time-to-twitch peak, velocity and power output were observed. In en- Biosciences, Ohio State University, Columbus, OH, USA. durance and HRV tests, KI-TnC-A8Vþ/þ mice had a lower running distance ca- The N-terminus of vertebrate fast-type myosin light chain 1 (MLC1F) contains pacity and their global autonomic, cardiovagal, and baroreflex activities were a conserved actin-binding domain, a nearby poly-alanine (poly-A) domain, and significantly reduced compared to WT. In spite of the increase in type I fibers an adjacent alanine-proline (A-P) repeat domain. The number of residues in the in soleus muscle of mice expressing c/ssTnC A8V, they display reduced endur- poly-A and A-P repeat domains vary considerably among vertebrate species. ance capacity. These findings suggest that the slow skeletal muscle is trying to We reported a strong correlation between the apparent mass of MLC1F and compensate for the cardiac dysfunction caused by the c/ssTnC A8Vþ/þ muta- adult body mass for nineteen mammalian species (Bicer and Reiser, Am. J. tion. Supported by NIH HL103840 (JRP). Physiol. Regul. Integr. Comp. Physiol., 292:R527-R534, 2007). The MLC1F sequence was then known for five of the nineteen species and the variation 2996-Pos Board B426 in MLC1F mass was determined to be due largely to the variation in the number Modelling the Calcium Dependent Actin-Myosin ATP-ase Cycle in of residues in the poly-A and the number of A-P repeats among these five spe- Solution cies. We have recently examined the reported MLC1F sequence of additional Srboljub M. Mijailovich1, Djordje Nedic2, Marina Svicevic2, mammalian species and of species of other vertebrate classes to determine Boban Stojanovic2, Michael Geeves3. the extent of the correlations between the number of poly-A residues and the 1Department of Chemistry and Chemical Biology, Northeastern University, number of A-P repeats and body mass among vertebrates, with a particular in- Boston, MA, USA, 2Department of Mathematics and Informatics, University terest in comparing species with different modes of locomotion (e.g., terrestrial of Kragujevac, Kragujevac, Serbia, 3Biosciences, University of Kent, walking/running versus swimming). We also examined the sequences of other Canterbury, United Kingdom. MLCs for the presence of systematic variations among species and possible We have implemented two computation versions of the solution actin-myosin correlations with species body mass. The results reveal that the numbers of ATPase cycle. A simple numerical model that simulates an eight step poly-A residues and A-P repeats increase with species body mass among terres- ATPase cycle and allows actin, myosin, substrate ATP and product ADP trial mammals. Furthermore, the MLC1F domains vary most consistently with & Pi concentrations to be fixed or varied. This model allows us to rapidly adult species body mass than any of the other examined MLCs. The results sug- assign realistic rate constants to each step of the cycle for any well- gest that MLC1F domains are likely involved with modulating the speed and/or defined myosin isoform. With this set of rate constants in place we can economy of locomotion among terrestrial mammals. Wednesday, February 11, 2015 595a

Cardiac Muscle Regulation II 3001-Pos Board B431 Cardiac Remodeling in the Mouse Model of Marfan Syndrome Develops 2999-Pos Board B429 Independently from Aortic and Valvular Abnormalities Cardiac Over-Expression of Creatine Kinase Improves Function in Failing Natalia Petrashevskaya, Hyun-Jin Tae, Shanon Marshall, Myocytes Melissa Krawczyk, Mark Talan. Carlo G. Tocchetti1, Michelle Leppo2, Yibin Wang3, Robert G. Weiss4, LCS, National Institute on Aging, Baltimore, MD, USA. Nazareno Paolocci4. Marfan syndrome (MFS) is a multisystem disorder of connective tissue caused 1Department of Translational Medical Sciences, Federico II University, by mutations in fibrillin-1. Heart involvement in the Marfan syndrome patients Naples, Italy, 2Medcine - Division of Cardiology, Johns Hopkins Medical includes aortic root dilatation, valvular insufficiency, and myocardial dys- Institutions, Baltimore, MD, USA, 3Division of Molecular Medicine, UCLA, function; it remains unclear, however, whether alterations in myocardium are Los Angeles, CA, USA, 4Medicine Division of Cardiology, Johns Hopkins triggered by valvular and aortic pathology or they develop independently. Medical Institutions, Baltimore, MD, USA. We evaluated the age-dependent cardiac remodeling and left ventricular þ Aims: Abnormal energy metabolism contributes to heart failure (HF) and the dysfunction in the mouse model of MFS known as Fbn1039G / mouse (Mar- failing heart is energy starved. Here we tested whether augmented CK energy fan HT mouse) using echocardiography, pressure-volume loop analyses and a metabolism improves myocyte dysfunction in experimental HF. number of histological and biochemical techniques. Methods and Results: We tested the response to the b-agonist isoproterenol Marfan HT mice of 2-4 month demonstrated a hypertrophic cardiac remodeling (2.5 nM, ISO) in cardiomyocytes isolated from wild-type (WT) mice and accompanied by predominant decline of diastolic function and increased TGF-b mice over-expressing cardiac myofibrillar and mitochondrial CK (CK-M canonical (p SMAD2/3) and non-canonical (pERK ½ and pMAPK38) signaling. and CK-mito) from sham and HF (8 wk transverse aortic constriction, Hypertrophic myocardium among older HT mice (6-14 months) was associated TAC) hearts, to dissect whether over-expressing CK-M or CK-mito might with two distinctly different phenotypes manifesting either dilated or constricted alter myocyte function at baseline or after an increase in energetic demand. LV chamber. Dilatation of LV chamber was accompanied by biochemical evi- At baseline, there were no differences in sarcomere fractional shortening dence of greater mechanical stress, including elevated ERK1/2 phosphorylation (FS) or whole Ca2þ transient amplitude in response to ISO among sham and brain natriuretic peptide expression in comparison with constricted heart. WT, CK-M or CK-mito myocytes. However, ISO impact on FS, Ca2þ tran- Diastolic dysfunction in the older HT mice was combined with significant sys- sient, time to 50 Ca2þ decay, and sarcomere re-lengthening were all reduced tolic impairment. The aortic valve regurgitation was registered in 20% of con- in WT TAC hearts, consistent with prior reports. Conversely, over-expressing stricted group and 60% of dilated, while mitral insufficiency was observed in CK-M or CK-mito rescued ISO-induced inotropy in TAC myocytes. No 40% of constricted group and 100% of dilated. In Marfan HT mice, extracellular sizable differences in ISO response were noticed in cells obtained from matrix abnormalities were not associated with the increase of interstitial fibrosis sham WT, CK-M or CK-mito hearts. To test whether over-expressing and non-myocyte proliferation. In the mouse model of fibrillin-1 haploinsuffi- CK-M or CK-mito confers a degree of protection against acute oxidative ciency the early onset of hypertrophic cardiac remodeling and dysfunction is stress, non-TAC myocytes were exposed to H O (50 mM for 10 min). The not consequent to functional valvular abnormalities, but it is likely to result 2 2 from deficient mechanosensing and transmission of mechanical forces. interval between the beginning of H2O2 superfusion and the appearance of an irreversible arrhythmia was measured. WT and CK-M myocytes showed 3002-Pos Board B432 5 5 ¼ a similar response (359 87s vs. 370 60s, n 5), whereas in CK-mito this Transgenic Over-Expression of Carbonic Anhydrase III in Cardiac 5 interval was prolonged (580 74s). Muscle Demonstrates a Mechanism to Resist Acidosis Conclusions: Over-expressing CK-M and CK-mito under failing-TAC condi- Hanzhong Feng, Jian-Ping Jin. tions improves myocyte contraction and relaxation, likely through preserved 2þ Physiology Department, Wayne State University, Detroit, MI, USA. Ca handling; however, only the up-regulation of CK-mito can effectively Carbonic anhydrase III (CAIII) is an abundant protein in skeletal muscle, liver buffer ROS, especially those of mitochondrial origin. and adipose cells. A cytosolic enzyme that catalyzes conversions between CO2 - and HCO3 in regulating intracellular pH, its physiological function in muscle is 3000-Pos Board B430 unclear. Mice lacking CAIII showed lower than wild type intracellular pH in Acute Ablation of Cardiac Myosin Light Chain Kinase Decreases Cardiac skeletal muscle cells during fatigue treatment. To further understand the role Performance of CAIII in muscle functions and stress adaptation, we developed transgenic 1 2 2 1 Audrey N. Chang , Pavan Battiprolu , Joseph A. Hill , Kristine E. Kamm , mice overexpressing CAIII in the heart under the control of a cloned myosin James T. Stull1. 1 2 heavy chain promoter for phenotype comparisons with wild type mouse hearts Physiology, University of Texas Southwestern, Dallas, TX, USA, Internal that are CAIII negative. Three months old transgenic mice showed normal car- Medicine (Cardiology), University of Texas Southwestern, Dallas, TX, diac phenotypes under non-stress conditions. Cardiac function was examined USA. using ex vivo working heart preparations under normal and low pH conditions Cardiac myosin light chain kinase (cMLCK) phosphorylates a single site in the to investigate CAIII function in pH regulation of cardiac muscle. With equili- regulatory light chain (RLC) of myosin to increase Ca2þ sensitivity of myofil- bration of 5% CO2 generating pH 7.4 in normal Kreb’s perfusion buffer, 10% ament contractions. A constellation of contractile protein phosphorylations in CO2 was used to lower pH to 7.0. Functional data showed that transgenic and addition to RLC phosphorylation fine-tune actin-myosin myofibrillar force wild type hearts had similar pumping functions under normal pH. Perfused with development to modulate cardiac performance. In the normal beating heart low pH buffer, heart functions of both groups were decreased. In comparison RLC is significantly phosphorylated (~45%) which may play a constitutive with wild type controls at low pH, CAIII transgenic mouse hearts showed physiological role to enhance cardiac performance. Conventional cMLCK higher left ventricular pressure development and systolic and diastolic veloc- knockout mice have dilated hearts with severely compromised cardiac perfor- ities under both baseline conditions and increased afterload stress, indicating mance at 10 weeks of age and older. To determine if the dilated phenotype a better tolerance to acidosis. The results suggest that CAIII may function in caused by cMLCK knockout is preceded by a loss of cardiac performance compensating for intracellular pH under acidotic conditions, a tractive novel associated with decreased phosphorylation of RLC and other myofibrillar pro- approach to develop new treatment of chronic congestive heart failure. teins, we generated an acute model for the conditional knockout of cMLCK. We optimized the minimal amount of tamoxifen necessary for cMLCK ablation 3003-Pos Board B433 and assessed cardiac performance measured as fractional shortening by echo- Myocardial Interstitial Serotonin and its Major Metabolite, 5-Hydroxyin- cardiography. Hearts were then harvested for analyses of protein contents dole Acetic Acid Levels Determined by Microdialysis Technique in vivo and phosphorylations. Five consecutive daily i.p. injections of 0.5 mg tamox- Rat Heart ifen per mouse were sufficient to reduce cMLCK 8052% by two weeks after Cheng-Kun Du, Dong-Yun Zhan, Tsuyoshi Akiyama, Takashi Sonobe, the first tamoxifen injection. RLC phosphorylation was reduced to 1552% Tadakatsu Inagaki, Mikiyasu Shirai. while left ventricular internal diameter at end-diastole significantly increased National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan. by 1.450.3 mm and fractional shortening decreased 4756%. There was no ev- Aims: The aim of this study was to elucidate myocardial interstitial serotonin idence of compensatory hypertrophy. Troponin-I and myosin binding protein-C (5-HT) kinetics in the heart, including 5-HT reuptake and enzymatic degrada- phosphorylation at Ser23/24 and Ser282, respectively, did not significantly tion to 5-hydroxyindole acetic acid (5-HIAA) via monoamine oxidase (MAO). change. Both phosphorylations were reduced with propranolol treatment, Main methods: Using microdialysis technique in anaesthetized rats, we simulta- which had no effect on RLC phosphorylation. These results suggest that consti- neously monitored myocardial interstitial levels of 5-HT and its major metabolite, tutive RLC phosphorylation contributes to cardiac performance in the normal 5-HIAA, in the left ventricle and examined the effects of local administration of a beating heart. MAO inhibitor, pargyline, or a 5-HT uptake inhibitor, fluoxetine. 596a Wednesday, February 11, 2015

Key Findings: Pargyline increased dialysate 5-HT concentration from 1.8 5 0.3 size and heart-to-body weight ratios in both TG-55 and TG-64 mice. Left ventric- at baseline to 3.9 5 0.5 nM but decreased dialysate 5-HIAA concentration from ular diastolic functional parameters (isovolumic relaxation time and E-wave 20.7 5 1.0 at baseline to 15.8 5 1.4 nM at 60-80 min of administration. Fluox- deceleration time) showed a graded increase in TG-55 and TG-64 mice; how- etine increased dialysate 5-HT concentration from 1.9 5 0.4 at baseline to 6.5 5 ever, such effects were only significant in TG-64 mice, suggesting impaired 0.9 nM at 60-80 min of administration, but did not change dialysate 5-HIAA relaxation. Systolic functional parameters (stroke volume, ejection fraction concentration. Local administration of ADP (100 mM) increased dialysate 5- and fractional shortening) were unaffected in TG-55 mice, but significantly HT and 5-HIAA concentrations. Pargyline did not affect ADP-induced increase decreased in TG-64 mice. Thus, higher levels of chimeric TnT (64%) depressed in dialysate 5-HT concentration but suppressed ADP-induced increase in dialy- both diastolic and systolic function significantly in TG-64 mice. We will discuss sate 5-HIAA concentration during 60 min of ADP administration. Fluoxetine the link between the effects of the modified N-terminus of TnT on cardiac increased dialysate 5-HT concentration at 40-60 min of ADP administration, myofilament function and the resultant pathological remodeling of the heart. but did not affect ADP-induced increase in dialysate 5-HIAA concentration. Our findings have pathological relevance because a growing number of Significance: Simultaneous monitoring of myocardial interstitial 5-HT and disease-related mutations are found both in and near the NTE of cardiac TnT. 5-HIAA levels provides valuable information on 5-HT kinetics including reup- take and enzymatic degradation by MAO, which play a role in the regulation 3006-Pos Board B436 of myocardial interstitial 5-HT levels at baseline and when 5-HT levels are Functional Effects of the H1-Helix of Rat Cardiac Troponin T on Cross- elevated. bridge Detachment Rate is Differently Modulated by a- and b-Myosin Heavy Chain Isoforms 3004-Pos Board B434 John J. Michael, Murali Chandra. The Treatment Benefit of Ghrelin on a Mouse Model of Inherited Dilated Department of Integrative Physiology and Neuroscience, Washington State Cardiomyopathy Caused by Troponin Mutation University, Pullman, WA, USA. DongYun Zhan1, ChengKun Du1, Sachio Morimoto2, Tsuyoshi Akiyama1, The primary structure of the H1-helix of troponin T (TnT) varies among different Daryl O Schwenke3, Hiroshi Hosoda4, Kenji Kangawa5, Mikiyasu Shirai1. types of striated muscles. Moreover, these muscles also express different myosin 1Department of Cardiac Physiology, National Cerebral and Cardiovascular heavy chain (MHC) isoforms. Recently, we demonstrated that pseudo-phosphor- Center Research Institute, Osaka, Japan, 2Department of Clinical ylation of residue 204 (near the H1-helix) of cardiac TnT affected the functional Pharmacology, Graduate School of Medical Sciences, Kyushu University, state of the thin filament differently in fibers that expressed either a-orb-MHC Fukuoka, Japan, 3Department of Physiology, University of Otago, Otago, isoforms (Michael et al., Basic Res Cardiol, 109:442, 2014). In this follow-up New Zealand, 4Department of Regenerative Medicine and Tissue study, we investigated how the isoform-specific function of the H1-helix of car- Engineering, National Cerebral and Cardiovascular Center Research diac TnT was influenced by a-andb-MHC isoforms. We generated a mutant Institute, Osaka, Japan, 5Department of Biochemistry, National Cerebral and rat cardiac TnT (RfsH1) in which the cardiac H1-helix was replaced by the fast Cardiovascular Center Research Institute, Osaka, Japan. skeletal H1-helix. Recombinant RfsH1 was reconstituted into detergent-skinned The therapeutic effect of ghrelin has been reported in humans as well as in an- cardiac muscle fibers from either normal rats (expressing a-MHC) or propylth- imal models of chronic heart failure. However, little is known about the thera- iouracil treated rats (expressing b-MHC). Steady-state and dynamic measure- ments were carried out at sarcomere length 2.3 mm. Our results demonstrated peutic efficacy of ghrelin for the treatment of inherited forms of dilated 2þ cardiomyopathy (DCM). We aim to examine whether ghrelin is beneficial for that RfsH1 decreased Ca -activated maximal ATPase activity differently in the treatment of inherited DCM with a deletion mutation DK210 in the cardiac a-MHC (~33%) and b-MHC (~17%) fibers. Furthermore, RfsH1 decreased ten- troponin T (cTnT) gene using a knock-in mouse model. Ghrelin (150 mg/kg/day) sion cost (~31%) and crossbridge (XB) distortion dynamics (~25%) in a-MHC was administered subcutaneously to the mouse model of inherited DCM. but not in b-MHC fibers. Because the above mentioned parameters are indices The therapeutic effects were examined on the basis of survival and myocardial of the rate of XB detachment, our results suggest that the interplay between the remodeling. Ghrelin administration prolonged the life span of DCM mice RfsH1- and a-MHC-mediated effects on the thin filament modulates XB detach- compared to the saline-treated controls. Echocardiography data showed that ment kinetics. Our findings suggest that the conformational changes in the H1- ghrelin reduced left ventricular (LV) end-diastolic dimensions and increased helix of TnT are sensitive to MHC isoform-mediated changes in the thin filament. LV ejection fraction. Moreover, histoanatomical data revealed that ghrelin 3007-Pos Board B437 decreased the heart-to-body weight ratio, prevented cardiac remodeling and Engineering Cardiac Troponin C: Potential Therapeutic for Heart Failure fibrosis, and markedly decreased the expression of brain natriuretic peptide. Vikram Shettigar, Sean C. Little, Bo Zhang, Jianchao Zhang, Steve Roof, Telemetry recording and heart rate variability analysis showed that ghrelin sup- Zhaobin Xu, Elizabeth A. Brundage, Brandon J. Biesiadecki, pressed the excessive cardiac sympathetic nerve activity (CSNA) and recovered Noah Weisleder, Paul Janssen, Mark T. Ziolo, Jonathan P. Davis. the cardiac parasympathetic nerve activity. Ghrelin has therapeutic benefits for The Ohio State University, Columbus, OH, USA. the treatment of DCM with DK210 mutation in cTnT. Importantly, these cardio- We have engineered cardiac TnCs with increased (L48Q) or decreased (D73N) vascular benefits of ghrelin are likely linked to the suppression of CSNA and Ca2þ sensitivity. To express these proteins in the in vivo heart we utilized an recovery of cardiac parasympathetic nerve activity. adeno-associated virus serotype 9 (AAV-9). The Ca2þ desensitized D73N TnC recapitulated a dilated cardiomyopathy phenotype and depressed function 3005-Pos Board B435 as observed by echocardiography and isolated cardiomyocytes. On the other The Cardiac Troponin T Mutant Missing the N-Terminal Extension hand, AAV-9 containing the Ca2þ sensitized L48Q TnC did not cause any Causes Dose-Dependent Effects on Cardiac Function and Remodeling in disease phenotype or arrhythmias commonly associated with increased myofil- Transgenic Mice ament Ca2þ sensitivity. In healthy mice, L48Q TnC increased myocyte contrac- 1 2 1 Sampath K. Gollapudi , Joseph Maricelli , John J. Michael , tion and whole heart contractility with improved cardiovascular performance O. Lynne Nelson3, Dan B. Rodgers2, Murali Chandra1. 1 (increased V02max). Excitingly, L48Q TnC expressing mice were able to pre- Dept of Integrative Physiology and Neuroscience, Washington State serve higher contractility, ejection fraction, cardiac performance and decreased University, Pullman, WA, USA, 2School of Molecular Biosciences, 3 death rate even after undergoing trans-aortic constriction or myocardial in- Washington State University, Pullman, WA, USA, Internal Medicine and farction. Additionally, L48Q TnC was able to increase contractility, ejection Cardiology, Veterinary Teaching Hospital, Washington State University, fraction and cardiac performance in mice which expressed L48Q TnC after Pullman, WA, USA. having a myocardial infarction. In summary, engineered TnCs show potential The N-terminal extension (NTE; residues 42-73) of mouse cardiac troponin T to be used as treatment strategies against different cardiomyopathies. (TnT) desensitizes cardiac myofilaments to Ca2þ by stabilizing thin filaments in the blocked-state. We arrived at this conclusion using detergent-skinned mus- 3008-Pos Board B438 cle from transgenic (TG) mouse hearts that expressed 54% of chimeric TnT (res- Modeling the Response of Cardiac Troponin C to Calcium on the Thin idues 1-73 of mouse cardiac TnT were replaced by residues 1-41 of mouse fast Filament: Effects of Disease-Related and Post-Translational Modifications skeletal TnT). Here, we extended our investigation to include higher dose effects Jalal K. Siddiqui, Bin Liu, Shane D. Walton, Vikram K. Shettigar, of the modified TnT on cardiac myofilament function/phenotype using Andrew J. O’Neil, Grace A. Davis, Peeyush Shrivastava, Jonathan P. Davis. detergent-skinned fiber studies and echocardiography measurements in two Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA. different TG mouse lines (TG-55 and TG-64 that expressed 55% and 64% of Calcium binding to and dissociation from cardiac troponin C (TnC) are essential chimeric TnT, respectively). Both TG-55 and TG-64 mice showed a similar in- steps leading to cardiac muscle contraction/relaxation. It is well documented þ crease in myofilament Ca2 sensitivity at sarcomere lengths (SL) of 1.9 and 2.3 that the calcium binding properties of TnC are not constant, but are sensitive to þ mm. However, Ca2 -activated maximal tension increased significantly only in complex interactions between the additional thin and even thick filament proteins. TG-64 mice at either SL. There was a progressive decrease in the overall heart There is a growing body of evidence that protein modifications/mutations within Wednesday, February 11, 2015 597a different subunits of the troponin complex (troponin T, troponin I) exert their mediated phosphorylation in skinned cardiac myocytes. In addition, incorpora- effects by altering the apparent TnC calcium sensitivity/exchange kinetics. There tion of unphosphorylated cTnI sped rates of force development, which suggests are a number of potential mechanisms that could alter the calcium binding proper- less cooperative thin filament activation and recruitment of non-cycling cross- ties of TnC, potentially the most significant being the ability of the regulatory bridges into the pool of cycling cross-bridges, a process that would tend to domain of TnC to bind the switch peptide region of TnI. We have developed a depress myocyte force and power. Regarding cMyBP-C, PKA treatment of rather simple mathematical model that can simulate the steady-state and kinetic slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but calcium binding properties of a wide assortment of disease-related and post- not TnI) and yielded faster loaded shortening velocity and an ~30% increase translational protein modifications in the isolated troponin complex and reconsti- in power output. These results add novel insight into the molecular specificity tuted thin filament. We propose that roughly half of the studied modifications do by which the beta-adrenergic system controls myofibrillar contractility and how not alter any of the intrinsic TnC calcium binding constants but rather alter the abil- attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may ity of TnC to ‘‘find’’ TnI in the presence of calcium. Considering TnI is essentially contribute to ventricular pump failure. tetheredtoTnCand cannotdiffuse away in the absence ofcalcium and that TnI also binds to actin, we suggest that the apparent calcium binding properties of TnC are 3011-Pos Board B441 highly dependent upon an ‘‘effective concentration’’ of TnI available to bind TnC. Length-Dependent Contractile Dynamics are Blunted Upon Ablation of Cardiac Myosin Binding Protein-C 3009-Pos Board B439 Ranganath Mamidi, Kenneth S. Gresham, Julian E. Stelzer. The Contribution of Myosin Binding Protein-C and Troponin I Phosphor- Department of Physiology and Biophysics, Case Western Reserve University, ylation to the b-Adrenergic Acceleration of Left Ventricular Contraction Cleveland, OH, USA. and Relaxation Enhanced cardiac contractile function due to increased sarcomere length (SL) is, Kenneth S. Gresham, Julian E. Stelzer. in part, mediated by a decrease in the radial distance between myosin heads and Case Western Reserve University, Cleveland, OH, USA. actin. The radial disposition of myosin heads relative to actin is modulated by Activation of the b-adrenergic signaling pathway in the heart leads to an cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C con- increased rate of pressure development in early systole and an acceleration of tributes to the length-dependent activation (LDA) in the myocardium. However, relaxation in diastole to increase cardiac output. These effects are mediated by the precise role of cMyBP-C in modulating cardiac LDA is unclear. To deter- protein kinase A (PKA), which primarily targets myosin binding protein-c mine the impact of cMyBP-C on LDA, we measured isometric force, myo- (MyBP-C) and troponin I (TnI) in the sarcomere for phosphorylation. However, filament Ca2þ-sensitivity (pCa50) and length-dependent crossbridge (XB) the relative contributions of MyBP-C and TnI phosphorylation to the b-ad- contractile dynamics in skinned ventricular muscle fibers isolated from the renergic-mediated increases in ionotropy and lusitropy remains unclear. To hearts of wild-type (WT) and cMyBP-C knockout (KO) mice, at SL’s 1.9mm investigate, we used mice expressing non-phosphorylatable TnI (TnIAla2), or 2.1mm. Our results show that maximal force was not significantly different be- non-phosphorylatable MyBP-C (MyBP-C3SA), and mice expressing non- tween KO and WT fibers. pCa50 was not significantly different between WT and phosphorylatable TnI and MyBP-C (TnI Ala2/MyBP-C3SA), as well as control KO fibers at long SL (5.8250.02 in WT vs. 5.8750.02 in KO), whereas pCa50 WT mice. Pressure-volume loop analysis was performed to measure early sys- was significantly different between WT and KO fibers at short SL (5.7150.02 in tolic pressure development and ventricular relaxation at baseline and in response WT vs. 5.8050.01 in KO; p<0.05). The rate of force redevelopment (ktr), to dobutamine (a b-agonist) administration. At baseline there was no difference in measured at submaximal Ca2þ-activation, was significantly accelerated at short systolic pressure development between the groups, but the acceleration of SL in WT fibers (8.7450.43s-1at 1.9mm vs. 5.7150.40s-1at 2.1mm, p<0.05). dp/dtmax after dobutamine administration was blunted in MyBP-C3SA and TnI Furthermore, the rates of stretch-induced XB relaxation (krel) and XB recruit- Ala2/MyBP-C3SA mice (mmHg/s; 14,41451,016 in WT, 12,9215982 in TnIAla2, ment (kdf) were accelerated by 32% and 70%, respectively at short SL in WT 7,8305899 in MyBP-C3SA, and 8,80351,001 in TnI Ala2/MyBP-C3SA after fibers. In contrast, ktr was not significantly different between both SL’s in KO dobutamine; p<0.05). We examined elastance (E) decay from 75% to 25% of fibers (8.0350.54s-1at 1.9mm vs. 8.9050.37s-1at 2.1mm). Furthermore, KO fi- maximal E (T75-25; normalized to cardiac cycle duration) to measure ventricular bers did not exhibit length-dependent differences in krel and kdf -suggesting that relaxation. At baseline there were no differences between the rate of relaxation in LDA is severely depressed in KO fibers. Collectively, our data indicate that any of the groups; however, after dobutamine administration MyBP-C3SA and cMyBP-C plays a central role in modulating cardiac LDA. TnI Ala2/MyBP-C3SA showed a significantly longer relaxation time compared 3012-Pos Board B442 to WT and TnIAla2 (6.850.3% in WT, 6.950.3% in TnIAla2, 8.350.2% in Cardiac Troponin I Ser-23/24 and Tyr-26 Phosphorylation Crosstalk MyBP-C3SA, and 8.550.4 in TnI Ala2/MyBP-C3SA after dobutamine; p<0.05). 1 1 1 These results show that MyBP-C phosphorylation is necessary to fully accelerate Hussam E. Salhi , Shane D. Walton , Nathan C. Hassel , 1 2 1 contraction and relaxation in response to b-adrenergic signaling. Elizabeth A. Brundage , Pieter P. de Tombe , Paul M.L. Janssen , Jonathan P. Davis1, Brandon J. Biesiadecki1. 3010-Pos Board B440 1Physiology and Cell Biology, The Ohio State University, Columbus, OH, Molecule Specific Effects of PKA-Mediated Phosphorylation on Myofi- USA, 2Cell and Molecular Physiology, Loyola University, Chicago, brillar Function Maywood, IL, USA. Laurin M. Hanft1, Brandon J. Biesiadecki2, Craig A. Emter3, The troponin complex is a critical molecular switch involved in transducing the Kerry S. McDonald1. calcium activating signal into contraction. Troponin I (TnI), the inhibitory sub- 1Medical Pharmacology and Physiology, University of Missouri, Columbia, unit of the complex, is phosphorylated as a key regulatory mechanism to alter MO, USA, 2Physiology and Cell Biology, The Ohio State University, the calcium regulation of contraction. Recent work identified a novel phosphor- Columbus, OH, USA, 3Biomedical Sciences, University of Missouri, ylation of TnI at Tyr-26 that is decreased from its normal basal level in heart Columbia, MO, USA. failure with unknown functional effects. Similar to the desensitizing TnI Ser- Increased cardiac myocyte contractility by the beta-adrenergic system is an 23/24 phosphorylation, TnI Tyr-26 is located in the unique cardiac TnI N-ter- important mechanism to elevate cardiac output to meet greater hemodynamic minal extension. Employing TnI containing actual phosphate at Tyr-26 and load and this process is often depressed in failing hearts. While increased Tyr-26 phosphomimetics, we demonstrate that TnI Tyr-26 phosphorylation de- contractility involves augmented myoplasmic calcium transients, the myofila- creases thin filament calcium sensitivity and accelerates deactivation. To assess ments also adapt to boost the transduction of the calcium signal. Accordingly, the functional integration of TnI Ser-23/24 and Tyr-26 phosphorylation, we ventricular contractility is tightly correlated with PKA-mediated phosphoryla- generated recombinant TnI with phosphomimetic substitution at all three resi- tion of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) dues. Calcium sensitivity measurements demonstrate no additional effect on and cardiac troponin I (cTnI), implicating these two proteins as important trans- calcium binding to troponin C nor calcium sensitive force development of ducers of hemodynamics to the cardiac sarcomere. Consistent with this, we the triple phosphomimetic TnI. However, the integration of Tyr-26 with Ser- have previously found that phosphorylation of myofilament proteins by PKA 23/24 pseudo-phosphorylation further accelerated thin filament deactivation. (a downstream signaling molecule of the beta-adrenergic system) increased The kinase responsible for TnI Tyr-26 phosphorylation remains to be eluci- force, slowed force development rates, sped loaded shortening, and increased dated. Considering that the Src kinase recognition sequence (EEXY) is similar power output in rat skinned cardiac myocyte preparations. Here, we sought to that surrounding TnI Tyr-26 when Ser-23/24 are phosphorylated (S(p)S(p) to define molecule-specific mechanisms by which PKA-mediated phosphoryla- NY), we hypothesize that Ser-23/24 phosphorylation would exhibit signaling tion modulates these contractile properties. Regarding cTnI, the incorporation crosstalk to alter the rate of Tyr-26 phosphorylation. Current efforts are of thin filaments with a majority of unphosphorylated cTnI (as observed in directed towards determining the ability of Ser-23/24 phosphorylation to modu- some models of late stage heart failure) decreased isometric force production late Src-family kinase phosphorylation of Tyr-26. Our findings suggest that TnI at any given activator [Ca2þ] and these changes were reversed by PKA- Tyr-26 phosphorylation in isolation functions similarly to Ser-23/24 N-terminal 598a Wednesday, February 11, 2015 phosphorylation to decrease myofilament calcium sensitivity and accelerate Cytoskeletal-based Intracellular Transport myofilament relaxation. Furthermore, Tyr-26 phosphorylation can buffer the desensitization of Ser-23/24 phosphorylation while further accelerating thin 3015-Pos Board B445 filament deactivation upon their integration. Therefore, the functional integ- Control of the Initiation and Termination of Kinesin-1-Driven Transport ration of TnI phosphorylation may be a common mechanism to modulate by Myosin-Ic and Non-Muscle Tropomyosin Ser-23/24 phosphorylation function. Betsy B. McIntosh1,2, Erika L.F. Holzbaur1,2, E. Michael Ostap1,2. 1Physiology, University of Pennsylvania, Philadelphia, PA, USA, 3013-Pos Board B443 2Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Cardiac Myosin Essential Light Chain N-Terminus Regulates Motor PA, USA. Step-Size Intracellular transport is largely driven by processive microtubule- and actin- Yihua Wang1, Katalin Ajtai1, Katarzyna Kazmierczak2, Danuta Szczesna- based molecular motors. Non-processive motors have also been localized Cordary2, Thomas P. Burghardt1. to trafficking cargos, but their roles are not well understood. Myosin-Ic 1Biochemistry, Mayo Foundation, Rochester, MN, USA, 2Department of (Myo1c), a non-processive actin motor, functions in a variety of exocytic events, Molecular and Cellular Pharmacology, University of Miami Miller School of although the underlying mechanisms are not yet clear. To investigate the inter- Medicine, Miami, FL, USA. play between myosin-I and the canonical long distance transport motor kinesin- Slow ventricular cardiac myosin (bmys) has three distinct unitary step-sizes, 1, we attached both motor types to lipid membrane-coated bead (MBC) cargo, nominally 3, 5, and 8 nm, that move actin with varying step-frequencies. Ventric- using an attachment strategy that allows motors to actively reorganize within the ular cardiac myosin essential light chain (vELC) has a 43 residue N-terminal membrane in response to the local cytoskeletal environment. We compared the extension that binds actin. The basis for the three unitary steps in cardiac myosin motility of kinesin-1-driven cargos in the absence and presence of Myo1c at en- was proposed to involve actin binding of the vELC extension wherein the predom- gineered actin/microtubule intersections. We found that Myo1c significantly in- inant 5 nm step does not engage the actin/vELC linkage, the slightly less likely 8 creases the frequency of kinesin-1-driven microtubule-based runs that begin at nm step engages actin/vELC linkage facilitating an extra ~19 degrees of lever-arm actin/microtubule intersections. Myo1c also regulates the termination of proces- rotation, and the minor 3 nm step is the unlikely conversion of the 5 nm step to the sive runs. Beads with both motors-bound have a significantly higher probability full actin/vELC bound 8 nm step. The hypothesis was tested using several cardiac of pausing at actin/microtubule intersections, remaining tethered for an average myosin constructs including a 17 residue N-terminal truncated vELC in porcine of 20 s, with some pauses lasting longer than 200 s. The actin-binding protein mys (D17) and a 43 residue N-terminal truncated human vELC expressed in trans- non-muscle tropomyosin (Tm) provides spatially-specific regulation of inter- genic mouse heart (D43). Mouse myosin heavy chain is the fast isoform (amys). actions between myosin motors and actin filaments in vivo; in vitro, we found Cardiac myosin step-size and relative step-frequency were measured using a high that Tm2 abrogates Myo1c-driven actin gliding. In the crossed-filament assay, throughput Qdot super-resolution in vitro motility assay. D17 showed a significant Tm2-actin abolishes Myo1c-specific effects on both run initiation and run termi- increase to the 5 nm step-frequency and coincident reduction to the 3 and 8 nm nation. Together these observations suggest Myo1c is important for the selective step-frequencies implying a reduction in probability for forming vELC/actin link- initiation and termination of kinesin-driven runs along microtubules at specific age. Nontransgenic (NTg) and human vELC expressed amys in transgenic mouse actin filament populations within the cell. heart had three unitary step-sizes like those in bmys although step-frequencies in NTg were redistributed to favor the 8 nm step. D43 showed a significant increase 3016-Pos Board B446 to the 5 nm step-frequency and a significant reduction to the combined 3 and 8 nm Visualizing Melanosome Transfer in vivo step-frequency again indicating lower overall probability for forming vELC/actin John A. Hammer, Xufeng S. Wu. linkage. Data strongly supports the hypothesis that the vELC extension is the basis Cell Biology and Physiology Center, NIH, Bethesda, MD, USA. for three step-sizes in cardiac myosin. Supported by NIH grants R01AR049277, Mammalian pigmentation is driven by the intercellular transfer of pigment- R01HL095572, and R01HL108343. containing melanosomes from the tips of melanocyte dendrites to surrounding keratinocytes. Using time lapse imaging of primary mouse melanocyte/keratino- 3014-Pos Board B444 cyte co-cultures prepared from transgenic mice (‘‘Holly’’ mice) in which the Human iPS Cell-Derived Cardiomyocytes Carrying MHC-R403Q Exhibit plasma membranes of melanocytes and keratinocytes are red and green, respec- Aspects of Hypertrophic Cardiomyopathy in vitro tively, we recently defined an intercellular melanosome transfer pathway that in- Eugenia Jones, Coby B. Carlson, Chad Koonce, Natsuyo Aoyama, volves the shedding by the melanocyte of melanosome-rich packages, which are Jun Wang, Benjamin Meline, Steven J. Kattman, Blake Anson. subsequently phagocytosed by the keratinocyte (Wu et al.; PNAS; 2012). Shed- Cellular Dynamics International, Madison, WI, USA. ding, which occurs primarily at the tips of the melanocyte’s dendrites, involves Hypertrophic cardiomyopathy (HCM) is an inherited disease of the heart mus- adhesion of the dendrite to the keratinocyte, thinning of the dendrite behind the cle that affects approximately 1 in 500 individuals. The condition is character- forming package, and an apparent self-abscission event that deposits a plasma ized by enlarged cardiac myocytes, thickened ventricular walls, non-compliant membrane-enclosed package of melanosomes on the surface of the keratinocyte. muscle structure, changes in blood pressure, electrical arrhythmias, and short- After a variable period of time, the keratinocyte then internalizes this package by ness of breath. The Arg-to-Gln switch at amino acid 403 of beta-myosin heavy phagocytosis, completely the intercellular transfer of pigment. To determine if chain (MHC-R403Q) is the most prevalent mutation associated with familial this shedding mechanism also drives melanosome transfer in vivo, we have per- HCM. Human induced pluripotent stem (iPS) cell-derived cardiomyocytes formed time lapse imaging of ear skin on anesthetized Holly mice. The bound- can be generated in highly pure and large quantities from both control and aries of red melanocytes and green keratinocytes are clearly visible in the skin, affected individuals to provide a path forward toward better mechanistic under- and the distribution and dynamics of black melanosomes inside both cell types standing of complex heart diseases. can be followed in three dimensions. Numerous events that occur along the sides Endothelin-1 (ET-1)-induced cardiac hypertrophy in human iPS cell-derived car- and tips of melanocyte dendrites, and that appear to be abortive shedding events, diomyocytes exhibits several classic hallmarks of cardiac hypertrophy including have been seen. To augment transfer, which is probably very infrequent in skin, up-regulation of the fetal gene expression program, cytoskeletal rearrangements, we have treated the animal’s ear skin with forskolin-containing cream to and an increase in cardiomyocyte cell size (Carlson et al., 2013). Similar to the augment pigment synthesis and transfer. Recent imaging yielded several transfer induced hypertrophic state, here we describe novel information regarding innate events that involved shedding of a melanosome-rich package by the melanocyte cardiac hypertrophy associated with MHC-R403Q. iPS cell-derived cardiomyo- and subsequent phagocytosis of the package by the keratinocyte, arguing that the cytes were generated from an MHC-R403Q-positive individual exhibiting the mechanism we defined previously in co culture also occurs in vivo. HCM phenotype. MHC-R403Q cardiomyocytes were generated at high purity (>95% TNNT-positive), exhibited the expected cardiac morphology, and 3017-Pos Board B447 showed autonomous contractile activity similar to the control cardiomyocytes The Effects of Wild-Type and Disease-Linked Tau Mutants on Cargo (non-MHC-R403Q). Transcript profiling demonstrated that basal gene expres- Transport in Cells sion of MHC-R403Q cardiomyocytes was similar to control cardiomyocytes Dezhi Yu1, Stuart Feinstein2, Megan T. Valentine3. that had been induced with ET-1 into a hypertrophic state. Similarly, basal 1Materials, University of California, Santa Barbara, Santa Barbara, CA, USA, BNP levels in MHC-R403Q cardiomyocytes were higher than those of uninduced 2Molecular, Cellular, and Developmental Biology, University of California, control cardiomyocytes. Interestingly, when treated with ET-1, cardiomyocytes Santa Barbara, Santa Barbara, CA, USA, 3Mechanical Engineering, from both backgrounds exhibited increases in BNP expression to similar levels. University of California, Santa Barbara, Santa Barbara, CA, USA. These data suggest that MHC-R403Q cardiomyocytes may have an innate predis- Tau proteins are expressed abundantly in human neurons, and are known to regulate position toward familial hypertrophic cardiomyopathy and underscore the advan- the dynamics and structures of cytoskeletal microtubule filaments. Tau mutations tages of modeling cardiovascular disease through the use of iPSC technology. and misregulation can cause severe neurodegenerative dementia diseases including Wednesday, February 11, 2015 599a frontotemporal dementia with parkinsonism-17, progressive supranuclear palsy, ginosa uses a bet-hedging strategy and a novel motility mechanism character- and corticobasal degeneration. We previously showed that 4RS wild-type tau pro- ized by counter-advection and lateral diffusion to explore flow streamlines and teins and 4RS tau mutants (R406W, P301L, DELTA_N296) can inhibit the effi- rapidly disperse through networks. This selective growth advantage enables P. ciency of kinesin-driven cargo transport along microtubules in a simplified in aeruginosa to self-segregate from competing pathogens, promoting the stable vitro environment. In this study, we used a combination of biophysical tools and coexistence of multiple species in distinct micro-environments. Guided by imaging techniques to investigate the effects of transient tau expression on lyso- our predictive dispersal model, we inhibited colonization and dispersal by some transport in COS-7 African green monkey kidney tissue cells, which are modifying surface chemistry. Our results show that flow and polarized motility natively tau-free. We found that the introduction of tau proteins (either wild-type structures define formation of mixed-species communities, suggesting novel or mutant tau) reduces the speed of lysosome translocation, and moreover, tau strategies for combating bacterial infections. expression induced abnormal aggregation and bundling of microtubules within the cell. Mutant specific effects and possible links to disease will be discussed. 3021-Pos Board B451 Spatial Regulators for Bacterial Cell Division Self-Organize into an 3018-Pos Board B448 Oscillator on a Flat Bilayer Environmental Influence on Microtubule-Based Bidirectional Cargo Anthony Vecchiarelli, Ling Chin Hwang, Min Li, Yeonee Seol, Transport Michiyo Mizuuchi, Keir Neuman, Kiyoshi Mizuuchi. Sarah Klein1,2, Cecile Appert-Rolland1, Ludger Santen2. NIDDK, NIH, Bethesda, MD, USA. 1LPT, Universite´ Paris-Sud, Orsay, France, 2Theoretical Physics, Saarland The E. coli Min system self-organizes into a cell-pole to cell-pole oscillator on the University, Saarbruecken, Germany. membrane to position cell division at mid-cell. We reconstituted and visualized For various intracellular cargos bidirectional motion is observed. Most times this the Min system on a flat lipid bilayer under protein depletion conditions and iden- transport is mediated by teams of molecular motors moving in opposite direction. tified a pattern we call ‘bursts’ - radially expanding bilayer binding centers of the It remains an open question how the cell can ensure efficient transport by relying MinD ATPase that are corralled and disassembled by a ring of the ATPase stim- on this biased stochastic process. We present a model which takes explicitly into ulator, MinE. Bursts share several features with in vivo dynamics such as the abil- account the elastic coupling of the cargo with each motor. In focus is here the anal- ity to oscillate. We propose a patterning mechanism whereby the Min system self- ysis of the influence of the cellular properties. By varying those parameter the cell organizes into an oscillator without the spatial confinement of a bacterial cell. can control the bias in a counter-intuituve manner. Furthermore, we observe in our one dimensional model sub- and superdiffusion on short and intermediate time 3022-Pos Board B452 scales, respectively, due to a temporal correlation in the cargo displacement. Positive Regulation Mechanism in Localizing Cell Division Proteins in Escherichia Coli 3019-Pos Board B449 Matthew W. Bailey, Jaan Ma¨nnik. A Single-Molecule View on Kinesin Motor-Protein Cooperation in Physics and Astronomy, University of Tennessee, Knoxville, TN, USA. Intraflagellar Transport in Living C. elegans How cellular components self-organize in space and time is a major open ques- Bram Prevo1, Pierre J.J. Mangeol1, Felix Oswald1, Jonathan M. Scholey2, tion of cell biology and biophysics. Escherichia coli is capable of localizing its Erwin J.G. Peterman1. cell division apparatus, the divisome, relative to the center of the cell with 1Physics and Astronomy, VU University, Amsterdam, Netherlands, remarkable accuracy and robustness [1]. So far, two molecular systems, the 2Molecular & Cellular Biology, UC Davis, Davis, CA, USA. Min system and nucleoid occlusion mechanism, have been identified as playing Intraflagellar transport (IFT) is an essential intracellular transport mechanism in a role in this localization process. Surprisingly, we found that E. coli without cilia, the hair-like, microtubule-based protrusions of eukaryotic cells with sensory the Min and nucleoid occlusion systems was still capable of accurately local- or motile functions. In the chemosensory cilia of the nematode C. elegans, IFT is izing its divisome with respect to the cell center in slow growth conditions. driven by the cooperative action of IFT-dynein (responsible for transport from In this localization process, the initial Z-ring assembly occurs over the center ciliumtip tobase) and two kinesinmotor proteins, Kinesin-II and OSM-3, (respon- of the nucleoid instead of nucleoid-free regions of the cell. Using live cell fluo- sible for transport in the opposite direction). Our goal was to understand why two rescent imaging we determined that divisome components track the Ter macro- kinesins are needed for IFT and what their respective roles are. To achieve this, we domain region of the chromosome within 50 nm accuracy throughout the cell generated mutant nematodes expressing fluorescent versions of the motor proteins cycle [2]. The Ter macrodomain region thus acts as a landmark for cell division at endogenous levels and subjected them to in vivo fluorescence microscopy with proteins. We furthermore found that the recognition of Ter region by divisome single-molecule resolution. Images obtained were analyzed using automated is mediated by MatP, ZapB and ZapA proteins - the Ter linkage. Unlike the Min kymograph and single-particle tracking analysis, providing unprecedented, quan- system and SlmA mediated nucleoid occlusion, the Ter linkage guides initial titative insight in the role of the kinesins in IFT. We find that the two kinesins fulfill assembly of the Z-ring through a positive regulatory mechanism. distinct roles in line with their distinct motility properties. Kinesin-II is the slower [1] Ma¨nnik J, Wu F, Hol FJH, Bissichia P, Sherratt DJ, Keymer JE, Dekker C and less processive motor. In IFT its key role is to load of IFT trains, to initiate the (2012) Robustness and accuracy of cell division in Escherichia coli in diverse transport of multiple, coupled motor proteins connected to cargo, and to effec- cell shapes. Proc Natl Acad Sci USA 109 (2012) 6957. tively traverse the transition zone, the semi-permeable protein barrier between [2] Bailey MW, Bisicchia P, Warren BT, Sherratt DJ, Ma¨nnik J (2014) Evi- cilium and rest of the cell. After successful crossing of the transition zone, dence for divisome localization mechanisms independent of the Min system Kinesin-II leaves the trains and the other, faster and more processive kinesin, and SlmA in Escherichia coli. PLOS Genetics 10 (2014) e1004504. OSM-3 binds and drives the longer-distance transport to the cilium tip. Our results provide insight in how cells use a combination of motor proteins to drive intracel- 3023-Pos Board B453 lular transport and demonstrate the power of single-molecule fluorescence micro- Cell Division Regulators MinC and MinD Form Polymers in the Presence scopy to unravel complex processes in the cells of living, multicellular organisms. of Nucleotide Joseph Conti, Marissa Viola, Jodi Camberg. University of Rhode Island, Kingston, RI, USA. Bacterial Mechanics, Cytoskeleton, and Motility The Min system of proteins, comprised of MinC, MinD and MinE, is essential for normal cell division in Escherichia coli. MinC forms an oscillating intracel- 3020-Pos Board B450 lular gradient near the poles that restricts placement of the division septum at Colonization, Competition, and Dispersal of Pathogens in Fluid Flow midcell by inhibiting polymerization of FtsZ, the major component of the septal Networks ring. MinC localization is directed by MinD, a Par-like ATPase that binds to the MinYoung Kevin Kim1, Albert Siryaporn2, Yi Shen3, Zemer Gitai2, membrane in the presence of ATP. MinE functions as a regulator of MinD by Howard A. Stone3. stimulating MinD ATP hydrolysis, which leads to membrane dissociation. Re- 1Department of Chemistry, Princeton University, Princeton, NJ, USA, sults using purified proteins in vitro show that MinC stabilizes a conformational 2Department of Molecular Biology, Princeton University, Princeton, NJ, change in MinD in the presence of ATP characterized by the formation of large USA, 3Department of Mechanical and Aerospace Engineering, Princeton oligomers, which are detectable in light scattering and ultracentrifugation University, Princeton, NJ, USA. assays. Electron microscopy of MinC-MinD complexes formed with ATP The mechanisms that shape the spread of bacterial infection in hosts are poorly show long polymers (300-500 nm) that are approximately 10-20 nm wide. understood. Single cells of Pseudomonas aeruginosa, an opportunistic pathogen Increasing the concentration of MinC in polymerization assays with MinD that colonizes fluidic pathways in plants and animals, move against fluid flow. and ATP enhances oligomer formation. MinCD complexes assemble in the But the effects on population-level behaviors such as colonization, competition, presence of the ATP analog ATPgS, but not ADP, suggesting that MinD is and dispersal have remained unclear. We explore P. aeruginosa population likely in an ATP-bound conformation. Using site-directed mutagenesis, we dynamics in flow networks that mimic host vasculature. We find that P. aeru- constructed a MinD mutant protein defective for ATP-dependent dimerization 600a Wednesday, February 11, 2015 and a truncated MinD mutant protein lacking the membrane targeting sequence in multiple bacterial species, developing a model of T6SS function. We present (MTS); both MinD mutant proteins are unable to polymerize with MinC. Addi- quantitative measurements of the dynamics of the secretion system - from tionally, the in vitro activity of MinC to prevent GTP-dependent FtsZ pelleting the assembly to contraction to disassembly - in conjunction with quantitative in sedimentation assays is impaired by MinD and ATP, suggesting polymeriza- measures of system function, including recipient cell lysis. tion with MinD may modulate MinC function. Finally, addition of MinE to MinC-MinD polymers induces concentration-dependent disassembly manner 3027-Pos Board B457 suggesting MinE competes with MinC for binding to MinD. Results suggest Role of Fumarate in the Operation of the Bacterial Flagellar Motor a novel nucleotide-dependent conformation of MinD with MinC and provides Jyoti Sharma, Vidhu Soman, Ravikrishnan Elangovan, Sunil Nath. mechanistic insight into the functional interactions of the Min proteins. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India. 3024-Pos Board B454 Fumarate has previously been identified as a ‘switch factor’ that can alter the Superresolution Investigation of the E. coli Cell Division Ring during switching frequency and bias of the sense of rotation of the bacterial flagellar Constriction motor; however, despite numerous studies during the past fifty years, the mo- Carla Coltharp, Jie Xiao. lecular mechanism by which fumarate causes these effects could not be under- Johns Hopkins University School of Medicine, Baltimore, MD, USA. stood. We studied the motility of Salmonella by bright field and by monitoring The E. coli cell division machinery are recruited to midcell by a ring-like struc- rotation in a tethered cell assay. We carried out a detailed computerized ture formed by the highly conserved FtsZ protein. This ‘‘Z-ring’’ scaffold can analysis of the swimming and rotational behavior in real time under various generate a membrane bending force that may be critical for cytokinesis. Several conditions of external pHs (5.0 5 8.0) and added fumarate concentrations mechanistic models for Z-ring force generation have been presented, but are (0 5 12 mM). Based on our experimental results, we make the novel proposal limited in detail by insufficient data regarding the arrangement of FtsZ fila- that the bacterial flagellar motor contains access channels permeable to the ments within the Z-ring and regarding the role of FtsZ’s GTPase activity in fumarate monoanion, and that it catalyzes the overall electroneutral transport constriction progression. of fumarate and protons. We further postulate that each ionic species provides Structural details about the Z-ring have recently become clearer due to ad- ~50% of the total energy for rotation of the bacterial flagellar motor. These pre- vancements in both optical and electron microscopy. Super-resolution fluores- dictions are applicable to both the normal (say CW) mode of motor rotation as cence microscopy and electron cryotomography studies support a loosely well as to the so-called ‘‘switched’’ (CCW) sense of rotation, depending on the bundled arrangement of FtsZ filaments, resulting in both sparse regions and direction of translocation of the ions. Hence previous proposals of fumarate as a dense clusters within the Z-ring. Super-resolution imaging also revealed that ‘switch factor’, are false, or at best incomplete, because fumarate participates in the Z-ring can adopt both a single-ring conformation at midcell and a the rotation of the flagellar motor in both normal and switched modes of oper- multiple-ring conformation reminiscent of a tight helix. ation. The new hypothesis extends to bacterial motility the essential and vital We have built upon these previous studies to determine the three-dimensional role played by dicarboxylic acid anions in oxidative phosphorylation and structure of the E. coli Z-ring in super resolution using Photoactivated Locali- photosynthesis according to Nath’s torsional mechanism of energy transduction zation Microscopy (PALM) and analyze its changes during constriction. We and ATP synthesis (Nath, S., Beyond the Chemiosmotic Theory, Journal of used quantitative analyses of PALM and time-lapse data to assess the plausibil- Bioenergetics and Biomembranes 2010,42, 301-309). ity of various FtsZ-centric constriction mechanisms. We found that the cell wall constriction rate is not correlated to Z-ring assembly/disassembly dynamics, 3028-Pos Board B458 density, or GTPase activity. These characterizations help refine and re-define A GTPase Deficient FtsZ Mutant Assembles Inefficiently and Impairs the role of FtsZ in cytokinesis. Cytokinesis in Bacillus Subtilis Cells Hemendra P.S. Dhaked, Anusri Bhattacharya, Saroj Yadav, 3025-Pos Board B455 Dulal Panda. Bacterial Growth and Shape Regulation by External Compression Biosciences & Bioengineering, IIT BOMBAY, MUMBAI, India. Fangwei Si, Bo Li, Sean X. Sun. FtsZ polymerizes to form a dynamic Z-ring at the mid-cell position and orches- Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA. trates the bacterial cell division. The perturbation of Z-ring has been found to Gram-negative Escherichia coli are rod-shaped and maintain a constant diameter be lethal to bacteria. Plumbagin and SB-RA-2001 were found to inhibit bacterial during their exponential growth phase. This shape has been shown to be critical cell proliferation by targeting FtsZ. Docking analyses indicated that both plumba- in regulating many fundamental biological functions including nutrient access, gin and SB-RA-2001 bind to the cleft region between H7 helix and C-terminal cell division, attachment, dispersal, and motility. Here, we experimentally domain of the BsFtsZ. Arg191 (R191) was identified as the common residue show that mechanical forces regulate growth and shapes of E. coli. By applying involved in H-bonding with these two compounds. To examine the importance precisely controllable compressive forces to growing rod-shaped E. coli, we of R191residue in the assembly of FtsZ, we constructed R191A-BsFtsZ. The sec- demonstrate that the cells no longer retain their rod shapes but grow and divide ondary structure of the mutated FtsZ (R191A-BsFtsZ) was similar to that of WT- with pancake-like shapes while these deformed cells can recover their shapes in BsFtsZ. The mutation (R191A) strongly diminished the GTPase activity of FtsZ. several generations after the forces are removed. Those cells are proved to have R191A-BsFtsZ exhibited less polymerization ability as compared to WT-BsFtsZ. comparable growth rate, proliferation rate, DNA replication, and protein synthe- It could also poison the assembly of WT-BsFtsZ in a concentration dependent sis to rod-shaped cells. Furthermore, the growth of compressed cells also helps manner. Moreover, R191A-BsFtsZ formed polymers of a different morphology reveal the biological and mechanical role of MreB in maintaining cell shape. than that of WT-BsFtsZ. In-silico study revealed that R191A BsFtsZ interacts with WT-BsFtsZ and forms a curved dimer while two WT-BsFtsZ monomers 3026-Pos Board B456 form a linear dimer. Further, we observed that when R191A-BsFtsZ was trans- Dissecting the Mechanism of Type VI Secretion System Effector Delivery formed into B. subtilis PL2084 strain, the B. subtilis cells became filamentous indi- by Fluorescence Cross-Correlation Microscopy cating that the mutation had a strong adverse effect on the division of B. subtilis 1 2 3 Jacqueline Corbitt , Michele LeRoux , Robin Kirkpatrick , cells. The results suggested that the GTPase activity of FtsZ plays an important Joseph Mougous4, Paul A. Wiggins1. 1 2 role in Z-ring formation. The study also suggested that designing antimicrobials Physics, University of Washington, Seattle, WA, USA, Molecular and targeting the Arg191 residue might help to curb the division process. Cellular Biology, University of Washington, Seattle, WA, USA, 3Biological Physics, Structure and Design, University of Washington, Seattle, WA, USA, 3029-Pos Board B459 4Microbiology, University of Washington, Seattle, WA, USA. Bacterial Motility Measured by a Miniature Chamber for High-Pressure The Type Six Secretion System (T6SS) is a bacterial toxin-delivery system tar- Microscopy geting bacterial cells which neighbor the donor, promoting recipient cell death. Masayoshi Nishiyama1, Seiji Kojima2. The T6SS is widely conserved among Gram-negative bacteria and may be a 1Department of Chemistry, Graduate School of Science, Kyoto University, central determinant in bacterial fitness in polymicrobial communities of partic- Kyoto, Japan, 2Department of Chemistry, Graduate School of Science, ular relevance to chronic infection. Sequence homology of secretion system Nagoya University, Nagoya, Japan. components to the T4 bacteriophage tail spike, cryoEM reconstructions of Hydrostatic pressure is one of the physical stimuli that characterize the environ- the secretion system and fluorescence imaging are all consistent with a dynamic ment of living matter. Many microorganisms thrive under high pressure and mechanism of secretion. The complex system, which is composed of at least 15 may even physically or geochemically require this extreme environmental con- proteins, forms a puncturing apparatus/delivery system which uses a donor pro- dition. In contrast, application of pressure is detrimental to most life on Earth; tein filament to puncture the recipient cell wall to deliver protein toxins. Using especially to living organisms under ambient pressure conditions. To study quantitative imaging analysis of multiple fluorescent fusions, we present a the mechanism of how living things adapt to high-pressure conditions, it is detailed characterization of T6SS system dynamics visualized in single cells necessary to monitor directly the organism of interest under various pressure Wednesday, February 11, 2015 601a conditions. Here, we report a miniature chamber for high-pressure microscopy the agar substrate leads to changes in topography, such as wrinkling, or to [1]. The chamber was equipped with a built-in separator, in which water pres- changes in material properties, such as polymer alignment. Recently, we found sure was properly transduced to that of the sample solution. The apparatus that elasticotaxis is not unique to M. xanthus; our preliminary results suggest developed could apply pressure up to 150 MPa, and enabled us to acquire many bacteria exhibit this behavior. Accordingly, we are examining whether bright-field and epifluorescence images at various pressures and temperatures. elasticotaxis is common to both gliding bacteria and swarming bacteria and We demonstrated that the application of pressure acted directly and reversibly how it may affect biofilm formation. on the swimming motility of Escherichia coli cells. The present technique should be applicable to a wide range of dynamic biological processes that 3032-Pos Board B462 depend on applied pressures [2, 3]. Atomic Force Microscope Spectroscopy: Progress toward Antibiotic Resis- [1] Nishiyama M. and S. Kojima. 2012. Bacterial Motility Measured by a Mini- tance and Biofilm Studies 1 2 3 ature Chamber for High-Pressure Microscopy. Int. J. Mol. Sci.13: 9225-9239. Mehrdad M. Tajkarimi , Albert M. Hung , Scott H. Harrison , 2 [2] Nishiyama M. et al. 2013. High Hydrostatic Pressure Induces Counterclock- Joseph L. Graves . 1Physical sciecne, Forsyth Tech, Winston-Salem,, NC, USA, wise to Clockwise Reversals of the Escherichia coli Flagellar Motor. J. Bacte- 2 tiol.195: 1809-1814. Nanoengineering, North Carolina A&T State University, Greensboro, NC, USA, 3Biological science, North Carolina A&T State University, [3] Okuno D. et al., 2013. Single-Molecule Analysis of the Rotation of F1- ATPase under High Hydrostatic Pressure. Biophys. J.105:1635-1642. Greensboro, NC, USA. The emerging field of live cell nanoscopy and force spectroscopy could revo- 3030-Pos Board B460 lutionize the way biologists explore the living cell at a molecular resolution. Bacterial Flagellar Switching: Hidden Markov Steps Revealed Atomic Force Microscope (AFM) and force spectroscopy analysis have been Henry G. Zot1, Javier E. Hasbun2, Nguyen Van Minh3. used to directly measure reversible physiochemical and specific binding inter- 1Biology, University of West Georgia, Carrollton, GA, USA, 2Physics, actions between cells. Stickiness is important biofilm formation stage that could University of West Georgia, Carrollton, GA, USA, 3Mathematics, Columbus also be measured at nN level. A significant source of foodborne illness results State University, Columbus, GA, USA. from biofilms. These are caused by microorganisms that attach to surfaces and Here we describe a Markov chain that accounts for hidden steps in the switching grow as highly organized multicellular communities. This study examines the mechanism of bacterial flagellar rotation. The bacterial flagellar motor appa- impact of silver resistance on bacterial adhesion and its viscoelastic formation. ratus switches between counter clockwise rotation (CCW) and clockwise rota- We present the first set of data that evaluates the elastic deformation of a bac- tion (CW) by a process that requires interactions between ligand-bound switch terial cell surface upon evolution of silver resistance in E. coli MG1655 using subunits of the rotor and motor units of the stator. If the ligand, CheY, binding AFM compared to controls in 200 generations. The adhesion stickiness and to switch subunits shifts equilibrium between CCW and CW, i.e. simplest pro- stiffness mean of the treated (evolved Ag resistant) and non-treated samples posed mechanism, the intervals between switches, dwell times, should be a were tested at nN level. The evolved samples had a significantly (P<0.05) random variable of an exponential distribution. Because dwell times are actu- higher stickiness ratio and value (from 0.015 0.04 nN to 0.065 0.02 nN) ally gamma distributed, the switching mechanism has evidence for multiple compared to the controls (non-resistant) strains (from0.015 0.02 nN Poisson steps, which is characteristic of a Markov process. We are proposing to0.045 0.02 nN). The highest difference of adhesion force happened on Gen- that the hidden steps of the switching mechanism are intermediates of the prin- eration 100. According to images of the bacteria in different generations, we ciple thermodynamic states, CCW and CW, generated by rotation. At steady- can see some major phenotype changes on the appearance at generation 100. state, the intermediates factor out of the derived state function, but both the ther- The MIC data for the non-evolved strains of E. coli MG 1655 through 200 gen- modynamic states and intermediates appear as statistical states of a Markov erations were also significantly lower 38.585 10.09 mg/l compare to evolved chain. The state function determines the bias and the conditional probabilities strains 272.25 5 153.94 mg/l (p<0.01). The experiment demonstrates impor- for all steps of the Markov chain except when rotation separates a switch sub- tant features of phenotype modulation resulting from the evolution of Ag resis- unit, which must have unit probability. To obtain a continuous time random tance that will be further studied by this group. walk, we applied the rotation rate to all conditional and unit probabilities of the discrete Markov chain. Published dwell time distributions were fit best by 3033-Pos Board B463 simulations that depend on bias alone regardless of the number of motor units Depletion-Mediated Pattern Formation in a Growing Bacterial Colony operating on a rotor. Because the bias in our model depends on the probability Pushpita Ghosh1, Jagannath Mondal2, Eshel Ben-Jacob1, Herbert Levine1. 1Center for Theoretical Biological Physics, Rice University, Houston, TX, that all motor units of a rotor interact with switch subunits that are ligand bound, 2 each motor unit contributes less to the free energy change required for a given USA, Chemistry, Columbia University, New York, NY, USA. bias as motor units increase. Yet the probability of a switch remains constant. Secretion of extracellular polymeric substances into growth medium of bacteria is the hallmark of forming biofilm-like structures. The morphological property 3031-Pos Board B461 of such systems might depend upon the physical interactions of cells with ex- Mechanical Stress Changes the Movements and Organization of Biofilm- tracellular polymeric substances(EPS). We have studied self-organization of Associated Bacteria nonmotile rod-shaped bacterial cells growing on solid substrate in presence of David J. Lemon1, Xingbo Yang2, Pragya Srivastava2, M. Cristina Marchetti2, self-producing EPS, secreted into the growth medium in expanding colony. In Anthony Garza1. our individual-based simulation model of bacterial cells and EPS, all the parti- 1Department of Biology, Syracuse University, Syracuse, NY, USA, cles interact mechanically via repulsive forces by pushing each other away as 2Department of Physics, Syracuse University, Syracuse, NY, USA. bacterial cells grow and divide consuming diusing nutrient and produce EPS. Historically, studies analyzing collective movements, biofilm formation, and We show that mechanical interactions control the collective behavior of the sys- the emergence of pathogenicity in bacteria have focused on chemical signals tem, particularly, we show that the presence of nonadsorbing EPS leads sponta- that elicit changes in cell behavior, while responses to physical cues have neous aggregation of bacterial cells by depletion attraction and generates phase only recently begun to garner attention. In contrast, the effects of changes in separated patterns in a nonequilibrium growing colony. This generic mechanism mechanical properties of the environment on eukaryotic cells have been studied powered by entropic forces could explain one of the possible ways to sponta- extensively. It is known, for instance, that substrate stiffness can guide the neous aggregated structure formation and spatial heterogeneity in a biofilm. migration of epithelial and endothelial cells and has profound effects on cell shape and division. Our research aims to examine how bacteria and bacterial 3034-Pos Board B464 colonies respond to changes in the physical properties of their environment Bacterial Chemotactic Tumble Angles Reduce Backtracking and (mechanical stress, stiffness, and surface topography) and to understand the Maximize Information Gathering molecular mechanisms behind the response. Previous work showed that the Jan H. Hoh1,2, William F. Heinz1. 1 Gram-negative, biofilm-forming bacterium Myxococcus xanthus responds to Physiology, Johns Hopkins School of Medicine, Baltimore, MD, USA, 2 tension and compression of its substrate’s surface by forming elliptical colonies Royal Institute of Technology, Stockholm, Sweden. that expand most rapidly perpendicular to the axis of compression. This Chemotaxing bacteria gather information from the environment and use that to behavior, dubbed ‘‘elasticotaxis’’, was initially hypothesized to be important control the balance between runs and tumbles in order achieve a biased motion for locating food sources. By combining physics-based modelling of the toward the source of a chemoattractant. We have examined the role of the tum- stresses in compressed substrates with experimental data showing the corre- ble angle on how effectively gradients are coupled into a bacterium’s trajectory. sponding change in shape of M. xanthus colonies, we have established a linear Chemotaxis was simulated using the ZBP program, and the average tumble correlation between the mechanical stress in the substrate and the degree of the angle varied from 0 to 180 degrees in the presence and absence of the normal elasticotaxis response. To identify the physical changes in the substrate that angle variance and/or rotational diffusion. 100,000 step (0.1 mm/step) trajec- elicit the elasticotaxis response, we are investigating whether compression of tories from these simulations where analysed using the k-space information 602a Wednesday, February 11, 2015 metric to quantify how much information was reflected in the paths. For wild- 3037-Pos Board B467 type bacteria the information in the trajectories is essentially constant across all Coupling Scheme of the Rotary Motor Thermophilic F1 tumble angles (and several gradients). However, if either the angle variance or Kengo Adachi1, Kazuhiro Oiwa2, Masasuke Yoshida3, the rotational diffusion is set to zero, distinct minima appear in the trajectory Kazuhiko Kinosita Jr1. information appear at 0, 90, and 180 degrees - and there are broad maxima 1Dept. of Physics, Waseda Univ., Tokyo, Japan, 2Adv. ICT Res. Inst., NICT, around 70 and 110 degrees. In simulations where both the angle variance Kobe, Japan, 3Dept. of Mol. Biosci., Kyoto Sangyo Univ., Kyoto, Japan. and the rotational diffusion are set to zero, the trajectory information exhibits Thermophilic F1 (TF1) is an ATP-driven rotary molecular motor driven by sequen- several more prominent minima, notably at 135, 60, and 45 degrees. We sug- tial hydrolysis of ATP in three catalytic sites. Rotation occurs in steps of 120 per gest that these minima arise because angles that are small integer fractions of ATP, and the 120 step is further resolved into 80-90 and 40-30 substeps. In the 180 or 540 degrees increase the likelihood of backtracking - thus reducing standard coupling scheme, ATP binding starts rotation from an ATP-waiting angle the new space explored by the bacterium. When a bacterium does tumble, it at 0,andat~200 the ATP is cleaved into ADP and Pi, and the ADP is released should do so in a way as to explore as much new space as possible in order around 240 after a third ATP is bound. Pi release is at 200 or 320, yet unsettled. to optimize information gathering. Notably that is not 90 degrees, but one With human mitochondrial F1 (HF1), Suzuki (2014) has indicated different angle maximum is close to the normal tumble angle of 68 degrees for E. coli. dependence: cleavage occurs at 210 and Pi release at 305. A peculiar finding in HF1 was that supposedly slowly hydrolyzed ATPgS not only lengthened the dwell 3035-Pos Board B465 at 210 but to much greater extent the 305 dwell, implying that the thio-Pi release Antibodies Change the Mechanics of Adhesion Fimbriae - a Case Study is much slower than the ATPgS cleavage. We thus re-examined under a micro- of CS20 Fimbriae Expressed by Enterotoxigenic Escherichia Coli scope how ATPgS affects TF . With fluorescently (Cy3) labeled ATPgSwe 1 1 2 1 Narges Mortezaei , Bhupender Singh , Bernt Eric Uhlin , observed a remarkably long dwell at only 200 after its binding. With unlabeled 3 4 1 Stephen J. Savarino , Esther Bullitt , Magnus Andersson . ATPgS mixed in ATP, we observed only one long dwell per ATPgS binding, 1Department of Physics, Umea˚ University, Umea˚, Sweden, 2The Laboratory compared to two consecutive long dwells in HF1. The long dwell with unlabeled for Molecular Infection Medicine Sweden (MIMS) and Department of 1 3 ATPgS, presumably at 200 , comprised two reactions with rates 460 s and 30 Molecular Biology, Umea˚ University, Umea˚, Sweden, Enteric Diseases s1, compared to 2400 s1 and 820 s1 with regular ATP. We have yet to decide Department, Infectious Diseases Directorate, Naval Medical Research which corresponds to cleavage and (thio-)Pi release, but kinetic difference from Center, Silver Spring, MD, USA, 4Department of Physiology and Biophysics, HF1 is obvious: either (thio-)Pi release occurs at 200 in TF1, or thio-Pi release Boston University School of Medicine, Boston, MA, USA. is not slow in TF1. Nonhydrolyzable Cy3-AMPPNP halted rotation at 200 after Enterotoxigenic Escherichia coli (ETEC) express a variety of fimbriae that binding, implying that ATP cleavage occurs at 200 or before, a conclusion previ- mediate adhesion to host epithelial cells. It has been shown that the ability of a ously drawn on the assumption of slow cleavage of ATPgSandamutant. fimbriated bacterial cell to attach and stay attached to host cells does not merely depend on the adhesin expressed distal of the fimbriae but also the biomechanical properties of the fimbriae are vital for sustained adhesion. Fimbriae can signi- Energy Transduction, Electron and Proton ficantly extend under a constant force when exposed to an external force and therefore reduce the load on the adhesin, which is believed to help bacteria to Transfer, and Light Harvesting withstand external forces applied by various body defense systems. Thus, it is thought that the fimbrial shaft and adhesin have co-evolved for optimal function 3038-Pos Board B468 when bacteria attach to host cells. To investigate if antibodies, normally found in Exploring the Staphylococcus Epidermidis Respiratory Chain the intestines, affects the biomechanical properties of fimbriae, we exposed CS20 Cristina Uribe Alvarez1,2, Natalia Chiquete-Fe´lix1,3, fimbriae expressed by ETEC to anti-fimbrial antibodies and measured these pro- Salvador Uribe-Carvajal1, Antonio Pen˜a1. 1 2 perties using optical tweezers force spectroscopy. Our data show a change in Inst FIsiol Celular, UNAM, Mexico, Mexico, Molec Genetics, INS FISIOL 3 the force required to extend the fimbriae and that the elasticity is significantly CELULAR, UNAM, Mexico City, Mexico, Molec genetics, INST FISIOL reduced by the presence of antibodies. The reduced elasticity, likely due to CELULAR, UNAM, Mexico city, Mexico. cross-linking of fimbrial subunits, could thus be another assignment for anti- Staphylcoccus epidermidis does not invade healthy tissues. However, it has been bodies; in addition to their mission in marking bacteria as foreign, our data indi- identified as a major cause of nosocomial infections because of its ability to infect cate that antibodies physically compromise fimbrial function. To further confirm polymer surfaces such as catheters or intra-cardiac valves, forming biofilms and interaction of antibodies to their specific target we performed western blot anal- evoking a reaction from the host that eventually leads to removal of prosthesis. S. ysis, transmission electron microscopy and immunofluoresence microscopy. In epidermidis is responsible for 50-70% of catheter-related infections and 30 to the presence of antibodies, we suggest that our assay and results will be a starting 43% of perioperative implant infections. S. epidermidis can survive in a wide point for further studies aimed at inhibiting bacterial adhesion by antibodies. range of environments and oxygen concentrations ([O2]). It may grow in atmo- spheric oxygen levels, in micro-aerobic environments, including normal tissues 3036-Pos Board B466 with [O2]¼ 3 to 5% and even in pathologically altered tissues where O2 tensions Single Cell Dynamics Drive Turbulent Flow in the Collective Motion may reach zero. S. epidermidis increases its propensity to form biofilms as [O2] of Bacteria decreases. Bacteria may contain different terminal oxidases that allow them to Alex Hamby, Charles Wolgemuth. cope with different [O2]. Also, respiratory chain-branching aids the cell to sur- University of Arizona, Tucson, AZ, USA. vive in the presence of toxic substances. Understanding the plasticity of the res- In most areas of biology, the principal confounding factor is the complexity. piratory chain is necessary to understand the physiology and pathogenicity of S. Biology from the cellular level to the ecosystem involves the action of a multi- epidermidis. In this species, we found two terminal oxidases expressed during tude of individuals that come together and achieve specific tasks. For example, at growth in different [O2]: bo is always present, while aa3 is expressed in aerobic the single cell level, the binding and unbinding of cytoskeletal proteins conspire conditions but not in microaerophilic conditions nor in KCN plus a non- to allow a cell to move across surfaces or through the extracellular matrix. At the fermentable carbon source. In Oxygen consumption experiments, the preference tissue level, these motile cells act together to heal wounds or form cancer metas- for different substrates varied depending on [O2] exposure during growth. tases. Tissues come together to form organisms, which form societies, and so on. Here we use dense communities of swimming bacteria to understand how collec- 3039-Pos Board B469 tive behavior arises out of the actions of an individual. At high density, rod- New Perspectives on Quinol Binding Motifs at the bc1 Complex Based on shaped bacteria produce complex fluid flows that include vortices and jets. These MD Simulations flows arise partially due to the dipole forces that each bacterium exerts on the Angela M. Barragan1,2, Abhishek Singharoy2, Anthony R. Crofts3,4, surrounding fluid. By confining Bacillus subtilis or Escherichia coli within Klaus Schulten1,2, Ilia A. Solov’yov5. Hele-Shaw cells of controllable depth, we probe how individual biophysical pa- 1Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA, rameters, such as shape, speed, external drag, and chemotaxis, affect the result- 2Beckman Institute for Advanced Science and Technology, University of ing collective behavior in this system. We then compare our results to Illinois at Urbana–Champaign, Urbana, IL, USA, 3Biochemistry, University of predictions from a two-phase fluid model that is based on the single-cell physics Illinois at Urbana-Champaign, Urbana, IL, USA, 4Center for Biophysics and of a swimming bacterium. Comparison of our experimental and simulation re- Computational Biology, University of Illinois at Urbana–Champaign, Urbana, sults show that the collective behavior in this system is largely determined by IL, USA, 5Physics, University of Southern Denmark, Odense, Denmark. the biophysics of the single organism. The physics of these dense communities The bc1 complex is a central player in the conversion of energy into ATP synthe- has many similarities to actomyosin systems, as well as to collective systems of sis in photosynthesis and respiration, and its overall mechanism, the Q-cycle, is epithelial cells. Therefore, our results are likely broadly applicable to a wide well known. However, the quinol-protein interaction that initiates Q-cycle at range of problems in cell migration, from the single cell to the collective. the Qo-binding site have not yet been described. Employing classical MD Wednesday, February 11, 2015 603a simulations in tandem with DFT calculations, the quinol binding motifs to the Qo- dielectric constant of the medium also plays a role in modulating the properties site of bc1 complex is investigated for a range of Qo-site protonation states. The of heme a through H-bonding of its formyl group. computations revealed a novel configuration of the key side groups at the Qo-site site, such as H156, Y147 and E295, that stabilize the reaction complex and pro- 3042-Pos Board B472 vide an optimal configuration prior to the charge transfer reactions between quinol Theoretical Investigation ofthe PrimaryEvent inProteorhodopsinActivation and iron-sulfur cluster of the Rieske protein. Re-arrangements in the E295 and Choongkeun Lee, Blake Mertz. Y147 side chains were observed in all our simulations, showing intermediate Chemistry, West Virginia University, Morgantown, WV, USA. a bridging hydrogen bonding between quinol and E295, not observed before. Sim- Retinal proteins are -helical transmembrane proteins that have the potential for ulations were extended to study cytochrome c2 docking to cytochrome c1, applications such as memory storage (bacteriorhodopsin) and optogenetics (chan- required for the removal of electrons from the bc1 complex. We found, the dock- nelrhodopsin). Proteorhodopsin (PR) is a recently discovered microbial retinal ing interface is characterized by a semicircular arrangement of electrostatic resi- proteinwhich acts asa proton pump, similar tobacteriorhodopsin. Initial activation of PR occurs when the covalently bound chromophore, retinal, absorbs a photon dues that draws the cytochrome c2 and bc1 complex into an encounter complex, a / hydrophobic minimal core to facilitate electron transfer, and mobility mismatch and undergoes an all-trans 13-cis isomerization. Despite several similarities between the bound surfaces to induce post-electron transfer undocking. between bacteriorhodopsin and PR, the details of the activation process remain un- clear. We investigated the photoisomerization of retinal in PR using a hybrid quan- 3040-Pos Board B470 tum mechanical (QM) and molecular mechanics (MM) approach. Two Glutathione S-Transferase Kappa 1 Knockdown Exacerbates Complex- photoexcited states were identified. Both retinal structures have a 13-cis,15-anti III-Mediated ROS Production in H9c2 Cardiac Cells conformation. However, one does not involve a hydrogen bond between the Schiff Kyriakos N. Papanicolaou, Agnieszka Sidor, Jasma Rucker, base and a water molecule, while the other structure does, dependent on the rota- Brian O’Rourke, D. Brian Foster. tional direction of the isomerization. The former is more stable than the latter by Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. about 0.9 eV and the former also has a lower energy barrier of the photoisomeri- Background: Mitochondria produce reactive oxygen species (ROS) that are zation by about 0.2 eV than the latter. Upon photoisomerization, PR reaches the scavenged by local antioxidant enzymes. Glutathione (GSH) is key interme- former excited state, subsequently proceeding through a slow rearrangement to diate in many of these reactions and its availability determines the antioxidant form the hydrogen bond between the Schiff base and water and generate the latter capacity of mitochondria. Glutathione S-transferases (GSTs) are known to excited state. We believe the former structure to be indicative of the K state, with consume GSH during xenobiotic detoxification but their involvement in ROS the latter structure more characteristic of an L-like state. This L-like state is spec- scavenging is less clear. Gstk1 was originally identified as a novel mitochon- troscopically silent and most likely extremely transient, due to the low energy bar- drial GST but its role there remains unknown. rier (0.1 eV) of the proton transfer from the Schiff base to the proton acceptor, D97. Objective: To examine whether loss of Gstk1 affects the [GSSG/GSH] ratios in A detailed understanding of the initial events inthe activation process is essential to cytosol and mitochondria of the cardiac-derived cell line H9c2. utilizing PR as a light-driven component in potential technological applications. Methods: Knockdown of Gstk1 (Gstk1-KD) in H9c2 cells was achieved by 3043-Pos Board B473 transfection with siRNA. Changes in [GSSG/GSH] were monitored using The Electron Transfer in Ferredoxins genetically-encoded ratiometric sensors that localize to cytosol or mitochon- Kelly N. Tran, Toshiko Ichiye. dria. Global oxidative stress was induced with hydrogen peroxide (HP). Chemistry, Georgetown University, Washington, DC, USA. Mitochondrially-targeted oxidative stress was induced by the superoxide gener- The 2[4Fe-4S]-ferredoxins are ubiquitous water-soluble electron shuttles found in ator di-methoxy-naphtho-quinone (DMNQ) and by antimycin-A (AA) which all bacteria. The intramolecular ‘‘self-transfer’’ electron transfer between the two interacts specifically with mitochondrial complex III. [4Fe-4S] clusters is an ideal model system for studying electron transfer indepen- Results: Treatment with HP elevatedcytosolic [GSSG/GSH] ratios equally in dent of association of thedonorand acceptorand may be consideredasthelimit of a both control and Gstk1-KD cells. Mitochondrial oxidative stress elicited by very tightly bound complex. Thus, the effects of environmental factors on electron HP or DMNQ likewise increased mitochondrial [GSSG/GSH] and was unaf- transfer rates can be focused on. Clostridium acidurici ferredoxin (CaFd) is a small fected by Gstk1-KD. Inhibition of Complex III by AA decreased mitochondrial (5.9 kDa, 55 residues), pseudo-symmetric protein containing two [Fe S (SR) ]2- [GSSG/GSH] persistently in control cells. However, in Gstk1-KD cells ex- 4 4 4 redox sites which are separated by ~12 A˚ , the typical distance of biological electron posed to AA, the decrease was transient and was followed by a sustained in- transfer. Molecular dynamics simulations of CaFd are performed using different crease in [GSSG/GSH]. force fields to test their performance. The environmental reorganization energy Conclusions: Here we examine for the first time the role of Gstk1 in ROS scav- l is calculated from the simulations and compared to experiment. enging in cardiac cells. While our data suggest that Gstk1 does not participate in the general response against exogenous ROS, they indicate that low levels of 3044-Pos Board B474 this enzyme associate with increased superoxide production from mitochon- Internal Switches Modulating Electron Flow in bc1 Complex drial complex III. Our findings may provide insights for experimental models Muhammad Hagras, Alexei A. Stuchebrukhov. of cardiac disease where Gstk1 expression is downregulated. Biophysics, University of California, Davis, Davis, CA, USA. Ubiquinol-cytochrome c oxidoreducase (BC1) protein complex is a smart ma- 3041-Pos Board B471 chine which employs an internal signal transduction network, regulated by the Evaluation of Heme Peripheral Groups Interactions in Low-Dielectric binding of natural ligands to either Qo or Qi sites, to modulate electron transfer Constant Media rates between different redox pairs. Binding of natural ligands or some inhib- Jose F. Cerda, Alaina T. Stockhausen, Nicolette D. Wilkes, itors leads to local conformational changes which propagate through protein Kathleen R. Silva, Allyson R. Langley, Mary C. Malloy, and control the conformation of key residues involved in the electron tunneling Brady O. Werkheiser. pathway. Aromatic-aromatic interactions are highly utilized in this internal Chemistry, Saint Joseph’s University, Philadelphia, PA, USA. network since the key residues are aromatic in nature. Molecular dynamics sim- In this study, we measured the contributions of the ionization of the heme pro- ulations of native BC1, natural ligand and inhibitor-bound BC1 homo-dimers in pionates to the reduction potentials of heme b and heme a (bis)N-methylimida- membrane were performed to investigate and compare the dynamics of those zole complexes in various low-dielectric constant conditions. Additionally, we key residues in their respective environments. In addition, molecular dynamics measured the effects of H-bond to the heme a formyl group on the reduction simulations reveals the transduction pathway induced by the binding of a ligand potential of the heme. The performed electrochemical measurements show or inhibitor at their sites. Electron tunneling calculations show that there is a that ionization of the heme propionates lead to the largest redox change in di- substantial correlated change of the electron transfer rates between different chloromethane with no electrolyte. The measured reduction potential changes redox pairs depending on the binding of natural ligands or inhibitors. for heme b and heme a were - 55 and 47 mV (510 mV) per ionized propi- onate, respectively. For heme a, the study demonstrates how the dielectric con- 3045-Pos Board B475 stant of the medium is important in the magnification of the apKa upon redox- Energetics of Lateral Membrane Proton Diffusion linked ionization of the heme propionates and their roles in the proton pump of Ewald Weichselbaum, Denis Knyazev, Peter Pohl. cytochrome c oxidase. Additionally, we carried out a detailed study on the H- Molecular and Membrane Biophysics, Institute of Biophysics, Linz, Austria. bonding properties of heme a model compounds (copper mono- and di-acetyl The diffusion of protons along biological membranes is vitally important for porphyrins) and the effects of the dielectric constant of the solvent on the cellular energetics. The weak dependence of both migration speed and span measured dissociation constant (Kd) between various H-bond donors and the on lipid composition suggests that proton migrate along water (Springer et copper porphyrins. Our measurements show that H-bonds between the copper al., 2011). However, it is unclear how to reconcile the apparently high proton porphyrins and TFA or phenol are significant in benzene and dichloromethane, affinity to the phase boundary (Zhang et al., 2012) with the poor proton accept- but extremely weak in the presence of acetonitrile. This study shows how the ability of water, i.e. with the low pK(¼0) value of water. Here we monitored the 604a Wednesday, February 11, 2015 diffusion of excess protons along the phosphatidylcholine lipid bilayer/water conformational heterogeneity. Generally, the introduction of the ε-Ade group interface at different temperatures. The kinetics of proton arrival from a distant leads to much faster excited state decay in the reduced state. To investigate spot of proton release to lipid-anchored fluorescent pH-sensitive dyes indicated this further, the evolution of the excited state was probed in the visible, near- that the in vitro Gibbs activation energy DGz for proton surface-to-bulk release UV, and UVB spectral regions to determine whether PET was responsible harbours only a minor enthalpic constituent. We observed that more than 2/3 of for the short-lived excited state. The significance of these results to the role DGz are entropic in origin, which explains the high proton affinity to mem- of the FAD adenine in DNA photolyase are discussed. branes in the absence of a potent proton acceptor. This work was supported by Grant P25981 from the Austrian Science Fund (FWF) to P.P. 3049-Pos Board B479 1. Springer, A., Hagen, V., Cherepanov, D.A., Antonenko, Y.N., and Pohl, P. High-Resolution Electronic Structure of the Primary Electron Acceptor A0 of Photosystem I (2011). Protons migrate along interfacial water without significant contribu- 1 2 2 3 tions from jumps between ionizable groups on the membrane surface. Proc. Stuart Smith , Jaya Tripathi , Sergey Milikisiyants , Sijie Hao , John H. Golbeck3, K.V. Lakshmi2. Natl. Acad. Sci. U. S. A. 108, 14461-14466. 1 2. Zhang, C., Knyazev, D.G., Vereshaga, Y.A., Ippoliti, E., Nguyen, T.H., Department of Physics & The Baruch ‘60 Center for Biochemical Solar Energy Research, Rensselaer Polytechnic Institute, Troy, NY, USA, Carloni, P., and Pohl, P. (2012). Water at hydrophobic interfaces delays proton 2 surface-to-bulk transfer and provides a pathway for lateral proton diffusion. Department of Chemisty & The Baruch ‘60 Center for Biochemical Solar Proc. Natl. Acad. Sci. U. S. A. 109, 9744-9749. Energy Research, Rensselaer Polytechnic Institute, Troy, NY, USA, 3Department of Biochemistry and Molecular Biology, Pennsylvania State 3046-Pos Board B476 University, University Park, PA, USA. Assessing the Protonation State and Dynamics of His37 in the Influenza M2 The Type I photosynthetic reaction center, Photosystem I, is an exquisitely tuned Proton Channel using Raman Spectroscopy protein complex comprised of multiple polypeptide subunits and protein-bound Michael D. Tentilucci, Matthew G. Romei, Casey H. Londergan. electron-transfer (ET) cofactors. Upon illumination, the special chlorophylls, Chemistry, Haverford College, Haverford, PA, USA. P700, are photoexcited which results in the rapid formation of the charge- þ - The influenza M2 protein is known to shuttle protons into the virion, facilitating separated state, P700 A0 . In order to prevent charge recombination, the electron replication and maturation. Histidine 37 is thought to provide ‘‘shuttling’’ ac- is transferred to the phylloquinone cofactor, A1, and subsequently to three four tivity to those protons, but the protonation states of the four His37 residues iron-four sulfur [4Fe4S] clusters, FA,FB, and FX, respectively. Through the ET in the tetrameric channel has not been definitively established. We use Raman reactions of PSI, the reducing equivalents that are required for the carbon fixation spectroscopy to collect the frequency of a C2-D probe group on His-37 in an reactions are generated and stored as NADPH or ‘biohydrogen’. The PSI reac- isotopically labeled version of the M2 transmembrane peptide. The C2-D fre- tion center displays pseudo-C2 symmetry, such that the ET cofactors, A0 and quency reports directly on the protonation state of selected His residues in a A1, are duplicated in what is termed the A- and B-branch. Although ET is bidi- way that other experiments cannot. The data are used to identify His37 proton- rectional along the A- and B-branch, it has been demonstrated that ET in the ation states in various pH conditions and to report directly on the activity and A-branch is highly preferred. This is thought to be due to the tuning of the dynamics of His37 with protons and water in its local environment. redox-potential of the ET cofactors by smart matrix effects from the surrounding protein environment. This research explores the electronic structure of the 3047-Pos Board B477 primary electron acceptor A0 in the A-branch (A0A) through the use of two- Effects of Laser Spot Sizes in Laser Driven Proton Therapy dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy Tung-Chang Liu1, Xi Shao1, Chuan-Sheng Liu1,2, Catherine Zhuang1,3, in tandem with density functional theory calculations. The application of 2D Bengt Eliasson1,4, Jyhpyng Wang5,6, Shih-Hung Chen6. HYSCORE spectroscopy allows for the identification of the 14N atoms and pro- 1 2 - University of Maryland, College Park, College Park, MD, USA, University tons that are magnetically interacting with the paramagnetic center of A0A . The of Macau, Taipa, Macao, 3Walt Whitman High School, Bethesda, MD, USA, 14N and 1H hyperfine parameters obtained here provide a direct measure of the 4 5 - Strathclyde University, Glasgow, United Kingdom, Academia Sinica, electron spin density distribution of A0 in the A-branch of PSI. These are then be Taipei, Taiwan, 6National Central University, Taoyuan, Taiwan. compared with simulations of the electronic structure of the A-branch of Photo- We present a numerical study of the effect of the laser spot size of a circularly system I in order to better understand the mechanism of electron transfer from polarized laser beam on the energy of quasi-monoenergetic protons suitable for P700 to the primary electron acceptor, A0A. particle cancer therapy in laser proton acceleration using a thin carbon- hydrogen foil. The energy dosage distributions of impinging the generated pro- 3050-Pos Board B480 ton beams into human tissues are also simulated, compared and analyzed. The Environmental Coupling and Population Dynamics in the PE545 used proton acceleration scheme is a combination of laser radiation pressure Light-Harvesting Complex 1 2 3 and shielded Coulomb repulsion due to the carbon ions. We observe that the Mortaza Aghtar , Johan Stru¨mpfer , Klaus Schulten , 1 spot size plays a crucial role in determining the net charge of the electron- Ulrich Kleinekatho¨fer . 1School of Engineering and Science, Jacobs University Bremen, Bremen, shielded carbon ion foil and consequently the efficiency of proton acceleration. 2 Using a laser pulse with fixed input energy and pulse length impinging on a Germany, 2Center for Biophysics and Computational Biology and Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA, carbon-hydrogen foil, a laser beam with smaller spot sizes can generate higher 3 energy but fewer quasi-monoenergetic protons. We studied the scaling of the Center for Biophysics and Computational Biology and Beckman Institute, proton energy with respect to the laser spot size and obtained an optimal University of Illinois at Urbana-Champaign, Urbana, IL, USA. spot size for maximum proton energy flux. Using the optimal spot size, we Experimentally long-lived quantum coherences have been found in the Fenna- can generate an 80 MeV quasi-monoenergetic proton beam containing more Matthews-Olson (FMO) complex of green sulfur bacteria as well as in the than a hundred million protons using a laser beam with power 250 TW and en- phycoerythrin 545 (PE545) photosynthetic antenna system of marine algae. ergy 10 J and a target made of 90% carbon and 10% hydrogen, capable of treat- In the latter system the long-lived coherences are clearly visible also at ambient ing tumor cells with depth up to 5 cm in human bodies. temperatures. A combination of classical molecular dynamics simulations, quantum chemistry and quantum dynamical calculations is employed to deter- 3048-Pos Board B478 mine the excitation transfer dynamics in PE545. To be able to described the Characterization of Excited State Etheno-Fad: A Probe of the Role of excitation transfer and dephasing phenomena the spectral density is a property Adenine in DNA Photolyase a key importance. To this end a time series of the vertical excitation energies of Kimberly Jacoby, Vijay R. Singh, Madhavan Narayanan, Robert J. Stanley. the individual pigments is determined. In a subsequent step. quantum dynam- Chemistry, Temple University, Philadelphia, PA, USA. ical simulations are performed using the earlier QM/MM calculations as input . Using femtosecond transient absorption spectroscopy , we have characterized Employing an ensemble-averaged classical path-based wave packet dynamics, flavin 1,N6 - ethenoadenine dinucleotide (ε-FAD), an analog of flavin adenine the excitation transfer dynamics between the different bilins in the PE545 com- dinucleotide (FAD), to understand the role of adenine in the photoinduced elec- plex is determined and analyzed. Furthermore, the nature of the environmental tron transfer (PET) reaction between reduced FAD (*FADH-) and thymine di- fluctuations determining the transfer dynamics is discussed. mers during its repair in DNA photolyase. The adenine in FAD may serve as a [1] M. Aghtar, J. Stru¨mpfer, C. Olbrich, K. Schulten, and U. Kleinekatho¨fer, J. virtual or real intermediate in the PET reaction. ε-FAD was used to modify the Phys. Chem. Lett. 5, 3131-3137 (2014) driving force between the adenine and its partners, providing data on parame- [2] M. Aghtar, J. Stru¨mpfer, C. Olbrich, K. Schulten, and U. Kleinekatho¨fer, J. ters and processes that dictate the kinetics and pathways of electron transport. Phys. Chem. B 117, 7157-7163 (2013). The neutral oxidized and the fully reduced anionic states of ε-FAD, FAD, and [3] C. Olbrich, T.L.C. Jansen, J. Liebers, M. Aghtar, J. Stru¨mpfer, K. Schul- flavin mononucleotide (FMN) in free solution exhibited multi-exponential ten, J. Knoester, and U. Kleinekatho¨fer, J. Phys. Chem. B 115, 8609-8621 decay kinetics that reflect their excited state quenching pathways and possible (2011). Wednesday, February 11, 2015 605a

[4] C. Olbrich, J. Stru¨mpfer, K. Schulten, and U. Kleinekatho¨fer, J. Phys. DNA is a unique nanoscale material that enables the design and synthesis of Chem. Lett. 2, 1771-1776 (2011). nanoscale structures of prescribed shape and functionality via programmable [5] C. Olbrich, J. Stru¨mpfer, K. Schulten, and U. Kleinekatho¨fer, J. Phys. self-assembly. Angstrom-level control over the location and orientation of pro- Chem. B 115, 758-764 (2011). grammed Watson-Crick basepairs offers the ability to scaffold and spatially organize optically active materials to yield novel optical and photonic proper- 3051-Pos Board B481 ties. Here, we present two distinct strategies for designing and synthesizing The Co-Assembly of Collagen-Mimetic Peptides and Natural Proteins DNA-based photonic materials, and emphasize the roles of computational 1,2 3 4 Kenneth N. McGuinness , Kathryn E. Drzewiecki , Michael J. Kopka , modeling in this process. First, we use computational modeling to design me- 4 4 3 2,5 Arpita A. Patel , Robert A. Niederman , David I. Shreiber , Vikas Nanda . chanically stiff nanoscale DNA ‘‘molds’’ that have user-specified three-dimen- 1Computational Biology and Molecular Biophysics, Rutgers, Piscataway, NJ, 2 sional cavity with a nucleating gold seed, which grows in solution to fill and USA, Center for Advanced Biotechnology and Medicine, Piscataway, NJ, replicate the cavity. We demonstrate the capability of producing nanoparticles USA, 3Biomedical Engineering, Rutgers, Piscataway, NJ, USA, 4Molecular 5 of various shapes and materials that exhibit plasmonic properties that are Biology and Biochemistry, Rutgers, Piscataway, NJ, USA, Biochemistry consistent with electromagnetism simulations. In silico design of stiff molds and Molecular Biology, Robert Wood Johnson Medical School, Rutgers of nearly arbitrary geometric cavities with resulting optical properties that University, Piscataway, NJ, USA. are prescribed a priori enable a property-by-design framework for producing We are developing synthetic peptides that non-covalently attach to natural inorganic particles with prescribed functional properties. Second, we use proteins, augmenting their properties and developing novel hybrid functional DNA to program the scaffolding of chromophores into complex three- materials. Using a set of hydrophobic-residue containing collagen-mimetic dimensional assemblies, enabling controlled energy transfer at the nanoscale. peptides that self-assemble into nanodiscs, we aim to encapsulate membrane This strategy is inspired by nature, where cells use organized assemblies of proteins, increase protein hydrogel hydrophobicity, and create novel materials chromophores to capture photons and funnel the resulting excitons toward using structural proteins. Preliminary results suggest nanodisc interactions with the reaction center where they are converted to chemical energy. In particular, the reaction center-light harvesting complex I (RC-LH1) could elucidate addi- the close-packing of chromophores results in the emergence of quantum coher- tional RC-LH1 structural and functional information, useful for the develop- ence that can strongly affect exciton transport across the structure. Using Fo¨r- ment of next generation solar cells. Furthermore, electron micrographs of ster energy transfer modeling, as well as a hybrid molecular dynamics and nanodiscs embedded in collagen type I (COL I) hydrogels provide evidence quantum mechanical approach, we demonstrate how emergent excitonic and for the enhancement of hydrophobic properties of COL I, and the potential light-harvesting properties of diverse DNA-dye assemblies can be elucidated. for sequestering hydrophobic molecules for drug delivery. Lastly, nanodisc induced assembly of the structural protein tropomyosin is explored with the 3054-Pos Board B484 intent of enhancing the structural properties of COL I hydrogels. EPR and X-Ray Spectroscopy Characterization of Reported Mono- 3052-Pos Board B482 Ruthenium Water Splitting Catalysts 1 2 3 4 Studying the Structural and Electronic Configurations during Photocata- Vatsal Purohit , Dooshaye Moonshiram , Lifen Yan , Igor Alperovich , Yulia Pushkar3. lytic Activation of O2 at a Diiron(II) Complex 1 1 2 2 Interdisplinary Life Sciences program, Purdue University, West Lafayette, Dooshaye Moonshiram , Ally Aukauloo , Frederic Avenier , 2 3 1 1 1 IN, USA, Argonne National Laboratory, Lemont, IL, USA, Physics, Purdue Steve Southworth , Carl Lehmann , Antonio Picon . 4 1Physics, Argonne National Laboratory, Lemont, IL, USA, 2Inorganic University, West Lafayette, IN, USA, Research Center for Nanoscale Chemistry Laboratory, Universite´ Paris-Sud 11, Orsay, France. Structure of Matter, South Federal University, West Lafayette, IN, USA. Several mono and carboxylate-bridged diiron nuclear enzymes are critical in acti- Technology to convert Solar energy into fuel is being developed to search for vating dioxygen in biological processes such as DNA synthesis, hydrocarbon alternate energy sources. This includes electrochemical cells based on artificial metabolism and cell proliferation1. Iron-containing enzymes such as cytochrome water splitting catalysts. By studying their water splitting mechanism we P450, peroxidases, catalases and methane monooxygenase (MMO) have been can hope to optimize the energy conversion process and also learn more natural water splitting complexes like Photosystem II. In this study, we analyzed two shown to activate dioxygen by using two electrons and protons to produce iron II 2þ reported single site Ruthenium based catalysts, [Ru (bpy)(tpy)H2O] and (IV) oxo intermediates2. The remarkable efficiency of these enzymes is attributed II þ II 2þ to formation of iron(IV) cations which serve as active oxidants in enzymatic re- [Ru (bpy)(tpy)Cl] . [Ru (bpy)(tpy)H2O] is a widely studied water splitting V ¼ 3þ actions and can attack C-H bonds of a wide range of hydrocarbon substrates. It catalyst for which [Ru (bpy)(tpy) O] has been detected as a rate limiting intermediate. However, using EPR, we see that this intermediate is not present is widely postulated that ferryl-oxo species are key intermediates in the mecha- IV nism of cytochrome P450 and MMO. However due to the complexity of protein when the sample is measured with Ce in D2O or HNO3. Using, Ru-K edge ¼ IV environments in biological iron enzymatic systems, monitoring the structural EXAFs, we also see that the Ru O distance measured after adding Ce is IV ¼ 2þ changes occurring during dioxygen activation is a complex undertaking. more consistent with its assignment as a [Ru (bpy)(tpy) O] intermediate V¼ This project serves to study the light driven activation of a well characterized and not with Ru O. We also found that [RuII(bpy)(tpy)Cl]þ is not a catalyst and that its oxidation past artificial analogue of an diiron MMO enzyme, (m-peroxo) (m-carboxylato) diir- III þ Ru is impeded by a lack of proton coupled electron transfer. Using L-edge and on(III) complex, [Fe2(m-O2)(N-EtHPTB)(m-PhCO2)]2 in a chromophore/ II diiron complex assembled unit. Such types of assemblies provide new avenues K-edge XANES spectra, we show that the edge position of the [Ru (bpy)(tpy) Cl]þ spectra is more consistent with the presence of [RuIII(bpy)(tpy)Cl]þ. for study of catalytic reaction mechanisms. They are promising examples of II þ artificial molecular systems leading to dioxygen activation as visible light is EXAFS analysis of [Ru (bpy)(tpy)Cl] also show shortening of the Ru-Cl ˚ ˚ II III efficiently absorbed at the chromophore, and light energy is in turn converted bond (2.41 A to 2.34 A), implying a transition from Ru to Ru . This is further into a chemical potential via an electron relay through charge accumulation tested in an Oxygen evolution assay where the sample is incubated in H2O for different time periods. We show that samples that are not incubated for long processes at the iron metal center. Formation of high valent iron peroxo species - times show no activity, hence implying that a Cl to H2O ligand exchange is have been shown by UV-Vis, EPR and Resonance Raman spectroscopy as well II þ as X-ray spectroscopic analysis. XANES and EXAFS revealed formation of an necessary for [Ru (bpy)(tpy)Cl] to show catalytic activity. These studies iron peroxo and oxo species with shorter bond distances and different coordi- give us crucial insight into the water splitting mechanism of these catalysts. nation numbers. (1) Do, L. H.; Hayashi, T.; Moenne-Loccoz, P.; Lippard, S. J. J.Am.Chem.Soc. 3055-Pos Board B485 2010, 132, 1273. Photosynthesis in a Single Protein 1 1 2 (2) Lawrence Que, J.; Y.N.Ho, R. Chem.Rev 1996, 96, 2607. Eskil M. Andersen , Ronald L. Koder , Andrew C. Mutter . 1Department of Physics, City College of New York, New York, NY, USA, 3053-Pos Board B483 2Department of Physics, Harvard University, New York, NY, USA. Programming Nanophotonic Materials with DNA We report the optimization of a self-assembling artificial protein reaction center E´ tienne Boulais1, Wei Sun2, Nicolas Sawaya3, Yera Hakobyan1, capable of nearly quantitative charge separation with microsecond lifetimes us- Weili Wang2, Amy Guan2, Keyao Pan1, Alan Aspuru-Guzik3, Peng Yin2, ing a phthalocyanine primary donor as well as a variety of natural and synthetic Mark Bathe1. electron donor and acceptor cofactors. We will further discuss the fusion of this 1Department of Biological Engineering, Massachuetts Institute of protein domain into a chimera with the diflavin domain of cytochrome P450, Technology, Cambridge, MA, USA, 2Wyss Institute, Harvard University, with the intention of creating a single-chain protein capable of light-powered Boston, MA, USA, 3Department of Chemistry and Chemical Biology, NADH production. The goal is a single gene which can make any micro- Harvard University, Cambridge, MA, USA. organism photosynthetic. 606a Wednesday, February 11, 2015

Mitochondria in Cell Life and Death breaking of the outer membrane. Isotonicity was restored by adding a hypertonic solution. A potassium selective current was recorded with a mean conductance of 280 5 2 pS in symmetrical 150 mM KCl solution. The channel was activated 3056-Pos Board B486 2þ New Fluorescence Probes for Visualizing Cell Structures and Function by Ca at micromolar concentrations and inhibited irreversibly by paxilline, an Yuning Hong. selective inhibitor of the BKCa channel. The substances known to modulate School of Chemistry, University of Melbourne, Parkville, Australia. BKCa channel activity were found to influence the bioenergetics of mitochon- Organic fluorogens with aggregation-induced emission (AIE) characteristics dria, isolated from human dermal fibroblast cells. In isolated mitochondria, 10 have demonstrated their potential to be ideal candidates for live cell imaging. mM NS1619 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. This effect was blocked by 20 mM paxilline. Opposite to conventional organic dyes, the AIE luminogens are non- 2þ luminescent when molecularly dissolved but highly emissive upon aggregation. Our findings indicate presence of the large conductance Ca -regulated potas- As small molecules, the AIE luminogens normally enter cells through diffusion, sium channels in the inner mitochondrial membranes of human fibroblast. accumulate in the target location, and generate light emission. Inherently, they This study was supported by a grant MERIS PBS1/B8/1/2012 from the possess large Stokes shift (> 100 nm) with appreciable brightness and they National Centre of Research and Development. are resistant to photo-bleaching and blinking, owing to the formation of aggre- 3059-Pos Board B489 gates inside the cells. In addition, they are structurally simple and synthetically Identification of the ATP Regulated Potassium Channel in Mitochondria of accessible: the excitation/emission wavelengths as well as the functionalities can Fibroblast Cells be fine-tuned via structural modification. In this study, a series of AIE dyes have Adam Szewczyk1, Piotr Bednarczyk1,2, Anna Kicinska 3, been constructed and their applications for specific imaging of different organ- Wies1awa Jarmuszkiewicz3. elles, tracking dynamics of mitochondria as well as sensing intracellular environ- 1Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental ment in physiological and pathological conditions will be introduced. Biology, Warsaw, Poland, 2Department of Biophysics, Warsaw University of 3057-Pos Board B487 Life Sciences - SGGW, Warsaw, Poland, 3Laboratory of Bioenergetics, Monitoring Mitochondrial Membrane Potential with Mitoview 633: A New Adam Mickiewicz University, Poznan, Poland. Molecular Probe The transport of potassium ions into the mitochondrial matrix can trigger the Jarod Benowitz1,2, Qince Li2, KahYong Goh2, Chih-Chang Wei2, protection of the injured cardiac and neuronal tissues. A growing body of evi- Lufang Zhou2,3. dence suggests that ischemic preconditioning, short episodes of ischemia that 1Physics, Utah State University, Logan, UT, USA, 2Cardiovascular Disease, increase tissue tolerance to lethal insults, could be mimicked by the administra- University of Alabama at Birmingham, Birmingham, AL, USA, 3Biomedical tion of openers of mitochondrial potassium channels. Hence, the identification Engineering, University of Alabama at Birmingham, Birmingham, AL, USA. of ion channels present in the inner mitochondrial membrane of fibroblasts is Background: Noninvasive mitochondrial inner membrane potential (DJm) important in distinguishing possible protective mechanisms in these cells. measurement has been largely constrained to the use of fluorescent probes. In our research, inner membrane mitochondrial ion channels of the human These probes accumulate in the mitochondrial inner membrane according to fibroblast cell line were investigated using a patch-clamp and biochemical tech- the Nernst equation. In this manner, DJm can be measured indirectly by the niques. The single channel activity of mitochondrial potassium channels were intensity of the fluorescent dye. investigated using a patch-clamp technique. In the inner mitochondrial mem- Objective: MitoView 633 is a new fluorescent probe in the far-red spectrum, brane of fibroblast we detected ATP-regulated potassium channel (mitoKATP which has great potentials in bioenergetics studies. However, its application channel) with mean conductance equal to 100 5 3 pS. The activity of this in live cell imaging has not been well characterized. We aimed to determine channel was inhibited by complex of ATP/Mg2þ and activated by the potas- the spectrum of MitoView using a spectrophotometer and characterized its dy- sium channel opener BMS191095 and diazoxide. The influence of substances namics in cardiomyocyte during live cell imaging. modulating ATP-regulated potassium channel activity on the bioenergetics, ox- Method: H9C2 cells or adult rat cardiomyocytes were stained with MitoView ygen consumption and membrane potential, of isolated human dermal fibro- or TMRM. The mitochondrial uncoupler FCCP was used to induce mitochon- blast mitochondria was also studied. drial depolarization and the change of DJm was recorded using a confocal mi- Our findings indicate presence of the ATP-regulated potassium channels in the croscope. Imagines were processed using ImageJ and the dynamics of inner mitochondrial membranes of human fibroblast. MitoView and TMRM were compared. In some experiments, cells were co- This study was supported by a grant MERIS PBS1/B8/1/2012 from the Na- stained with MitoSOX for simultaneous ROS and DJm recording. tional Centre of Research and Development. Results: Spectrum analysis showed that MitoView emission could be detected at 3060-Pos Board B490 660þ50 nm. In addition, MitoView perfectly located in mitochondrial matrix, as Suppression of Dynamin-Related Protein 1 by Eicosapentaenoic Acid Ame- demonstrated by its colocalization with Mito-eYFP or TMRM in H9C2 cells and liorates Palmitate-Induced Lipotoxicity in Differenciated H9C2 Myocytes rat cardiomyocytes. Furthermore, the photobleaching of MitoView was insig- Atsushi Sakamoto, Masao Saotome, Terumori Satoh, Daishi Nonaka, nificant and comparable to that of TMRM and the FCCP-induced decay of Tsuyoshi Urushida, Hideki Katoh, Hiroshi Satoh, Hideharu Hayashi. MitoView fluorescence was faster than TMRM, indicating its superior sensi- Internal Medicine 3, Hamamatsu University School of Medicine, tivity to DJm. Finally, MitoView could be co-stained with MitoSOX, allowing Hamamatsu, Japan. for simultaneous measurement of ROS and DJm in live cell imaging. Objective: Although previous clinical investigations have reported many pref- Conclusion: We showed that MitoView is very sensitive to DJm. Due to its erable effects of eicosapentaenoic acid (EPA; n-3 polyunsaturated fatty acid) minor photobleaching effect, high sensitivity to DJm, and far-red emission against cardiovascular disease, the precise mechanisms are not fully clarified. spectrum, MitoView could be an idea molecular probe to monitor DJm alone Since aberrant mitochondrial dynamics plays key roles for the pathogenesis or together with other indicators in live cell imaging. of cardiovascular disease, we investigated the relationship between EPA- 3058-Pos Board B488 mediated cardiac protection and mitochondrial dynamics. Biophysical and Biochemical Properties of the Large Conductance Methods and Results: When differentiated H9c2 myocytes were exposed to Potassium Channel in Fibroblast Mitochondria palmitate (PAL; 400 mM, saturated fatty acid) for 24 hours, the PAL-treated Piotr Bednarczyk1,2, Anna Kicinska 3, Wies1awa Jarmuszkiewicz3, myocytes exhibited depolarized mitochondrial membrane potential (DJm: Adam Szewczyk2. measured with JC10 ratio; 2.150.4 vs. 4.550.3 of control, P<0.01) and 1Department of Biophysics, Warsaw University of Life Sciences – SGGW, activated caspase3/7 (measured with luminescent assay; 4.150.7 fold increase Warsaw, Poland, 2Laboratory of Intracellular Ion Channels, Nencki Institute from control, P<0.01), suggesting the PAL-induced lipotoxicity. The of Experimental Biology, Warsaw, Poland, 3Laboratory of Bioenergetics, PAL-treated myocytes also showed dynamin-related protein 1 (Drp1) up- Adam Mickiewicz University, Poznan, Poland. regulation and fragmented mitochondria (75.355.0 % of cells vs. 9.253.2 Potassium channels have been found in the inner mitochondrial membranes of % of control, P<0.01), indicating aberrant mitochondrial dynamics by PAL. various cells. The activation of these channels is cytoprotective. Hence, the When the PAL-treated myocytes were co-incubated with EPA (50 mM), the identification of ion channels present in the inner mitochondrial membrane caspase3/7 activation by PAL (1.550.3 fold increase vs. PAL, P<0.01) was of fibroblast cells is important in distinguishing possible protective mechanisms attenuated and the DJm depolarization by PAL was restored (2.950.2 vs. in these cells. PAL, P<0.01). EPA also suppressed the PAL-induced Drp1 expression and In our study single channel activity of a large conductance Ca2þ-regulated mitochondrial fragmentation (37.656.4% vs. PAL, P<0.01). In addition, potassium channel was measured by patch-clamp of mitoplasts isolated from EPA alone reduced the Drp1 expression and exhibited elongated mitochondria. fibroblast cell line. Mitoplast were prepared by addition to a hypotonic solution Conclusion: We conclude that EPA ameliorates the PAL-induced lipotoxicity causing unfolding of the cristae of the inner membrane and consequently by the regulation of mitochondrial dynamics through Drp1. Wednesday, February 11, 2015 607a

3061-Pos Board B491 tions, while the ANT-CK-VDAC and ANT-VDAC-HK complexes allow the Cardiolipin Reorganization and Phase Transition Induced by Dynamin- application of a part of the sum of the corresponding ‘‘kinase voltage’’ and Related Protein 1 Facilitates Mitochondrial Membrane Fission the inner membrane potential to the outer membrane. The developed computa- Natalia Stepanyants, Patrick Macdonald, Rajesh Ramachandran. tional models demonstrate a high probability of generation of the Physiology and Biophysics, Case Western Reserve University, Cleveland, metabolically-dependent OMP with magnitudes high enough to close free OH, USA. VDACs. We suggest that in case of cardiac cells, the CK-VDAC-mediated Cardiolipin (CL) is a unique, dimeric phospholipid essential for mitochondrial channeling of mitochondrial energy, earlier postulated by Saks and Wallimann, dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a member of leads to the metabolically-dependent generation of a high OMP during systole, the dynamin superfamily of large GTPases, maintains the balance of mitochon- thus causing electrical closure of free VDACs and essentially avoiding the drial division and fusion by rapidly catalyzing mitochondrial fission. Although mitochondrial ATP usage by non-contraction system consumers. In case of recent studies have indicated a role for CL in stimulating Drp1 self-assembly as cancer cells, that have a very high percentage of hexokinase attached to mito- well as GTPase activity on the mitochondrial surface, the exact mechanism by chondria, the electrical closure of free VDACs by OMP allows the preferential which CL functions in membrane fission remains unclear. Here we use a variety VDAC-HK-mediated channeling of the mitochondrial ATP to initiate the aer- of fluorescence spectroscopic and imaging approaches, together with model obic glycolysis by an anti-turbo mode. (Research grants #111852128625 and membranes, to demonstrate that Drp1 and CL function cooperatively in effect- #520154531565, Colciencias). ing membrane fission in three distinct steps: (i) Drp1’s preferential association with unconstrained, fluid-phase CL molecules located at a high spatial density 3064-Pos Board B494 in the membrane bilayer, (ii) CL’s reorganization in concert with Drp1 self- Alpha-Synuclein Blocks VDAC Suggesting Mechanism of Mitochondrial assembly, and (iii) CL’s rapid phase transition from a lamellar, bilayer structure Regulation and Toxicity in Parkinson Disease 1,2 2 2,3 to an inverted hexagonal, non-bilayer configuration in the presence of Drp1 and Philip A. Gurnev , Tatiana K. Rostovtseva , David P. Hoogerheide , Olga Protchenko4, Thai Leong Yap5, Jennifer C. Lee5, Sergey M. Bezrukov2. GTP, resulting in the creation of localized membrane constrictions that are 1 2 primed for fission. We propose that Drp1 thus catalyzes mitochondrial division. Physics Department, UMass-Amherst, Amherst, MA, USA, Section on Molecular Transport, PPB/NICHD/NIH, Bethesda, MD, USA, 3Center for 3062-Pos Board B492 Neutron Research, NIST, Gaithersburg, MD, USA, 4Liver Diseases Branch, VDAC3 Forms Typical Voltage-Gated, Anion-Selective, and Tubulin- NIDDK/NIH, Bethesda, MD, USA, 5Laboratory of Protein Conformation and Sensitive Channels Dynamics, NHLBI/NIH, Bethesda, MD, USA. Oscar Teijido Hermida1, Adam J. Kuszak2, Susan K. Buchanan3, Alpha-synuclein (a-syn), an intrinsically disordered neuronal protein, is impli- Sergey M. Bezrukov1, Tatiana K. Rostovtseva1. cated in the etiology of Parkinson disease (PD) and a number of other neurode- 1Program on Physical Biology, NICHD, NICHD, National Institutes of generative dementias. Though recent research demonstrates the involvement of Health (NIH), Bethesda, MD, USA, 2NIDDK, National Institutes of Health a-syn in a variety of mitochondrial dysfunction in neurodegeneration, the mo- (NIH), Bethesda, MD, USA, 3Program on Physical Biology, NICHD, lecular mechanism of a-syn toxicity and its effect on neuronal mitochondria NIDDK, National Institutes of Health (NIH), Bethesda, MD, USA. remain vague. We demonstrate a functional interaction between a-syn and The Voltage Dependent Anion Channel (VDAC) forms the major pathway for the voltage-dependent anion channel (VDAC), the major conduit for ATP metabolites across the mitochondrial outer membrane (MOM). Mammalian and other bioenergetics metabolites in the mitochondrial outer membrane. mitochondria have three isoforms of this protein: VDAC1, VDAC2, and We found that at nanomolar concentrations, the full-length a-syn, its 45 amino VDAC3 which may play different roles in the regulation of mitochondrial func- acid C-terminal truncated mutant, as well as b- and a-synuclein isoforms tions. VDAC1 is the most studied isoform, since it is the major protein of the induce reversible and highly voltage-dependent blockage of VDAC reconsti- MOM in most cells, followed by VDAC2. Both, VDAC1 and VDAC2 recon- tuted into planar lipid bilayers. Binding parameters varied with each isoform, stituted into planar membranes form voltage-dependent anion-selective chan- revealing the key role of the negatively charged C-terminal of synucleins in nels whose conductance is modulated by dimeric tubulin. As counterpart, the blocking a positively charged pore of VDAC. Synuclein-blocked states of poor channel-forming ability of VDAC3 together with its low proficiency in VDAC differ in ionic selectivity, implying the existence of several conforma- restoring normal growth in pore-less yeast led to hypothesize that this isoform tion states of synuclein molecules in the channel pore. We propose a model of does not form channels and could be just a modulator of the other two isoforms. a-syn interaction with VDAC, in which the negatively charged C-terminus of We developed an improved protocol of purification and refolding of mouse re- a-syn enters the net-positive channel pore, providing a steric block for ATP combinant VDAC3 that allowed us to fully characterize channels formed by flux. Experiments with a yeast strain deficient in VDAC1 demonstrate that VDAC3 reconstituted in planar lipid membranes and to compare its channel a-syn toxicity in yeast depends on VDAC, revealing a-syn interaction with properties with those of VDAC1 and VDAC2. We observed the typical the channel in living cells. Thus, our findings show the long-sought physiolog- VDAC channel behavior of VDAC3, such as high conductive open state ical and pathophysiological roles for monomeric a-syn, which reconcile previ- (3.5-4.1 nS in 1M KCl) and multiple low conductive states at potentials R ous observations of various synuclein effects on mitochondrial bioenergetics. - þ 540mV, selectivity for anions with PCl /PK ¼ 1.7, and typical voltage- gating. This suggests a structural homology of VDAC3 with the other two iso- 3065-Pos Board B495 forms. Interestingly, the most distinct characteristic of VDAC3 is its signifi- Mitochondrial DNA: The Heart of the Matter 1 1 1 1 cantly lower sensitivity to the blockage by tubulin, which would lead to an Meagan McManus , Martin Picard , Alessia Angelin , Prasanth Potluri , Jagat Narula2, Douglas Wallace1. increased permeability of this channel to ATP and other metabolites. This 1 2 might explain why the down-regulation of VDAC3 expression among the three CHOP/UPENN, Philadelphia,, PA, USA, Mt Sinai, New York, NY, USA. VDAC isoforms in some cancer cells causes the most drastic decrease in their Cardiac conduction defects and cardiomyopathy are commonly reported in pa- mitochondrial membrane potential, or why VDAC3 is predominantly ex- tients with inborn errors of metabolism harboring pathogenic mutations in pressed in the short-term high energy demanding cells, such as spermatozoa. either mitochondrial or nuclear DNA (mtDNA and nDNA, respectively). We hypothesized that the pathophysiology of inborn errors of metabolism varies 3063-Pos Board B493 due to synergistic heterozygosity between the two genomes. We examined a Channeling of Mitochondrial Energy in Cardiac and Cancer Cells by the 13-generation Mennonite pedigree with autosomal recessive cardiomyopathy Metabolically-Dependent Outer Membrane Potential due a mutation in the adenine nucleotide translocator-1 (ANT1). Substantial Victor V. Lemeshko. variability in the progression of heart disease segregated with maternal lineage, Escuela de Fisica, Universidad Nacional de Colombia, Medellin, Colombia. and the severity of cardiomyopathy correlated with the mtDNA haplogroups Mitochondria are the main source of energy in eukaryotic non-proliferating aer- (Strauss, et al 2013). To determine the causative nature of this correlation, obic cells. In cancer cells, significant part of the energy metabolism is sup- we examined the influence of inherited mtDNA mutations on ANT1- ported by the aerobic glycolysis. Oxidative phosphorylation metabolites cardiomyopathy in the mouse. We introduced homoplasmic mtDNA ND6 or cross the mitochondrial outer membrane through the voltage dependent anion COI missense mutations into the mouse female germ line, generating mice channel (VDAC). The possibility of the voltage-gating functioning of VDAC with complex I or IV deficiency, respectively, and analyzed Ant1-dependent under physiological conditions and proposed mechanisms of generation of cardiomyopathy on the different mtDNA backgrounds. On wt mtDNA back- the outer membrane potential (OMP) require further study. In this work, we ground, the Ant1-/- mice developed a distinctive concentric dilated cardiomy- present a computational thermodynamic analysis of a possible generation of opathy, characterized by substantial myocardial hypertrophy and ventricular OMP by the creatine kinase(CK)-VDAC and VDAC-hexokinase(HK) com- dilation. Both COI and ND6 mtDNA mutations accelerated Ant1-/- age- plexes, as well as by their intermembrane complexes with the adenine nucleo- dependent cardiomyopathy, as evidenced by ultrastructrural abnormalities, bio- tide translocator (ANT). The CK-VDAC and VDAC-HK complexes function energetic defects, mtROS production, sensitized mitochondrial permeability as direct steady-state voltage generators, using the free energy of kinase reac- transition, increased mtDNA damage, and heart failure, which ultimately 608a Wednesday, February 11, 2015 attenuated the lifespan of Ant1-deficient mice. Our results prove mtDNA dic- 3068-Pos Board B498 tates the penetrance of age-related cardiomyopathy and mammalian lifespan. EPR Data Support the Existence of a Symmetric BH3-in-Groove Homo- Therefore, therapeutics that most effectively preserve mitochondrial DNA dimer in Oligomeric BAK and bioenergetics will provide the most promise for healthy aging. Tirtha Mandal, Kyoung Joon Oh. Biochemistry and Molecular Biology, Rosalind Franklin University of 3066-Pos Board B496 Medicine and Science, North Chicago, IL, USA. Activation of the Mitochondrial Permeability Transition Pore Leads to the The BAX or BAK oligomeric pore formation in the mitochondrial outer membrane Increase in Amount of C-Subunit of ATP Synthase Associated with Channel- is a critical step in apoptosis, yet their structures are not clearly understood. Czabotar Forming Complex of Polyhydroxybutyrate and Inorganic Polyphosphate et al. (Cell 2013, 152, 519) reported a crystal structure of a water-soluble tetramer 1 1 1 Pia A. Elustondo , Alexander Negoda , Alejandro M. Cohen , of the BAX fragment (helices a2-a5) fused to the green-fluorescent protein (GFP), 2 Evgeny Pavlov . in which the a2-a3 extended helices and the a5 helix, respectively, were juxtaposed 1Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada, 2 to each other, in an anti-parallel orientation, forming a symmetric ‘‘BH3-in-groove Department of Basic Sciences, College of Dentistry, New York Universty, homodimer (BGH).’’ We have constructed a GFP-BAK fusion protein using the New York, NY, USA. a2-a5 helices of mouse BAK, designated as GFP-BAKa2-a5, which also forms Low permeability of the mitochondrial inner membrane is critical for maintain- a soluble tetramer. To determine whether the BGH exists in the BAK oligomers ing the mitochondrial electrochemical potential - the driving force for ATP pro- in the membrane or not, we spin labeled the C-terminal hexahistidine-tagged solu- duction. Acute stress conditions, lead to the increase in the mitochondrial inner ble form of mouse BAK (helices a1-a8) at residues 84, 122, 128 and 135 and the membrane permeability due to the opening of the permeability transition pore corresponding residues in GFP-BAKa2-a5. We then compared the continuous (PTP). PTP allows free movement of ions and small molecules leading to mito- wave (CW) EPR spectra of the spin-labeled residues from the tetrameric GFP- chondrial depolarization, ATP depletion and cell death. Recent studies suggest BAKa2-a5 with those from the oligomeric BAK in membrane. Spin labeled resi- that C-subunit of the mitochondrial ATP synthase plays a central role in PTP. due 122R1, located in the loop interconnecting helices a4anda5 in the homology Previous work in our laboratory showed that mitochondria contain non-protein model of the GFP-BAKa2-a5 tetramer, displayed a mobile lineshape. The corre- complex composed of polyhydroxybutyrate, inorganic polyphosphate and cal- sponding residue in the oligomeric BAK also had a remarkably similar lineshape, cium that forms an ion channel with properties resembling PTP. Here we indicating that the two residues are in similar structural environments. Residues explore the possibility of interactions between these non-protein components 84R1, 128R1 and 135R1, located at the anti-parallel helical interfaces in the and C-subunit during the induction of PTP. BGH also had remarkably similar immobile lineshapes both in the GFP-BAKa2- To induce PTP,isolated energized mitochondria were treated with calcium. a5 tetramer and in the oligomeric BAK in membrane, further strengthening the Control mitochondria were treated with calcium either in the presence of ruthe- above conclusion. The intra-dimer distances between 84R1 spin label pairs in the nium red, inhibitor of calcium uptake or Cyclosporin A, inhibitor of PTP. This GFP-BAKa2-a5 and the oligomeric BAK, determined by the double electron was followed by a water-free chloroform extraction of channel forming fraction electron resonance (DEER) method, also support this interpretation. of PTP. Components of the extract were analyzed using immunoblot analysis. We found significantly increased amount of C-subunit associated with channel 3069-Pos Board B499 forming fraction extracted from mitochondria with activated PTP. In contrast, Modulation of Membrane Interactions of Anti-Apoptotic Regulator Bcl-xL C-subunit was not detectable in the extract when Ruthenium Red was present by Lipids and significantly decreased in the presence of Cyclosporin A or in the absence Mauricio Vargas-Uribe, Mykola V. Rodnin, Alexey S. Ladokhin. of calcium. Biochemistry and Molecular Biology, KUMC, Kansas City, KS, USA. These results show that C-subunit is likely an interacting partner of the pore- The Bcl-2 family of proteins (e.g., pro-apoptotic Bax and anti-apoptotic Bcl-xL) forming complex of polyphosphate, calcium and polyhydroxybutyrate. We hy- regulates the mitochondrial outer membrane permeabilization during the early pothesize that fully functional PTP requires calcium-induced formation of the stages of apoptosis. The prevalent Embedded Together Model of Bcl-2 action pore made of both the complexed polymers and the c-subunit of ATP synthase suggests that the membrane environment is critical for their proper functional interactions, consistent with the increasing evidence of lipids being involved 3067-Pos Board B497 in the regulation of apoptotic response. In this study, we apply a collection of Small-Molecule PKD Inhibitor Prevents Mitochondrial Fragmentation fluorescence-based methods to investigate the effect of various lipids on the and Dysfunction during Gq-Protein Coupled Receptor Stimulation in Car- pH-triggered membrane interactions of Bcl-xL. The initial membrane association diac Cells was studied using a FRET assay with donor-labeled Bcl-xL and acceptor-labeled Bong Sook Jhun, Xiaole Xu, Jyotsna Mishra, Stephen Hurst, Jin O-Uchi, vesicles, while the insertion/refolding of Bcl-xL into the membrane was moni- Shey-Shing Sheu. tored using the environment-sensitive probe NBD selectively attached in the mid- Thomas Jefferson University, Philadelphia, PA, USA. dle of hydrophobic helix a6. Our results demonstrate that the lipid composition Regulation of mitochondrial morphology and dynamics is crucial for the main- affects the pH-dependence of both initial membrane association and subsequent tenance of various cellular functions in cardiomyocytes. Abnormal mito- insertion/refolding of Bcl-xL. We found that a linear correlation exists between chondrial morphologies concomitant with mitochondrial dysfunction are the membrane surface potential created by anionic lipids and the pKa of mem- frequently observed both in human heart failure (HF) and in animal HF models. brane binding, suggesting that the initial step is controlled by an electrostatic However, it is still unclear which cardiac signaling pathways regulate mitochon- mechanism. The effect of lipids on the membrane insertion/refolding step is drial morphology and function under pathophysiological conditions. Recent more complex and appears to be influenced by the size of the lipid headgroup. reports suggest that Gq-protein coupled receptor (GqPCR) signaling pathways The kinetics of both the membrane association and membrane insertion/refolding are critical for the development and progression of HF. Therefore, we hypoth- is affected by the presence of non-bilayer forming lipids commonly found in esize that GqPCR stimulation induces mitochondrial fragmentation and dysfunc- mitochondria. While the presence of phosphatidylethanolamine accelerated the tion, which initiates cardiomyocyte death. We found that protein kinase D process, addition of lysophosphatidylcholine had the opposite effect, suggesting (PKD) activated by GqPCR signaling was translocated to outer mitochondrial that mechanical properties of the bilayer also play a role. Taken together our membrane (OMM) observed by Western blot analysis of cytosolic and results indicate that lipids can modulate the membrane interactions of Bcl-xL mitochondria-enriched fractionated proteins and by live cell imaging of fluores- in multiple ways, providing an additional regulatory mechanism that ensures cence resonance energy transfer (FRET). We also found that GqPCR-mediated proper control of a complex cascade of apoptotic reactions leading to cell death PKD activation induced mitochondrial fragmentation, leading to increased or survival. NIHGM-069783, Fulbright-CONICYT, BRTP. reactive oxygen species (ROS) generation as well as increased mitochondrial permeability transition pore (mPTP) opening, which initiates apoptotic 3070-Pos Board B500 signaling activation and cardiomyocyte death. These morphological and func- Binding of Pro-Apoptotic Protein Bax to Cytoprotective UDCA and TUDCA tional changes in cardiac mitochondria were mediated via PKD-dependent phos- Taˆnia Sousa1, Ana Coutinho2, Soojay Banerjee3, Rui Castro4, phorylation of mitochondrial fission protein, Dynamin-Like Protein 1 (DLP1) at Richard Youle3, Cecı´lia Rodrigues4, Manuel Prieto1, Fa´bio Fernandes1. S637. Moreover, pretreatment with a novel potent PKD inhibitor CRT0066101 1Centro de Quı´mica-Fı´sica Molecular and Institute of Nanoscience and effectively inhibited GqPCR-mediated PKD translocation to OMM, DLP1 Nanotechnology, Instituto Superior Tecnico, Universidade de Lisboa, phosphorylation at S637, mitochondrial fragmentation, ROS generation and Lisbon, Portugal, 2Dep. Quı´mica e Bioquı´mica, Faculdade de Cieˆncias da 3 mPTP activation. In conclusion, we demonstrate that GqPCR stimulation Universidade de Lisboa, Ins, Lisbon, Portugal, NINDS, National Institutes induces mitochondrial fragmentation and dysfunction through PKD-dependent of Health, Bethesda, MD, USA, 4Faculdade de Farma´cia da Universidade de phosphorylation of DLP1 at S637, which likely contributes to cardiomyocyte Lisboa, Lisbon, Portugal. injury. Thus, small-molecule PKD inhibitor may become a novel and potent Hydrophobic bile acids, such as deoxycholic acid (DCA), strongly induce therapeutic for preventing cardiac cell injury and death during HF. apoptosis in both hepatic and non-hepatic cells while hydrophilic bile acids, Wednesday, February 11, 2015 609a like ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA), tion, overexpression of these MCU-YFs failed to increase mitochondrial Ca2þ are cytoprotective and inhibit cell death. The mechanisms associated with these uptake in response to cytosolic Ca2þ elevation by thapsigargin, whereas wild- distinct effects are not entirely clear. However, the effect of hydrophilic bile type MCU transfection dramatically accelerates mitochondrial Ca2þ uptake acids seems to be related with the blockage of a series of processes that compared to non-transfected cells. In summary, MCU contains Pyk2-specific converge on mitochondrial damage. Bax is a pro-apoptotic protein that belongs phosphorylation site(s) and Pyk2-dependent tyrosine phosphorylation of MCU to the superfamily of the Bcl-2 proteins and is involved in mitochondrial pore can modulate its channel functions and regulate mitochondrial Ca2þ uptake. formation. Submicellar concentrations of cytoprotective bile acids have been 3073-Pos Board B503 shown to modulate Bax concentration in mitochondria, suggesting that these 2D molecules may interact directly with the protein. In this study, our objective Cardioprotective Roles of Neuronal Ca Sensor-1 during Stress Tomoe Y. Nakamura-Nishitani1, Shu Nakao1, Shigeo Wakabayashi2. was to evaluate the affinity of bile acids to recombinant Bax protein, making 1 2 use of fluorescence spectroscopy (FRET and fluorescence anisotropy), as Molecular Physiology, Natl.Cer.Cardiovasc.Ctr., Suita, Japan, Cardiac Physiology, Natl.Cer.Cardiovasc.CtrWakabayshi, Suita, Japan. well as Fluorescence Correlation Spectroscopy (FCS). Our results show that 2þ the cytoprotective bile acids UDCA and TUDCA associate with recombinant Dysregulation of Ca homeostasis in cardiomyocytes often results in heart fail- ure. Identifying molecular targets that regulate cardiomyocyte survival is of ther- Bax protein with high affinity, while the cytotoxic bile acid DCA only seems 2þ 2þ to be able to adsorb to the protein with much lower affinity. Notably, the bind- apeutic importance. Neuronal Ca -sensor-1 (NCS-1) is an EF-hand Ca - binding protein, which is important for excitable cell functions. We previously ing site for UDCA seems to be located in a hydrophobic pocket of the protein. / This interaction could be responsible for the disruption of Bax translocation to found that NCS-1-deficient (Ncs1 ) mice had excess neonatal mortality the mitochondrial outer membrane in the presence of UDCA and/or TUDCA. (Circ. Res. 2011). The aim of the present study is to examine whether NCS-1 plays Supported from FCT/Portugal (Projects PTDC/QUI-BIQ/119494/2010 and beneficial roles in cardiac survival during stress and the possible mechanisms un- RECI/CTM-POL/0342/2012). derlying these effects. Neonatal mouse ventricular myocytes or whole hearts from wild-type (WT) and Ncs1/ mice were subjected to stressors, and the resistance T.S. and F.F. acknowledges FCT grants SFRH/BD/92398/2013 and SFRH/ / BPD/64320/2009 to stress was evaluated. Ncs1 mouse hearts were more susceptible to stress induced by oxidative impairment and ischemia-reperfusion injury. Stress- 3071-Pos Board B501 induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, a major MAC Inhibitors Neutralize the Pro-Apoptotic Effects of Tbid survival pathway, was substantially reduced in the Ncs1/group, and overex- Pablo M. Peixoto1, Oscar H. Teijido2, Laurent M. Dejean3, Evgeny Pavlov4, pression of NCS-1 or the constitutive active form of Akt increased the survivalrate Bruno Antonsson5, Kathleen W. Kinnally4. of Ncs1/myocytes. Cellular ATP levels, as well as mitochondrial respiration 1 2 Natural Sciences, Baruch College - CUNY, New York, NY, USA, National rates (both basal and maximal O2 consumption) were significantly depressed in Institute of Child Health and Human Development, Baltimore, MD, USA, Ncs1/ myocytes; especially with oxidative stress. Furthermore, intracellular 3Chemistry, California State University of Fresno, Fresno, CA, USA, 4New Ca2þ handling was more easily dysregulated in stressed Ncs1/ myocytes York University College of Dentistry, New York, NY, USA, 5Merck Serono, than WT myocytes. Since NCS-1 levels were increased by stress, the data suggest Geneva Research Center, Geneva, Switzerland. that NCS-1 is a survival-promoting factor, which is upregulated by stress stimuli. Since our initial characterization of the iMACs, different di-bromocarbazole Interestingly, however,supra-physiological NCS-1 expression was toxic to cells. derivatives with anti-apoptotic function have been developed and tested in Taken together, our data suggest that moderate NCS-1 expression during stress several mouse models of brain injury and neurodegeneration [13-21]. Owing promotes cardiomyocyte survival by maintaining proper Ca2þ handling, which to the increased therapeutic potential of anti-apoptotic di-bromocarbazole de- is required for activation of Akt survival pathways and mitochondrial function. rivatives, we sought to expand our knowledge of the mechanism of action of these small molecule inhibitors. We investigated the kinetics of MAC inhibi- 3074-Pos Board B504 tion in mitochondria from wild type, Bak, and Bax knockout cell lines using Initiation of Electron Transport Activity and a Decrease of Oxidative patch clamp electrophysiology, fluorescence microscopy, ELISA, and quantita- Stress Occur Simultaneously during Embryonic Heart Development tive western blot analyses. Our results show that iMACs work through at least Gisela Beutner, George A. Porter, Jr. two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to Pediatrics, University of Rochester Medical Center, Rochester, NY, USA. mitochondria and 2) by disassembling Bax oligomers in the outer membrane. Mitochondria in early embryonic hearts are not thought to produce ATP, yet A comparison of the inhibitory effects over channel conductance and cyto- they do produce reactive oxygen species (ROS) that regulate myocyte differen- chrome c release suggests that the iMACs interacted with both Bax and Bak tiation. To assess changes in ATP and ROS generation in the developing heart, with similar kinetics. Interestingly, wild type mitochondria were more suscep- we measured mitochondrial oxygen consumption, the activity of the complexes tible to inhibition than the Bak or Bax knockouts. A quantitative western blot (Cx) 1 and 2 of the electron transport chain (ETC), ETC supercomplex assem- analysis showed that wild type mitochondria had lower steady state levels of bly, and ROS in embryonic mouse hearts. At embryonic day (E) 9.5, mitochon- Bak, which suggests an uneven stoichiometry of the MAC components. drial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the ETC complexes are present. We show that Cx-1 is able to 3072-Pos Board B502 accept electrons from the Krebs cycle, but enzyme assays that specifically mea- Tyrosine Phosphorylation of Mitochondrial Ca2D Uniporter Regulates D sure electron flow to ubiquinone or Cx-3 show no activity at this early embry- Mitochondrial Ca2 Uptake onic stage. At E11.5, mitochondria appear functionally more mature; ETC Jin O-Uchi, Stephen Hurst, Jyotsna Mishra, Xiaole Xu, Bong Sook Jhun, activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, Shey-Shing Sheu. the assembly of highly efficient respiratory supercomplexes containing Cx 1, Depariment of Medicine, Center for Translational Medicine, Jefferson 3, and 4, ubiquinone, and cytochrome c begins at E11.5, the exact time Medical College, Thomas Jefferson University, Philadelphia, PA, USA. þ þ when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS Mitochondrial Ca2 has a critical role for balancing cell survival and death. Ca2 of embryonic heart mitochondria are indistinguishable from adult mitochon- influx into mitochondrial matrix is mediated primarily by the mitochondrial cal- dria. In contrast, generation of reactive oxygen species (ROS), as measured cium uniporter (MCU). However, the signaling pathways that regulate MCU with Amplex Red, is high at E9.5 and drops significantly by E11.5, coinciding channel functions via post-translational modifications of MCU are completely with activation of the ETC. In summary, our data suggest that between E9.5 and unknown. Here we show that adrenergic signaling induces MCU tyrosine phos- E11.5 dramatic changes occur in the mitochondria of the embryonic heart, 2þ phorylation and accelerates mitochondrial Ca uptake in cardiac cells. Adren- which result in a decrease of ROS generation and an increase in OXPHOS ergic signaling induces activation of proline-rich tyrosine kinase 2 (Pyk2) and due to the activation of Cx-1 and the formation of supercomplexes. translocation into the mitochondrial matrix; enhancing the interaction between Pyk2 and MCU, which subsequently accelerates mitochondrial Ca2þ uptake via 3075-Pos Board B505 Pyk2-dependent MCU tyrosine phosphorylation. MCU contains 15 tyrosine res- The Stoichiometry between MICU1 and MCU Determines the Different 2D idues (5 in the N-terminus, 0 in the pore-forming region, 4 in transmembrane do- Mitochondrial Ca Uptake Phenotypes in Heart and Liver mains and 6 in the C-terminus), which are conserved across all eukaryotic Melanie Paillard, Gyo¨rgy Csorda´s, Tu¨nde Golena´r, Cynthia Moffat, species. Among them, only 3 of these tyrosine residues (Y157 at N-terminus, Erin Seifert, Gyo¨rgy Hajno´czky. Y288, and Y316 at C-terminus in mouse MCU) remained as potential phosphor- MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, PA, ylation candidate sites for protein tyrosine kinases using phosphorylation predic- USA. 2þ tion programs. We mutated these tyrosine residues to phenylalanine and Mitochondrial Ca uptake is central to oxidative metabolism and cell death generated non-phosphorylation mimetic MCU mutants (MCU-YFs). We signaling. The first clues to its molecular mechanism have emerged from the 2þ confirmed that only two tyrosine sites were phosphorylated in response to adren- recent identification of the mitochondrial Ca uniporter’s pore forming protein ergic stimulation in situ using cell lines stably expressing MCU-YFs. In addi- (MCU) as well as its regulators. Among the regulators, MICU1 shows striking 610a Wednesday, February 11, 2015 co-expression and co-evolution with MCU. MICU1 has been demonstrated to be [Ca2þ]c (from 5558to9158 nM) at the same time. When the cells were incu- aCa2þ-sensing protein, which both sets a threshold for low Ca2þ concentration bated in a Ca2þ-free medium and were pretreated with thapsigargin to prevent while it assures cooperative activation during high Ca2þ rises. Mitochondrial Ca2þ entry and intracellular Ca2þ mobilization, respectively, H2O2 continued Ca2þ uptake shows tissue specific differences and interestingly, mRNA level to inhibit the mito-motility dose-dependently without any changes in [Ca2þ]c. for MICUs and MCUs also displays tissue specificity. We set out to investigate These results indicate that H2O2 can cause suppression of mito-motility if the stoichiometry between MICU1 and MCU could account for the previously through a Ca2þ independent mechanism we are currently analyzing. described differences between heart and liver in mitochondrial Ca2þ uptake. Immunoblotting showed higher expression for all MICU1, MICU2 and MCU 3078-Pos Board B508 in mouse liver versus heart mitochondria, and a 4.5 fold higher MICU1 to Miro1 is Dispensable for Calcium-Mediated Inhibition of Mitochondrial MCU ratio in liver. In fluorometric measurements of mitochondrial Ca2þ uptake, Movement heart mitochondria displayed a decreased threshold and lesser cooperativity David B. Weaver1, Agnieszka Lewandowska2, Tammy T. Nguyen2, compared to liver mitochondria. Additionally, NAD(P)H elevation was detect- Valentina Debattisti1, Janet M. Shaw2, Gyorgy Hajnoczky1. 2þ 1MitoCare Center, Dept of Pathology, Anatomy and Cell Biology, Thomas able after exposure to moderate [Ca ] elevations only in heart mitochondria. 2 Overexpression of MICU1 in the heart using AAV9-MICU1 tail-vein injection Jefferson University, Philadelphia, PA, USA, Biochemistry, University of significantly increased the MICU1 protein level without any changes of Utah School of Medicine, Salt Lake City, UT, USA. MICU2 or MCU. This increased the MICU1 to MCU ratio in the heart and led Miro1 and 2 are (Rhot1 and 2) are two highly similar GTPases that are bound to to increased thresholding and cooperativity, reproducing the liver-like mito- the surface of mitochondria and possess EF-hand calcium binding motifs. chondrial Ca2þ uptake phenotype. Vice versa MICU1 downregulation in the liver Several groups have reported that Miro is involved in mitochondrial motility has been shown to lower the threshold and cooperativity of mitochondrial Ca2þ and inheritance, and particularly its calcium regulation, but the roles of the two uptake in hepatocytes. Thus, heart and liver mitochondria show different levels isoforms have not been established. Genetic deletion of Miro1 in mouse is lethal of Ca2þ threshold and cooperative activation of Ca2þ uptake, which seem to at birth (Nguyen et al., 2014) and fibroblasts (MEFs) derived from Miro1 KO em- result from differential quantitative relationship between MICU1 and MCU. bryos show abnormal mitochondrial distribution, but the calcium-dependent in- hibition of motility is unaffected and the respiratory and calcium buffering 3076-Pos Board B506 capacities are normal. Neuron-specific knockout of Miro1 leads to progressive ER Calcium Release is Tuned by Mitochondrial Redox Nanodomains deficits of upper motor neuron function, however mitochondria in processes of David M. Booth1, Bala´zs Enyedi2, Miklo´s Geiszt2,Pe´ter Va´rnai2, cortical neurons from Miro1 KO and wild-type embryos showed comparable Gyo¨rgy Hajno´czky1. calcium-sensitive motility inhibition. While no significant increase in Miro2 pro- 1MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, tein was observed in Miro1 KO MEFs, these data raise two possibilities: Miro1 PA, USA, 2Department of Physiology, Semmelweis University, Budapest, and 2 are interchangeable with regard to calcium regulation of mitochondrial Hungary. motility or Miro2 is the key player in this regard. To finally resolve this question, Spatially and temporally controlled increases of H2O2 emerge as an intracellular we are in the process of generating Miro2 KO and Miro1/2 KO cell lines. signal. We hypothesized that ROS and Ca2þ interact locally, in the restricted vol- ume of the ER-mitochondrial interface. These physically tethered structures host 3079-Pos Board B509 enrichments of ion transport proteins such as the IP3 receptor, which support Mitochondrial Fusion Dynamics in the Heart elevated nanodomains of Ca2þ during signalling events and are sensitive to Veronica Eisner1, Ryan Cupo1, Erhe Gao2, Gyo¨rgy Csorda´s1, Lan Cheng3, H O . We used the genetically encoded H O sensor HyPer incorporated into Jessica Ibetti2, J. Kurt Chuprun2, Walter J. Koch2, Gyo¨rgy Hajno´czky1. 2 2 2 2 1 an inducible linker system to probe the redox environment at the ER- MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, 2 mitochondrial interface in HepG2 cells. We found a moderately elevated H O PA, USA, Center for Translational Medicine, Temple University School of 2 2 3 nanodomain which developes into a H2O2 transient following IP3 receptor- Medicine, Philadelphia, PA, USA, Center for Translational Medicine, mediated ER Ca2þ release and mitochondrial Ca2þ uptake. Pharmacological in- Thomas Jefferson University, Philadelphia, PA, USA. hibition showed that the transient was dependent upon ER Ca2þ, mitochondrial Heart physiology depends on oxidative metabolism that likely requires dy- membrane potential and functional electron transport chain. HyPer measure- namic and permanent reorganization of mitochondria by fusion and fission. ments of the mitochondrial intermembrane space revealed significantly elevated To directly evaluate mitochondrial fusion dynamics in cardiomyocytes (CM), H2O2 within this volume. Using electron microscopy we found that HepG2 mito- mitochondrial matrix-targeted photoactivatable GFP and DsRed were intro- chondria possess a cohort of dilated cristae, which disappeared following IP3- duced either in vitro or in vivo by adenovirus and were followed by confocal linked Ca2þ release. Paxilline that inhibits mitochondrial BKCa channels microscopy. Four conditions were analyzed: 24 to 48 h cultured neonatal and blocked the cristae reshaping and also abolished the H2O2 transient at the inter- in vitro transduced adult CM, and CM from in vivo infected rat hearts. In the face. Furthermore, paxilline caused suppression of the IP3-linked calcium signal, latter case, CM were isolated 7-10 days after infection and were imaged whereas interface targeted killer red, a photoactivated H2O2 source, induced promptly or 24-48 h post harvesting. Neonatal CM mitochondria form a highly sensitization to the IP3-linked agonist. We conclude that the intermembrane/ connected network, whereas both in vitro and in vivo transformed cultured CM cristae volume of mitochondria represents an oxidized pool fed by the electron displayed only some connectivity. Impressively, in vivo transduced adult CM transport chain. Ca2þ-uptake stimulates expansion of the mitochondrial matrix that were imaged promptly after harvesting, unveiled a significantly higher con- via Kþ and concomitant water uptake, squeezing the oxidized volume of the nectivity among mitochondria than the 24-48h cultured adult CM. Furthermore, cristae to the interface. Here, a transient H2O2 nanodomain sensitizes IP3 recep- fusion events (f.e./75 square micrometers/min) were almost absent in cultured tors to further stimulation. We demonstrate a physiological setting where Ca2þ in vitro transduced CM, meanwhile in vivo transduced and cultured CM 5 release may autoregulate using mitochondrial H2O2 released from the cristae. showed 0.4 0.2 f.e./min, whereas isolated, freshly-imaged CM displayed 1.45 0.1 f.e./min. Imaging in perfused whole heart ex vivo, showed consider- 3077-Pos Board B507 able mitochondrial continuity and fusion activity in ventricular CM. To study Reactive Oxygen Species (ROS) Suppress Mitochondrial Motility more directly the role of CM’s contractile activity in mitochondrial fusion, Valentina Debattisti, Masao Saotome, Sudipto Das, Gyorgy Hajnoczky. CM were incubated with Verapamil (10mM), that blocked spontaneous contrac- MitoCare Center, Thomas Jefferson University, Philadelphia, PA, USA. tion and partially suppressed the fusion activity of mitochondria. Also, mito- Mitochondrial distribution and transport play pivotal roles for many cellular chondrial fusion activity appeared to be higher after spontaneous contraction functions, including cell differentiation, cell division to ensure proper inheri- or short term field stimulation in isolated freshly-imaged CM. Thus, mitochon- 2þ tance, apoptosis, ATP supply at the local sites of demand, Ca buffering for dria are dynamic in both neonatal and adult CM, but under culture conditions, 2þ intracellular Ca homeostasis. adult CM lose mitochondrial fusion activity. This might be at least in part, We previously showed that mitochondrial motility (mito-motility) is regulated because cardiomyocyte contractile activity is altered in culture and contractions 2þ 2þ by the cytosolic Ca concentration ([Ca ]c), providing the basis for a likely provide some factors to support mitochondrial fusion activity. homeostatic circuit in which the organelles decrease their movements along microtubules to locally buffer high [Ca2þ]c and contribute to ATP supply. Mito- 3080-Pos Board B510 chondria are also a major site for production and scavenging of ROS that serve Mechanistic Characterization of the Thioredoxin System in the Removal of as both a messenger and regulator of calcium signaling and are particularly rele- Hydrogen Peroxide vant for the control of mitochondrial function. Here we tested the hypothesis Venkat R. Pannala, Ranjan K. Dash. that ROS target mito-motility to control mitochondrial function. H9c2 myoblast Bioengineering and Biotechnology, Department of Physiology, Medical cells were transfected with a mitochondrial matrix targeted YFP and then loaded College of Wisconsin, Wauwatosa, WI, USA. with fura2, to monitor the mito-motility simultaneously with [Ca2þ]c. H2O2 The thioredoxin system plays a critical role in the defense against oxidative (100 mM) caused a decrease in mito-motility (6458 %) and an elevation in stress by removing harmful hydrogen peroxide (H2O2). Specifically, Wednesday, February 11, 2015 611a thioredoxin (Trx) donates electrons to peroxiredoxin (Prx) to remove H2O2 and superoxide anion. In yeast, SOD1 is required to protect VDAC1 from oxidation then thioredoxin reductase (TrxR) maintains the reduced Trx concentration but also from carbonylation induced by ROS [3]; in addition, yeast strains with NADPH as the cofactor. Despite a great deal of kinetic information gath- devoid of endogenous SOD1 show down-regulated VDAC1 and TOM40 ered on the removal of H2O2 by the Trx system from various sources/species, a levels, and VDAC shows a significantly less pronounced voltage dependence mechanistic understanding of the associated enzymes is still not available. We and conductance [4]. To unravel SOD1 metabolic role in relation to address this issue by developing a thermodynamically-consistent mathematical VDAC1-mediated metabolism, we expressed human SOD1 in yeast devoid model of the Trx system which entails mechanistic details and provides quan- of endogenous VDAC (Dpor1). While Dpor1 strain cannot grow in the presence titative insights into the kinetics of the TrxR and Prx enzymes. Consistent with of a not-fermentable carbon source, possibly due to altered mitochondria, our experimental studies, the model analyses of the available data show that both results indicates that the overexpression of hSOD1 in Dpor1 strain relieves enzymes operate by a ping-pong mechanism. The proposed mechanism for the growth defect, suggesting that SOD1 participates in the mitochondrial TrxR, which incorporates substrate inhibition by NADPH and intermediate metabolic intersection with the cytosol. protonation states, well describes the available data and accurately predicts Acknowledgments: PRIN 2010CSJX4F (VDP) and ARISLA (AM). the bell-shaped behavior of the effect of pH on the TrxR activity. Most impor- [1] Messina et al, 2012, BBA 1818, 1466-1476 tantly, the model also predicts the inhibitory effects of the reaction products [2] De Pinto et al, 1989, BBA 987, 1-7 (NADPþ and Trx(SH)2) on the TrxR activity for which suitable experimental [3] Tomasello et al, PlosOne, 2013, 8, e81522 data are not available. The model analyses of the available data on the kinetics [4] O’Brien et al, 2004, JBC 279, 51817-51827 of Prx from mammalian sources reveal that Prx operates at very low H2O2 con- [5] Karakitos et al, 2009, FEBS Lett 583, 449-455 centrations compared to their human parasite counterparts. Furthermore, the model is able to predict the dynamic overoxidation of Prx at high H2O2 con- 3083-Pos Board B513 centrations, consistent with the available data. The integrated Prx-TrxR model The Role of Complex I in Mitochondrial Reactive Oxygen Species simulations show that the coupling of TrxR- and Prx-dependent reduction of Formation in Cochlear Sensory and Supporting Cells during Ototoxic H2O2 allowed ultrasensitive changes in the Trx concentration in response to Aminoglycoside Exposure 1 1 2 changes in the TrxR concentration at high Prx concentrations. Danielle Desa , Michael G. Nichols , Heather Jensen Smith . 1Physics, Creighton Univeristy, Omaha, NE, USA, 2Biomedical Sciences, 3081-Pos Board B511 Creighton Univeristy, Omaha, NE, USA. Higher Mitochondrial Membrane Potential Induces ROS Production in Aminoglycosides (AGs) are the most widely used class of antibiotics in the the Familiar Form of Frontotemporal Dementia with MAPT Mutations world despite causing permanent hearing loss by damaging inner ear sensory Noemi Esteras Gallego, Selina Wray, Elisavet Preza, Andrey Y. Abramov. cells. Although the mechanisms of cochlear sensory cell damage are not fully Molecular Neuroscience, Institute of Neurology, University College London, known, reactive oxygen species (ROS) are clearly involved. During normal London, United Kingdom. mitochondrial metabolism low levels of ROS, primarily superoxide, are pro- Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP- duced at complexes I and III in the electron transport chain. These levels can 17) is a neurodegenerative disorder caused by mutations in the MAPT gene en- increase when mitochondrial dysfunction occurs. Complex I-specific ROS for- coding tau protein. Mitochondrial alterations have been associated with mation was evaluated in acutely-cultured murine cochlear explants exposed neuronal death in several diseases. The objective of our study was to analyse to gentamicin (GM, 300 mg/ml), a representative ototoxic AG antibiotic. Mito- the mitochondrial function in human iPS cells from a patient of FTDP-17 car- chondrial membrane potential and pro-apoptotic signaling were measured using rying the 10þ16 MAPT mutation. In addition, we have selected three different Tetramethylrhodamine and apoptosis-inducing factor (AIF) labeling, respec- time points of the differentiation from pluripotent stem cells to cortical neurons tively. Fluorescence intensity-based measurements of nicotinamide adenine to study how mitochondrial alterations develop. dinucleotide (NADH) were used to detect changes mitochondrial metabolism. We have used fluorescence imaging techniques to examine the mitochondrial Relative amounts of superoxide and hydrogen peroxide produced during function: TMRM to measure the mitochondrial membrane potential (Dcm) acute GM exposure were measured using MitoSox Red and Dihydrorhodamine and dyhidroethidium (DHE) to measure the rate of reactive oxygen species 123, respectively. GM increased NADH fluorescence intensity in low- and (ROS) production. high-frequency sensory cells. The complex I inhibitor rotenone (250 nM) Dcm was higher in iPS-derived neurons from the patient bearing the MAPT mu- significantly increased superoxide, not hydrogen peroxide, in low- and high- tation (158.3530.2% of control). Higher Dcm was also found in non- frequency sensory cells (p < 0.01). GM significantly increased superoxide differentiated pluripotent stem cells (133.4510.1%) and in the neural rosettes, and hydrogen peroxide formation in low- and high-frequency sensory cells (p which represent an earlier stage of the differentiation (151.5512.4%). In < 0.05). Rotenone increased GM-induced superoxide formation but decreased contrast, mitochondrial mass was lower in mutant iPS-derived neurons GM-induced hydrogen peroxide formation. This effect was greatest in high- (85.153.9%), although it was similar in non-differentiated cells. frequency cells indicating fundamental differences in ROS formation in high- We have also found that the rate of ROS production, measured using DHE, was and low-frequency sensory cells exposed to ototoxic antibiotics. This project also higher in iPS-derived neurons from the patient (127513.9% of control). provides a base for understanding the underlying mechanisms of mitochondrial The increased rate of ROS production in these cells may be the consequence ROS production in cochlear cells during exposures to ototoxic antibiotics. of the enhanced membrane potential. Consistently, the rate of ROS production Supported by the National Institute on Deafness and Other Communication in non-differentiated cells and in neural rosettes was also significantly higher Disorders (NIDCD,RO3DC012109), and COBRe (8P20GM103471-09) to (123512.9% and 13056.9%, respectively). HJS and a Ferlic Undergraduate Research Scholarship to DD. Our study indicates that this MAPT mutation leads to a higher mitochondrial membrane potential, which induces a higher ROS production that may be a 3084-Pos Board B514 trigger for neurodegeneration. Mitochondrial Iron and Sphingosine Synergize Initiation of Hepatocyte Death by Augmenting Oxidative Stress 3082-Pos Board B512 Sergei A. Novgorodov1, Tatyana I. Gudz1, Andaleb Kholmukhamedov2, The Overexpression of Superoxide Dismutase 1 Restores Growth Defect in Raymond Deepe2, John J. Lemasters3,4. a Porin1-Less Yeast Strain and Improves Mitochondrial Metabolism 1Neurosciences, Medical University of South Carolina, Charleston, SC, USA, Andrea Magrı`1, Simona Reina1, Flora M. Tomasello2, Maria C. Di Rosa1, 2Drug Discovery & Biomedical Sciences, Medical University of South Angela Messina1, Vito De Pinto1. Carolina, Charleston, SC, USA, 3Biochemistry & Molecular Biology, 1Biological, Geological and Environmental Sciences - Section of Medical University of South Carolina, Charleston, SC, USA, 4Institute of Biochemistry and Molecular Biology, University of Catania, Catania, Italy, Theoretical & Experimentla Biophysics, Pushchino, Russian Federation. 2CNR – Institute of Biostructure and Bioimaging, Section of Catania, Hepatocytes exposed to ischemia/reperfusion (I/R) succumb to cell death after Catania, Italy. reperfusion. Sphingosine and ceramide profiles revealed substantial accumula- Metabolic exchanges between cytosol and mitochondria are made possible by tion of sphingosine after 4h of ischemia to rat hepatocytes, whereas other sphin- the presence of the pore-forming protein VDAC1 on the outer mitochondrial goid bases did not change. A lysosomotropic inhibitor of acid ceramidase membrane [1-3]. VDAC1 is directly involved in ATP/ADP, glucose and ions suppressed I/R-induced death, indicating a lysosomal origin of sphingosine. transportation, calcium homeostasis and apoptosis regulation. Moreover, it Addition of exogenous sphingosine to hepatocytes increased cell death, which shows high level of sequence conservation in all eukaryotes: the homologous was insensitive to the ceramide synthase inhibitor, fumonisin B1. This finding por1 in yeast S. cerevisiae shows 70% of identity and similar functional prop- indicates that accumulation of sphingosine, not ceramide formed from sphingo- erties [1]. Recent studies have highlighted the existence of a link between sine, promoted cell death. Exogenous sphingosine also inhibited complex IV VDAC1 and SOD1 enzyme, the most important cytosolic defense against (cytochrome oxidase), the terminal component of the respiratory chain, in 612a Wednesday, February 11, 2015 isolated mitochondria. Accordingly, we hypothesized that downstream respira- 3087-Pos Board B517 - tory inhibition by sphingosine leads to increased formation of O2 radicals Monte Carlo Simulation of Wnt Propagation by a Novel Transport Mech- after reperfusion, which by themselves have only a moderately harmful effect. anism Complementing a Joint Experimental Study However when Fe2Dredistributes from lysosomes into mitochondria during Claude Sinner1, Eliana Stanganello2, Anja I.H. Hagemann2, ischemia, Fenton chemistry occurs after reperfusion, leading to formation of Benjamin Mattes2, Dana Meyen3, Sabrina Weber2, Erez Raz3, highly reactive OH radicals, potent inducers of the mitochondrial permeability Steffen Scholpp2, Alexander Schug1. transition pore and cell death. This hypothesis was directly tested using bafilo- 1Steinbuch Centre for Computing, Karlsruhe Institute of Technology, mycin, which induces the release of Fe2D from lysosomes with subsequent up- Eggenstein-Leopoldshafen, Germany, 2Institute of Toxicology and Genetics, take into mitochondria. Indeed, bafilomycin potentiated sphingosine-induced Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany, cell death. The data highlight a novel mechanism mediating I/R injury, 3Institute of Cell biology, University of Mu¨nster, Mu¨nster, Germany. which involves sphingosine accumulation and uptake of lysosomal iron into Tissue development is a key process in living organisms. An essential compo- mitochondria during ischemia, leading to respiratory chain inhibition, iron- nent for these developmental processes - but also for tissue regeneration and dependent oxidative stress, mitochondrial permeability transition and cell death stem cell regulation - is the communication of cells by paracrine signaling. after reperfusion. DK073336, DK037034 and 14.Z50.31.0028 (JJL) and Following the French flag model, these processes are responsive to concentra- NS083544 (TIG). tion gradients of signal carrying molecules, so-called morphogens. The highly conserved family of Wnt proteins can act as morphogens and represents impor- Cellular Signaling and Metabolic Networks tant regulators of all these processes. After secretion, specific transport mecha- nism must ensure proper distribution of the morphogen. Experimental studies in 3085-Pos Board B515 zebrafish embryos and human kidney cells have given first evidence for a novel Guardian Function of Mitsugumin 53 in Cell Membrane Repair and short-range transport of Wnt morphogens from the Wnt active tissue towards Metabolic Syndrome receiving cells using cell protrusions, so-called filopodia, as mediating agent. Hanley Ma1,2, Jason Liu2,3, Zehua Bian2, Yuqi Cui2, Xinyu Zhou2, These specialized filopodia transmit signaling proteins between communicating Xuefeng Zhou2, Bo Zhang2, Timothy M. Adesanya2, Ki Ho Park2, Hua Zhu2. cells and allow a high degree of control of propagation speed, direction and con- 1Olentangy Liberty High School, Powell, OH, USA, 2Department of Surgery, centration of the transmitted ligand. The crucial question is how this novel Davis Heart and Lung Research Institute, The Ohio State University, short-range mechanism can result in a long-range gradient of morphogen mol- Columbus, OH, USA, 3Dublin Jerome High School, Dublin, OH, USA. ecules covering the complete responsive tissue. In order to give an answer to Mistugumin 53 (MG53) is an essential molecule in facilitating cellular mem- this question and address the theoretical feasibility of the new model we have brane repair. A member of the TRIM protein family, MG53 possesses a RING set up complementary Monte Carlo simulations. The simulation iteratively re- domain functioning as an E3 ligase to mediate the down-regulation of insulin produces ligand production, cell migration, and a slight ligand decay in concor- receptor substrate-1 (IRS-1) in insulin signaling. Conflicting results have been dance with experimentally measured boundary conditions. In a filopodia presented that elevated MG53 expression may serve as a causative factor for mediated transport system the major parameters are not anymore diffusion the development of metabolic syndrome. This study was designed to test whether rate, cell adhesion, and concentration of the ligand but length, angle distribu- high-fat diet (HFD) treatment alters the expression and function of MG53 within tion, and growth frequency of filopodia. During the simulation we were able mice models of metabolic syndrome. Western blotting showed that MG53 to identify key parameters of the underlying mechanism and quantitatively expression does not change within the skeletal and cardiac muscles of mice reproduce our experimental data. These results provide evidence that a filopodia subjected to HFD treatment. This data contradicts earlier findings presented based short-range transport system for Wnt has long-range signalling function. by Song et al. (Nature, 494: 375-379, 2013), who claimed that MG53 expression 3088-Pos Board B518 is markedly elevated in animal models of insulin resistance and metabolic A Genetically-Encoded FRET Sensor based on AMP-Activated Protein syndrome. Rather, we found that stress provoked by metabolic syndrome in Kinase Reports Allosteric Kinase Activation the mice models actually reduced MG53 levels in the serum. Immunohistochem- Uwe Schlattner1,2, Martin Pelosse1,2, Imre Berger3. ical analyses revealed that skeletal muscle fibers of HFD-induced mice experi- 1Laboratory of Fundamental and Applied Bioenergetics (LBFA), Joseph ence localization of intracellular MG53 around mitochondria. The reduced Fourier University - Grenoble 1, Grenoble Cedex 9, France, 2Inserm U1055, MG53 serum levels observed may explain the compromised tissue regenerative Grenoble, France, 3EMBL Grenoble, Grenoble, France. capacity of diabetic patients. Clustering of MG53 around mitochondria may AMPK is a multi-protein nanomachine that is activated by multiple, complex represent an adaptive response to metabolic stress. Overall, our study supports mechanisms, allowing fine tuning of AMPK activity in different situations of the guardian function of MG53 in cell membrane repair and metabolic syn- metabolic stress. Binding of adenine nucleotides to the gamma subunit plays drome. Therapeutic approaches for an elevation of MG53 expression in tissues a major role in either direct allosteric activation of AMPK or modulation of that bypasses its interaction with IRS-1 may be a novel approach to treating AMPK phosphorylation and dephosphorylation by upstream kinases and phos- human diseases with degenerative tissue repair functions. phatases. These activation mechanisms require crosstalk between AMPK sub- units by a nucleotide-induced conformational switch. We have engineered an 3086-Pos Board B516 AMPK complex that allows a direct, real-time readout of the AMPK conforma- Non-Invasive Interogation of Signaling Activated Gene Regulation tional state by fluorescence energy transfer (FRET). This molecular sensor con- Gregor Neuert. firms the exquisite sensitivity of AMPK to low micromolar concentrations of Vanderbilt University, Nashville, TN, USA. AMP, shows the exclusive ability of ATP, but not MgATP, to compete with Many signal transduction and gene regulatory pathways are highly dynamic AMP, and allows insight into the role of CBS domains for allosteric AMPK resulting in a variety of dynamic signaling and gene expression profiles. activation. It has potential applications as a tool for screening of allosteric ac- Currently, our biophysical understanding of these profiles relies on the manip- tivators of AMPK, and as a reporter of cellular energy state. ulation of specific genes or drug treatment of specific proteins. One drawback of this approach is that a gene of interest needs to be identified to affect the 3089-Pos Board B519 pathways dynamic. Another drawback is that such perturbations may result Integrated Omic Analysis of a Guinea Pig Model of Heart Failure and in a significant interference with the function of the cell. In order to avoid these Sudden Cardiac Death drawbacks, we developed a novel non-invasive perturbation approach to inves- D. Brian Foster, Ting Liu, Robert N. O’Meallly, C. Conover Talbot, Jr., tigate the dynamic properties of signaling and gene regulatory pathways, Robert N. Cole, Brian O’Rourke. without genetic manipulation or drug treatments. To demonstrate the feasibility Johns Hopkins School of Medicine, Baltimore, MD, USA. of this approach we choose to interrogate a stress response pathway in yeast, Mechanistic understanding of heart failure (HF) and sudden cardiac death which enables us to manipulate the intensity, duration and shape of the signal (SCD) has been hampered by the lack of suitable experimental models with transduction profile. By combining quantitative single cell and single molecule features of human disease. We have developed a guinea pig model of cardiac experiments with predictive modeling, we are able to quantify signal transduc- hypertrophy (HYP) and HF, characterized by predisposition to SCD (Liu tion activation, signal transduction saturation and gene expression activation et al. Circ. Res. 2014). Our objective was to refine guinea pig models of HF thresholds, which is not possible to quantify with any other currently available progression by integrating protein, metabolite and transcript levels. technology. We also found that the signaling dynamics is proportional to the Relative protein abundances from sham-operated, HYP and HF hearts were as- first time derivative of the external perturbation profile. Because this approach sessed using isobaric tags for relative and absolute quantification, prior to liquid is independent of the biological pathway or organism, it presents a general chromatography and tandem mass spectrometry (LC-MS/MS). Metabolites methodology to interrogate signaling and gene expression pathways non- were quantified by LC-MS/MS or gas chromatography coupled to MS. Tran- invasively without the need for genetic or drug perturbations. scriptome profiles were obtained from Affymetrix microarrays. Wednesday, February 11, 2015 613a

Theguinea pig HF proteome exhibited classic biosignatures of cardiac HYP, is the Warburg effect, which corresponds to an increase of anaerobic glycol- left ventricular dysfunction, fibrosis, cellular degeneration, inflammation and ysis vs mitochondrial oxidative phosphorylation to produce energy for extravasation. Fatty acid metabolism, mitochondrial transcription/translation cellular processes. However, the mechanisms related to these metabolic factors, and other mitochondrial processes, were downregulated. Processes/ switches are still a matter of debate. Bcl-2 family proteins contain both proteins upregulated in HF include fibrillogenesis, extracellular matrix remod- pro- (e.g. Bax), and anti-apoptotic (e.g. Bcl-2) members which are respec- eling, cytoskeletal proteins, the unfolded-protein response, and acute phase tively encoded by tumor suppressors and proto-oncogenes. Up-regulation of inflammation markers. Among metabolites, downregulation of acyl-carnitines the anti-apoptotic proteins Bcl-2 has been associated with Non-Hodgkin’s was observed in HYP, while fatty acids accumulated in HF. Levels of the lymphoma; and certain studies indicate that Bcl-2 plays a role in the regula- TCA cycle metabolite, citrate, and the potent inhibitor, 2-methylcitrate, tion of energy metabolism. However, the molecular players involved in this increased upon transition from HYP to HF. Among transcripts, downregulation regulation are still to be defined. We recently observed that Bcl-2 overexpres- of repolarizing Kþ channels, SERCA2a, phospholamban and ryanodine recep- sion led a significant increase of both glucose consumption and lactate pro- tor was noted, and upregulation of mRNAs for cyclic-nucleotide gated duction rates in a mouse pro-lymphocyte B cell line. This phenomenon was hyperpolarization-activated channels and TRPC6. associated with a stimulation of the lactate dehydrogenase (LDH) enzyme The biosignature of decompensation in the guinea pig HF/SCD model parallels specific activity; and a Bcl-2-mediated increase of the expression of the prior rodent models but spotlights metabolic bottlenecks. Fatty acid use is LDH-A subunit. Also, this phenotype was strongly attenuated if a Bcl-2 likely abrogated by impaired mitochondrial import and oxidation, while mutant of interaction with Bax (Bcl-2-G145E) was overexpressed instead compensation by glycolytic flux is predicted to be impaired, along with TCA of native Bcl-2. These data suggest that Bcl-2 expression levels may play cycle dysfunction and accumulation of inhibitors (e.g. citrate). Pathway anal- an active role in the stimulation of lactic fermentation commonly observed ysis suggests avenues for metabolic therapy beyond PPAR activation. in blood cancer cells; and that this effect may be dependent to the ability of Bcl-2 to physically interact with Bax. 3090-Pos Board B520 Cellular Signaling Networks Function as Generalized Wiener-Kolmogorov 3093-Pos Board B523 Filters to Suppress Noise Human Adipose Cell Response to Insulin: Analysis of Cellular Switch-Like Michael Hinczewski1, Devarajan Thirumalai2. Transformations and Distributions 1Department of Physics, Case Western Reserve University, Cleveland, OH, Vladimir A. Lizunov1, Paul S. Blank1, Karin G. Stenkula2, Monica Skarulis3, USA, 2Institute for Physical Science and Technology, University of Samuel Cushman4, Joshua Zimmerberg1. Maryland, College Park, College Park, MD, USA. 1NICHD, Laboratory of Cellular and Molecular Biophysics, National Cells process and transmit information about their environment through Institutes of Health, Bethesda, MD, USA, 2Experimental Medical Sciences, complex signaling cascades. The accurate transmission of the information is Lund University, Lund, Sweden, 3Diabetes, Endocrinology, and Obesity crucial for normal function, with defects in the cascades linked to a host Branch, National Institute of Diabetes and Digestive and Kidney Diseases, of cancers. As in many designed communications systems, these biological Bethesda, MD, USA, 4Experimental Diabetes, Metabolism, and Nutrition circuits must cope with noise that inevitably corrupts the signal. How to effi- Section, National Institute of Diabetes and Digestive and Kidney Diseases, ciently filter noise, to reconstruct the best estimate of the input, has been a key Bethesda, MD, USA. problem in engineering. One of the great advances in this area was a mathe- Insulin resistance is associated with decreased glucose transporter-4 matical framework developed by Norbert Wiener and Andrey Kolmogorov (GLUT4) translocation in adipose cells and muscles in response to insulin. during the Second World War, originally inspired by the need to filter noise Here we quantified, per cell, insulin’s effects on GLUT4 storage vesicle in the targeting of anti-aircraft systems. Remarkably, we show that cells effec- (GSV) tethering and fusion in adipose cells from human subjects with vary- tively implement the same mathematical solution encoded within the chemical ing insulin sensitivity index (SI). Basal GSV tethering and fusion rates were reaction network of a kinase phosphatase push-pull loop, a basic unit of distributed unimodally and did not vary significantly with SI. In contrast, signaling pathways. To demonstrate this, we generalize the ideas of Wiener after a maximal insulin challenge, both tethering and fusion rates were bimo- and Kolmogorov to deal with some of the challenges that arise in the bio- dally distributed, with two distinct subpopulations; the first subpopulation logical context, including signaling through discrete changes in molecular was indistinguishable from the basal distribution, while the second corre- populations, and the highly nonlinear relation between input and output. We sponded to a normal insulin response. Importantly, the fraction of cells in provide mathematically rigorous bounds on the performance of biochemical the two-subpopulations were strongly correlated with donor subject SI. noise filters, and highlight features of the system relevant for optimizing filter These data suggest that the loss of systemic SI is not due to a gradual efficiency, encoded in a single, measurable, dimensionless parameter. Our decrease in the insulin response of all cells, but rather an increase in the theory, which describes noise control in a large class of signal transduction fraction of cells that switch off their insulin response. This observed hetero- networks, is also useful both for the design of synthetic biochemical signaling geneity may be an important consideration in modeling the cellular transi- pathways, and the manipulation of pathways through experimental probes like tions and systemic changes associated with the onset of Type II diabetes. oscillatory input. Thus, isolated human adipose cells, when treated with high concentrations of insulin, exist in either the basal or fully insulin-stimulated state, and pop- 3091-Pos Board B521 ulation dose response curves reflect the proportion of adipose cells in these To Grow is not Enough: The Impact of Cell Response Time on Fitness two distinct states. Nash Rochman, Fangwei Si, Sean Sun. Johns Hopkins, Baltimore, MD, USA. 3094-Pos Board B524 Recent results showing the magnitude of fluctuations in individual cell division Accelerating Systems Biology Computation: Enhanced Sampling of times to be highly concerted across widely varying cell types and environmental Spatially Realistic Stochastic Models using the Weighted Ensemble conditions have been difficult to explain with current molecular cell cycle Approach models. Here we present a phenomenological model for the regulation of cellular Rory Donovan. division time distributions determining both bulk growth rate and ensemble fluc- University of Pittsburgh, Pittsburgh, PA, USA. tuations. A cellular ‘‘fitness’’ function is proposed which incorporates not only We apply the ‘‘weighted ensemble’’ (WE) simulation strategy, previously em- growth rate, which is maximized when fluctuations are minimized, but also ployed in the context of molecular dynamics and spatially homogeneous chem- ensemble response time to environmental stimulus which decreases for ical kinetics simulations, to a number of three dimensional spatially resolved increasing fluctuations. Single cell division data is collected on a population of stochastic systems-biology models. WE is relatively easy to implement, does isogenic cells subjected to varying environmental stimuli and compared to the not require extensive hand-tuning of parameters, does not depend on the details model. Our findings suggest that even cells exhibiting exponential growth do of the underlying simulation algorithm, and can facilitate the unbiased sam- not optimize their ‘‘fitness’’ through growth rate alone, but also response time. pling of extremely rare events. We study three systems of varying complexity and structure: a toy model in 3092-Pos Board B522 an artificial geometry with ~10^3 diffusing molecules, a spatially realistic Bcl-2 Overexpression Stimulates Glycolysis and Lactic Fermentation in a cellular model with ~10^6 molecules, and a spatially realistic neuromuscular Bax-Dependent Fashion junction model with time-varying rate ‘‘constants’’ containing ~10^6 mole- Bushra Mahmood, Jessica Wilson, Miriam Ahmad, Patricia Olino, cules. We are able to speed up the sampling of key events by many orders Justin King, Laurent Dejean. of magnitude and obtain unbiased estimates of event probabilities. Data is ob- Chemistry, California State University of Fresno, Fresno, CA, USA. tained using the publicly available ‘‘WESTPA’’ implementation of WE, It is now well established that shifts in energy metabolism are associated with which orchestrates MCell kinetic Monte Carlo trajectories of the various cancer development and progression. The most studied of these phenomena models. 614a Wednesday, February 11, 2015

3095-Pos Board B525 understanding of how scaffolds might influence signal transduction. The goal Accumulations and Energy Recycling Pathway in P. Falciparum of this work is to systematically determine how various properties of signaling Gametocyte-Infected Human Erythrocytes cascades are impacted in the presence or absence of scaffold proteins. Using Fuyuki Tokumasu1, Takeshi Q. Tanaka2, Suzumi Tokuoka1, rule-based modeling languages, we constructed three basic models of signaling Daichi Nakatani2, Shin-ichiro Kawazu2, Kiyoshi Kita3. based loosely on the network structure of the yeast pheromone signaling 1Dept. of Lipidomics, Graduate School of Medicine, The University of cascade. The first model lacks a scaffold; this solution cascade involves a series Tokyo, Tokyo, Japan, 2National Research Center for Protozoan Diseases, of Goldbeter-Koshland covalent modification cycles where each activated sub- Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan, strate is the kinase for a subsequent substrate. The second, machine, model con- 3Biomedical Chemistry, Graduate School of Medicine, The University of tains a scaffold that acts as a nucleation point for the hierarchical assembly of Tokyo, Tokyo, Japan. kinases into a discrete multi-subunit signaling complex. Finally, the ensemble Quick emergence of drug resistant phenotypes of Plasmodium falciparum model contains a scaffold to which kinases bind independently. This ensemble against new anti-malarial drugs have been a serious problem in the clinical network therefore exhibits considerably more combinatorial complexity in field fighting malaria. Since no effective malaria vaccine is available, terms of the numbers of possible signaling species compared to the others. transmission-blocking targeting gametocyte and gemtocytegenesis may be We performed a comparative analysis of these three paradigms, and found sig- an excellent alternative approach. Gamtocyte parasite exhibits lower sensi- nificant phenotypic differences. Notably, scaffold-based models exhibited far tivity against currently available anti-malarial drugs and characteristic biology less noise as cascade depth increases. Furthermore, only machine-like models that is quite different from that in asexual intraerythrocytic stages. However, are capable of preventing crosstalk in instances where two pathways share com- our understanding gametocyte biology is still premature for leading an effec- ponents. More generally, we observed that a machine-like cascade generally of- tive transmission-blocking drug to stop parasites moving to mosquito stages. fers greater functional robustness, though at the cost of evolutionary plasticity In this study, we focused on lipid metabolism, particularly lipid accumulation and time to signal response. The evolution of scaffold proteins, and the rules and recycling of the lipid storage. With fluorescence microscopy study, game- that govern the assembly of signaling complexes, has likely been driven by tocyte showed marked increases in the total lipid amount as well as lipid drop- the distinct functional roles that scaffold-based signaling complexes can play. lets in parasite cytoplasm while no significant increase in cholesterol, demonstrating a unique lipid environment is developed inside parasite. How- 3098-Pos Board B528 ever, hyperspectral imaging showed most of hemoglobin molecules were Glucose-Induced Cyclic-AMP Oscillations: Modeling Incretin Impact on consumed during gemetocytegenesis indicating its correlation with lipid pro- Pancreatic Beta Cell Secretion ductions and accumulations. Gametocytes are taken by mosquito and continue Bradford E. Peercy1, Richard Bertram2, Arthur Sherman3. 1 2 active stages where high amount of energy must be supplied. Therefore, we Mathematics and Statistics, UMBC, Baltimore, MD, USA, Florida State 3 hypothesized that accumulated lipids will be recycled through gluconeogen- University, Tallahassee, FL, USA, Laboratory of Biological Modeling, esis consuming triacylglycerides (TAG) as a supplemental energy source to National Institues of Health, Bethesda, MD, USA. obtain dihydroacetonephosphate that is involved in the normal glycolysis. Pancreatic beta cells modulate insulin secretion via incretin-induced amplifica- 2þ We used high-sensitivity LC/MS and quantified relative amount ratio of tion pathways of cytosolic calcium (Ca )-induced exocytosis, which may be TAG and glycerol, an intermediate product in gluconeogenesis, to compare help compensate for reduced insulin sensitivity in Type II Diabetes Mellitus. between asexual and gametocyte stages. One modulator is cAMP, which is activated by an incretin-triggered Gs protein cascade and sensitive to glucose levels. Interestingly, cAMP is found to be 3096-Pos Board B526 oscillatory, even in cases where Ca2þ is not, providing a possible mechanism Photo-Regulation of the Interaction between Ras and Ralgds using GTP for pulstile insulin secretion in the absence of Ca2þ oscillations. When Ca2þ Analogues Composed of Photochromic Molecules and cAMP both oscillate, the two may be either in-phase or out-of-phase Kaori Masuhara1, Seigo Iwata2, Nobuhisa Umeki3, Shinsaku Maruta2. with one another. To understand the cAMP oscillations and their relationship 1 Department of Bioinformatics, Soka University, Hachioji, Tokyo, Japan, to Ca2þ oscillations we use mathematical modeling. We model the cAMP pro- 2 Division of Bioinformatics, Soka University Graduate School of duction sensitivity to glucose metabolism with AMP sensitive adenylyl cy- 3 Engineering, Hachioji, Tokyo, Japan, Cellular Informatics Laboratory, clases and degradation with Ca2þ sensitive phosphodiesterases. These RIKEN, Wako, Saitama, Japan. modest additions to the Dual Oscillator Model for beta cells allow us to capture Small GTPases of the Ras family have significant roles of signal transduction in observed relationships between cAMP and Ca2þ. This provides further evi- eukaryotic cells. The G-proteins transmit the signals from a number of recep- dence for metabolic oscillations in beta-cells that are affected by, but that do tors to a variety of effectors. RalGDS is a guanine nucleotide dissociation stim- not require, oscillations in Ca2þ. ulator for Ral, which is a member of the Ras GTPase superfamily that regulates cellular proliferation, differentiation and transformation by mediating multiple 3099-Pos Board B529 signal transduction pathways. RalGDS can specifically promote the conversion Modeling HER2 Inhibition in Breast Cancer Cells from an inactive GDP-bound state to an active GTP-bound state for Ral. Pre- Marc Y. Fink, Danni Zhou. viously we have demonstrated that the GTPase activity of K-Ras modified Biomedical Sciences, LIU Post, Brookville, NY, USA. with thiol reactive azobenzene derivatives at the functional sites were photo- Cancers cells proliferate and survive in response to an oncogene-driven reversibly controlled upon ultraviolet and visible light irradiation. Aim of signaling network. Inhibition of a driver oncogene results in growth arrest or this study is to control the interaction of Ras with RalDGS photo-reversibly us- apoptosis, but some cells are able to adapt to oncogene loss and continue ing photochromic molecules. Recently we found that a fluorescent GTP growth. These varied responses are a result of differential signaling and gene analogue, 20(30)-O-[6-(N-(7- nitrobenz-2- oxa-1,3-diazol-4-yl)amino)hexa- responses, to oncogene inhibition, between cancer cells. Using the BT474 breast noic]-GTP (NBD-GTP) which has a fluorescent probe at 2’ or 3’ position of cancer cell as a model system, the HER2 oncogenic signaling network was in- ribose induced binding of Ras to RalGDS more significantly than regular hibited and subsequent responses at the phospho-proteomic and transcriptomic GTP. NBD-GDP did not induce binding of Ras to RalGDS. The fluorophore level were quantified. These measurements were combined with kinetic NBD might affect as an enhancer of the interaction between Ras and RalGDS. modeling of HER2 inhibition. These results identify several features of the In this study, we synthesized novel photo-responsive GTP analogues that have HER2 signaling network that may underlie heterogeneity at the cellular level. photochromic molecules at 2’ or 3’ position of ribose, 20(30) -O-Azobenzene- EDA-GTP (AB-EDA-GTP), 20(30) -O-Spiropyran-EDA-GTP (SP-EDA- 3100-Pos Board B530 GTP). Trans-isomer of AB-EDA-GTP induced more significant binding of Noncanonical Neurotransmission at the Neuromuscular Junction Ras to RalGDS than Cis-isomer. On the other hand, SP-EDA-GTP did not Huinan Li, Mark Lee Harlow. show the significant difference between the spiro and merocyanine isomers. Biology, Texas A&M, College Station, TX, USA. Large-scale proteomic analyses of heterogeneous CNS preparations, such as 3097-Pos Board B527 isolated CNS synaptic vesicles, have provided a wealth of information related Phenotypic Properties of Scaffold-Based Signaling Paradigms to synapse specific proteins, and synaptic signaling pathways. However, these Ryan Suderman, Addison Schauer, Eric J. Deeds. studies under-represent, or lack entirely, many vesicle and neurotransmitter Center for Bioinformatics, University of Kansas, Lawrence, KS, USA. signaling pathways. In particular the neuromodulatory adrenergic and cholin- Scaffold proteins are key components of signaling networks, recruiting and ergic pathways are often under-represented. In order to better understand the organizing other signaling proteins. Certain scaffolds have been extensively molecules and signaling pathways involved at cholinergic synapses, we have characterized and a number of hypotheses exist regarding general scaffold focused on the classic preparation of the electric lobe and electric organ of Tor- functions, including the linearization of dose-response behavior and the medi- pedo californica. The abundant macromolecular material available from this ation of feedback. Despite intense investigation, however, we lack a complete preparation has contributed greatly to our understanding of the synthesis, Wednesday, February 11, 2015 615a storage, and release of the neurotransmitter acetylcholine and its postsynaptic 3103-Pos Board B533 activation of the nicotinic acetylcholine receptor. Many lines of evidence sup- Spin-Labeled Uni-Lamellar Vesicles as an Oxygen Sensitive Analyte for port the idea that in addition to acetylcholine, additional neurotransmitters and/ Measurement of Cellular Respiration using Rat Dopaminergic Neuronal or neuromodulators are also released from cholinergic synapses. Utilizing a Cells single-vesicle imaging approach, we have found that individual synaptic vesi- Laxman Mainali, Jason W. Sidabras, Theodore Camenisch, cles from the electric ray possess neurotransmitter transporters for glutamate, Jeannette Vasquez-Vivar, James Hyde, Witold K. Subczynski. ATP, and acetylcholine. In addition to those transporters, cholinergic synaptic Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA. vesicles from the electric ray also possess an orphaned transporter from the Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphospha- bile acid transport family, SLC10A4. We are now expanding our research to tidylcholine (POPC) with 1% spin label of 1-palmitoyl-2-(16-doxylstearoyl) identify the molecules transported by SLC10A4 using multiple biochemical phosphatidylcholine (16-PC). POPC membranes are in the fluid phase in the approaches and a mouse knockout model. physiological temperature range, which ensures high solubility of oxygen in the membrane center where the 16-PC nitroxide moiety is located. The nitro- Magnetic Resonance Spectroscopy, Imaging, and xide moiety is isolated from cellular reductants and paramagnetic ions that might interfere with spin-label oximetry measurements. This analyte is stable EPR Spectroscopy in cell suspensions and can be used in studies as long as 400 min. The saturation recovery EPR approach, possibly for the first time, was used to study cellular 3101-Pos Board B531 respiration of rat dopaminergic neuronal cells. It is widely accepted that this Multi-Component Water Dynamics and Exchange in Brain Cortical Tissue approach is the most direct way to carry out oximetric studies. The preliminary Probed via In-Vitro D-T2 2D Correlation NMR experiments were carried out at X-band for closed chamber geometry. Oximet- Ruiliang Bai1,2, Peter J. Basser1. ric measurements were performed for cell concentrations from 750 to 5000 1National Institutes of Health, Bethesda, MD, USA, 2University of Maryland, cells in one mL of culture media. Results confirmed that the oxygen consump- College Park, MD, USA. tion rate was linearly proportional to the number of cells. Furthermore at the Water self-diffusion in brain cortical tissue has been shown to have two com- lower cell concentration, we found a breaking point in the rate which is not ponents by diffusion NMR measurement. This phenomenon is tissue due to diffusion limitation but is due to the change in cell metabolism because microstructure-related and is essential for understanding the diffusion NMR there is not enough oxygen, consistent with Michaelis-Menten kinetics. In the signals’ changes associated with some physiological processes, such as acute developed analyte, the same hydrocarbon environment, which dissolves oxy- stroke, neuronal activation, etc. However, the biophysical mechanisms explain- gen very well, always surrounds nitroxide moieties of spin-label molecules. ing multi-component diffusion inside cortical tissue and possible exchange Therefore, the partial pressure of oxygen influence the EPR spectrum of among components are still poorly understood. spin-labels in the analyte. Such microscopic analytes are readily and uniformly Here we propose using a Diffusion (D) – Transverse Relaxation (T ) 2D corre- 2 distributed within the sample, thus giving a rapid response to changes in oxygen lation NMR method to characterize the multi-component water dynamics in the partial pressure. The improved oximetry methodology developed here presents biological tissue. In the first dimension, the diffusion is measured by a spin echo a high impact opportunity for non-invasive assessment of disease mechanisms in a strong static magnetic field gradient, which can efficiently shorten the diffu- and response to therapeutic intervention. sion time to be less than 1.6ms, making the effect of exchange negligible. In the second dimension, a CPMG pulse sequence is used to measure the T2 of each 3104-Pos Board B534 diffusion component. Here, exchange will affect the NMR signal evolution dur- Probing the Protein-Protein Interactions between KCNQ1 and KCNE1 ing this long T2 encoding period. In this 2D D-T2 NMR method, each dynamics- using Electron Paramagnetic Resonance (EPR) Spectroscopy different water component can be characterized by its specific (D, T2) combina- Andrew F. Craig, Indra D. Sahu, Rongfy Zhang, Megan M. Dunagan, tion and the exchange process among different components can also be quanti- Kunkun Wang, Robert M. McCarrick, Gary A. Lorigan. fied simultaneously. Another unique aspect of this work is that our NMR data is Chemistry, Miami University, Oxford, OH, USA. obtained using a single-sided NMR system with permanent magnet. KCNQ1 is a membrane-bound protein composed of 676 amino acids with a mo- Furthermore, temperature-dependent D-T2 experiments were performed on lecular weight of 75 kD and contains 6 transmembrane helices. KCNQ1 is a cortical tissue from fixed rat brain. The self-diffusion constant of both the voltage-gated potassium channel primarily involved in the transport of potas- fast and slow diffusion components and the exchange between them are slowed sium in the human heart. The activity of KCNQ1 is modulated by the integral as the temperature decreases, whereas the slow diffusion component shows membrane protein KNCE1 (also known as MinK). This potassium modulation higher translational activation energy. More interesting and surprising, the rela- is critical for repolarization in the heart, specifically in the left ventricle. Muta- tive fraction of each component is also observed to have a strong dependence tions in the KCNQ1 and KCNE1 genes have been linked to genetic disorders on the temperature, which indicates this bi-component diffusion is diffusion- such as long QT syndrome, sudden infant death syndrome (SIDS), as well as distance dependent. deafness. However, most current studies between KNCQ1 and KCNE1 have been from an electrophysiological or functional standpoint, which leaves struc- 3102-Pos Board B532 tural and dynamic information on the proteins unknown. EPR spectroscopy is a Probing the Secondary Structure of Membrane Proteins with the Pulsed powerful structural biology tool that can be used to probe both protein struc- EPR Technique: Electron Spin Ehco Envelope Modulation (ESEEM) tures as well as protein-protein interactions between KCNQ1 and KCNE1. Lishan Liu, Gary Lorigan. Currently, we have overexpressed full length KCNE1 and the C-terminal Miami University, Oxford, OH, USA. domain of KCNQ1. In this study, we used both continuous-wave (CW) EPR Despite the importance and large number of membrane proteins, relatively and the pulsed EPR technique of double electron-electron resonance (DEER) limited structural information is known about them. New biophysical tech- to probe the structural and dynamic parameters of the interaction of KCNQ1 niques are needed to probe their structural properties. Their hydrophobic nature and KCNE1. and low expression yields cause difficulties for traditional structural techniques such as x-ray crystallography and solution NMR. ESEEM spectroscopy indi- 3105-Pos Board B535 rectly observes NMR transitions through an electron spin coupled to a nearby Characterization of a Bifunctional Spin Label for the Structure and NMR active nucleus. ESEEM can detect weak dipolar interactions between a Dynamics of a Membrane Protein using CW-EPR Spectroscopy NMR active nucleus and a spin label out to a distance of approximately 8 A˚ . Lauren M. Bottorf, Indra D. Sahu, Lishan Liu, Gary A. Lorigan. The modulation depth for weakly coupled nuclei is scaled by 1/r6. A novel Chemistry and Biochemistry, Miami University, Oxford, OH, USA. approach is being developed to probe the secondary structure of membrane pro- Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful struc- teins and peptides qualitatively utilizing the three-pulse Electron Spin Echo tural biology tool when combined with site-directed spin labeling (SDSL) Envelope Modulation (ESEEM) pulse sequence. In order to demonstrate the to study the structure and dynamics of peptides and proteins. The most widely practicality of this biophysical technique, the M2delta subunit of AChR (a-he- used spin label for SDSL is methanthiosulfonate (MTSL), however the flex- lical) and KIGAKI(b-sheet) peptides were incorporated into phospholipids bi- ibility of this spin label introduces greater uncertainties in the EPR measure- celle to probe their secondary structure with ESSEM spectroscopy. Utilizing ments obtained for determining structure, side chain dynamics, and backbone site-directed spin-labeling (SDSL) coupled with deuterated amino acid labeling motion of membrane protein systems. Most recently, a new bifunctional spin of the peptides, the corresponding ESEEM spectra reveal characteristic patterns label (BSL) 3,4-Bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-di- for a-helix and b-sheet structures. This ESSEM secondary structural approach hydro-1H-pyrrol-1-yloxy, has been introduced to overcome some of the lim- can be used with different deuterated amino acids and provide pertinent qual- itations associated with the MTSL spin label and has been invaluable in itative structural information on membrane proteins in a short period of time determining protein dynamics and inter-residue distances due to its restricted (10 minutes) with small amounts of sample (30 mg). internal motion and fewer size restrictions. While BSL has been successful in 616a Wednesday, February 11, 2015 providing information about the structure and dynamics of several proteins, in the unit cell is known. To this end high-resolution crystal structures (0.9 - 1.3 the details of its characterization is lacking. The goal of this research is to A˚ ) of spin labeled T4 lysozyme were taken at 100 K and are compared to data gain insights into the behavior of the BSL that has been so useful in providing deposited in the protein data bank. details to characterize a variety of protein systems. Fmoc solid phase peptide On this poster we will discuss the angular dependent line shape of the EPR synthesis (SPPS) has been used to generate a series of double cysteine spectra taken at room temperature with respect to the orientation of the spin la- mutants of the 23 amino acid a-helical membrane peptide, AChR M2delta bel as deduced from X-ray crystallography at 100 K. Particular emphasize will at i and iþ4 residue positions. BSLs bearing peptides were incorporated be on the ability of currently available models to describe the observed dy- into 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) bicelles and namics of the spin label and the implications of this for the line shape analysis multilamellar vesicles. The spectra were collected at different temperatures of disordered samples. utilizing continuous wave (CW)-EPR spectroscopy to examine the dynamics of the BSL within a membrane. The EPR spectral lineshape analysis will be 3108-Pos Board B538 conducted to obtain the static and dynamic EPR parameters. This study will Uncertainty Quantification in Deer Spectroscopy using Bayesian Statistical provide more accurate insights into the behavior of BSL in order to provide Inversion Methods the optimal conditions for other studies that may utilize this promising spin Thomas H. Edwards, Stefan Stoll. label. Chemistry, University of Washington, Seattle, WA, USA. Double electron-electron resonance (DEER) spectroscopy is a powerful elec- 3106-Pos Board B536 tron paramagnetic resonance experiment that provides 2-8nm distance mea- EPR Spectroscopic Study of the Voltage-Sensor Domain (VSD) of the surements between spin-labels in systems varying from small, soluble Human KCNQ1 Potassium Ion Channel proteins, to large, membrane-bound complexes. A key strength of DEER is Indra D. Sahu1, Brett M. Kroncke2, Megan M. Dunagan1, Rongfu Zhang1, that it reports a distance distribution corresponding to conformational ensem- Andrew Craig1, Kunkun Wang1, Avnika Bali1, Robert M. McCarrick1, bles in frozen solutions. However, in practice its usable information is often Charles R. Sanders2, Gary A. Lorigan1. limited to the dominant spin-spin distance. Peak widths and shapes, which 1Chemistry and Biochemistry, Miami University, Oxford, OH, USA, describe conformational heterogeneity, are highly sensitive to time-domain 2Biochemistry and Center for Structural Biology, Vanderbilt University, noise, regularization parameters, and spin-label conformations. A robust Nashville, TN, USA. method for quantifying uncertainty in DEER distributions does not currently KCNQ1 is a voltage-gated potassium channel modulated by members of the exist, complicating interpretation. Here we demonstrate the use of Bayesian KCNE protein family. KCNQ1 is essential to both the cardiac action poten- statistical inversion methods to generate conditional covariance estimates and tial that mediates heartbeat and Kþ homeostasis in the inner ear. KCNQ1 Bayesian credibility sets in DEER data analysis. To decouple true uncertainty dysfunction has been linked to multiple diseases, including various cardiac from spin-label conformations, we attached spin labels featuring varying flex- arrhythmias, congenital deafness, and type II diabetes mellitus. Human ibilities to maltose binding protein (MBP) and measured using DEER under KCNQ1 is a 676-residue protein consisting of 100-residue N-terminal cyto- varying conditions. By determining the contributions to DEER data from solic domain, followed by an ~260-residue channel domain containing six both the spin-label and uncertainty, we have taken an important step towards transmembrane (TMD) helices, and a 300-residue cytosolic C-terminus. robust assignment of distance distribution features to protein conformations. The first four TMD helices (S1-S4) form the voltage-sensor domain 3109-Pos Board B539 (VSD) that is linked to the pore domain (helices S5 and S6). Roughly Characterization of Calmodulin Binding to the Ryanodine Receptor by > 40% of the 200 reported disease-related mutations in the KCNQ1 gene Solution and Solid-State NMR result in amino acid substitutions in the VSD, making structural and dynamic Sarah E. Nelson1, Tata Gopinath2, David D. Thomas1, Gianluigi Veglia1. studies of this domain important in unraveling molecular mechanisms in hu- 1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, man pathophysiology. Site-directed spin labeling EPR is a very powerful Minneapolis, MN, USA, 2University of Minnesota, Minneapolis, MN, USA. structural biology technique to study the structural and conformational dy- Calmodulin (CaM) is a 16.7 kDa, Ca2þ-sensing protein that regulates a number namics of membrane proteins in membrane environment. In this study, we of cellular targets based on the amount of Ca2þ available in a cell. One of have successfully expressed the Cysteine substituted Q1-VSD in E. Coli, pu- CaM’s cellular targets is the ryanodine receptor (RyR), a tetrameric Ca2þ chan- rified into detergent micelles, labeled with MTSL spin labels, and collected nel involved in Ca2þ release from the sarcoplasmic reticulum (SR) in myo- CW-EPR spectra. We are developing several advanced EPR spectroscopic cytes. CaM’s regulation of the RyR has been shown to be disrupted due to techniques (such as double electron-electron resonance (DEER) and electron both oxidative modification and naturally occurring mutations in CaM, howev- spin echo envelope modulation (ESEEM)) for probing the structural and dy- er the structural aspects of CaM’s binding to the full RyR, and how this binding namic properties of Q1-VSD in a lipid bilayer environment. This study will is disrupted in disease states, has not been completely characterized. provide important mechanistic information on the Q1-VSD channel upon By applying a combination of solution and solid-state NMR techniques we are binding with KCNE1. able to characterize the structure and dynamics of CaM’s binding interaction with the RyR on a residue-specific basis in a biologically relevant environment. 3107-Pos Board B537 Solution-state NMR experiments provide an assessment of the changes in fast Combining Single Crystal Electron Paramagnetic Resonance and X-Ray timescale dynamics with exposure to the SR, while magic angle spinning Crystallography to Study the Orientation and Dynamics of MTSSL Spin (MAS) solid-state NMR in both extracted SR and reconstituted lipids charac- Labels in T4 Lysozyme terize conformational and topological changes with CaM in the presence of 1 2 3 1 Phillipp Consentius , Bernhard Loll , Ulrich Gohlke , Thomas Risse . the RyR. Our results indicate that CaM does exhibit changes in chemical shift 1Institute of Chemistry and Biochemistry, Physical Chemistry, Freie 2 and dynamics in the presence of the RyR, and that oxidation and disease mu- Universita¨t Berlin, Berlin, Germany, Institute of Chemistry and tations of CaM exhibit structural and dynamics changes compared to WT Biochemistry, Laboratory of Structural Biochemistry, Freie Universita¨t 3 CaM. These results are being applied toward the determination of a structural Berlin, Berlin, Germany, Max-Dellbru¨ck Zentrum, Berlin, Germany. model for CaM bound to the RyR, and structural models of how this interaction The line shape of cw electron paramagnetic resonance (EPR) spectra of spin is altered in the development of disease states. labeled proteins has been used extensively to analyze local dynamics, backbone fluctuations and structural rearrangements of these systems. The commonly 3110-Pos Board B540 used paramagnetic spin label MTSSL, a methanethiosulfonate derivative, Confined Space, Structural Biology, Biophysics and Drug Discovery attached to the protein via covalent disulfide coupling on a designated cysteine Brian Fuglestad1, Christine Jorge1, Bryan Marques1, Nathaniel V. Nucci2, residue serves as a reporter of these properties. Much effort has been made to Evan O’Brien1, Kathleen G. Valentine1, A. Joshua Wand1. extract structural and dynamic information as encoded in the observed line 1Biochemistry and Biophysics, University of Pennsylvania, Perelman School shapes. of Medicine, Philadelphia, PA, USA, 2Departments of Physics & Astronomy In this study we use single crystals of MTSSL labeled T4 lysozyme as a proto- and Biomedical and Translational Sciences, Rowan University, Glassboro, type example of an a-helical protein to investigate the structure and dynamics NJ, USA. of the paramagnetic side chain in detail by a combination of X-ray crystallog- High-resolution multi-dimensional solution NMR is unique as a biophysical raphy and EPR spectroscopy. X-ray diffraction is used to determine the orien- and biochemical tool in its ability to examine both the structure and dynamics tation of the unit cell with respect to the EPR sample tube, which in turn allows of macromolecules at atomic resolution. Conventional non-TROSY/ to determine the orientation of the spin labels for every EPR spectrum of an deuteration solution NMR approaches, however, are largely limited to exam- angular dependent series given that the structure and orientation of the protein inations of relatively small (< 25 kDa) molecules, mostly due to the Wednesday, February 11, 2015 617a spectroscopic consequences of slow rotational diffusion. Encapsulation of Electron Microscopy, Diffraction, and Scattering macromolecules within the protective nanoscale aqueous interior of reverse micelles dissolved in low viscosity fluids has been developed as a means Techniques through which the ‘slow tumbling problem’ can be overcome. This approach has been successfully applied to diverse proteins and nucleic acids ranging 3113-Pos Board B543 up to 100 kDa, considerably widening the range of biological macromole- Cryo-Electron Tomography and Sub-Tomogram Averaging of Isolated cules to which the comprehensive small protein solution NMR methodologies Z-Discs from Honeybee Flight Muscle can be applied. Recently, we have expanded the library of surfactants used Mara Rusu1, Dianne Taylor2, Kenneth Taylor2, John Trinick1. to encapsulate proteins and nucleic acids within the reverse micelle water 1School of Molecular and Cellular Biology, Leeds University, Leeds, United core with high structural fidelity. In addition, new applications have emerged Kingdom, 2Institute of Molecular Biophysics, Florida State University, including studies of protein hydration, dynamic nuclear polarization in Tallahassee, FL, USA. solution, structural characterization of integral membrane proteins and The Z-disc is the mechanical linkage that transmits the active and passive membrane-anchored lipidated proteins. The nanoscale interior of the reverse forces developed by muscle sarcomeres. It is also the location of proteins micelle water core also offers the potential for a range of novel applications involved in diverse other processes, such as stress sensing into signaling path- in drug discovery. With the emergence of fragment based drug discovery, ways controlling muscle growth and wasting. About 40 different proteins are the screening of protein targets for binding to weak ligands has become the now known to be in the Z-disc, some to which are transient and relocate to focus of some interest. NMR is well positioned to provide detailed site- other destinations such as the nucleus. The main route of force transmission specific information about ligand binding to proteins. The confined space is through pairs of antiparallel a-actinin Z-bridges; these link the overlapped of the reverse micelle allows a considerable reduction in ligand required ends of thin filaments from adjacent sarcomeres. The gross structure of the for detection of weak binding and can also significantly extend the detection Z-disc varies widely between different muscles and species. Thickness varies sensitivity into the low mM regime. Examples utilizing dihydrofolate reduc- in proportion to the number of registers of Z-bridges. The lattice of thin fila- tase and aldoketoreductase will be presented. Supported by NIH grants ment ends is hexagonal in invertebrates, whereas in vertebrates it is tetragonal. R01 GM107829, T32 GM071339, T32 GM008275, F32 GM087099 and However the structure of the Z-disc is known only in outline to ~ 7nm resolu- NSF grant MCB-115803. tion and the detailed layout of its components is mostly unknown. Methods to isolate Z-discs date back 50 years but such preparations have not been sub- 3111-Pos Board B541 jected to modern electron microscopy methods, such as cryo-EM, tomography Sara: A Software Environment Supporting Rapid Acquisition and or image processing. An advantage of isolated discs is they are thin, which Analysis of NMR Relaxation Rates with Accordion Spectroscopy obviates the need for sectioning for microscopy, which is damaging. We Bradley J. Harden, Dominique P. Frueh. have prepared Z-discs from honeybee flight muscle using high salt extraction Biophysics, Johns Hopkins University, Baltimore, MD, USA. and density gradient purification. Vitrified preparations were examined in We present SARA (Software for Accordion Relaxation Analysis), an interac- the 300 kV Krios microscope at the MRC Laboratory of Molecular Biology, tive and user-friendly MATLAB software environment designed for analyzing Cambridge, UK. Tilt series images were recorded to þ/ 70 with a Falcon relaxation data obtained with accordion spectroscopy. Accordion spectroscopy II direct electron detector. 3D reconstruction and sub-tomogram averaging can be used to measure nuclear magnetic resonance (NMR) relaxation rates used Protomo software more quickly than traditional methods, yet data analysis can be intimidating and no unified software packages are available to assist investigators. Hence, 3114-Pos Board B544 the technique has not achieved widespread use within the NMR community. Label-Free Molecular Observations of Membrane-Associated Species SARA offers users a selection of analysis protocols spanning those presented using Backscattering Interferometry in the literature thus far, with modifications permitting a more general applica- Michael M. Baksh1, Ashley Lockwood1, Christopher Richards2, M.G. Finn1, tion to crowded spectra such as those of proteins. We discuss the advantages David Heidary3. and limitations of each fitting method and suggest a protocol combining 1Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, the strengths of each procedure to achieve optimal results. In the end, SARA USA, 2Chemistry, University of Kentucky, Lexington, KY, USA, provides an environment for facile extraction of relaxation rates and should 3Chemistry, University of Kentucky, Lexingon, KY, USA. promote routine application of accordion relaxation spectroscopy. Membrane-associated proteins are integral components of cellular processes and disease pathogenesis. Quantitative observations of membrane protein inter- 3112-Pos Board B542 actions are extremely difficult; the membrane environment that is necessary to The Structure and Function of Supramolecular Self-Assembling Binary maintain appropriate structural and functional characteristics of such species Guanosine Gels interferes with or perturbs many analytical methods. In fact, typical assays to Alexander Bruening1,2, Stuart Smith2,3, Linda B. McGown1,2, observe such interactions require the species of interest to be isolated and K.V. Lakshmi1,2. removed from the native membrane environment, usually with covalent modi- 1 Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, fication. We will describe a label-free method to observe and evaluate mem- 2 NY, USA, The Baruch ‘60 Center for Biochemical Solar Energy Research, brane protein-ligand interactions in minimally-altered native membrane 3 RPI, Troy, NY, USA, Physics, Rensselaer Polytechnic Institute, Troy, environments. This strategy is based on the use of backscattering interferom- NY, USA. 0 etry (BSI) in which minute changes in the refractive index of the bulk solution Binary liquid crystals of guanosine (GUO) and guanosine-5 -monophosphate caused by cognate ligand-receptor interactions are observed and quantified (GMP) have been found to have unique and interesting properties in their without the need for extrinsic molecular labeling. Combined with a method ability to solubilize and selectively purify nanoparticles. The thermoassocia- to present membrane proteins in an isotropically-scattering matrix derived tive properties of these GMP-GUO g-gel phases have not been thoroughly from the native cellular environment, Ashlwe are able to observe a variety of explored and the mechanism of action is not understood. In the present study, cognate ligand-receptor interactions over a large range of equilibrium binding nuclear magnetic resonance spectroscopy is used to probe the anisotropic affinities. properties of binary liquid crystals by employing deuterium nuclei. Deuterons have a quadrupolar moment that is normally averaged out by rapid molecular 3115-Pos Board B545 reorientation. In liquid crystals, however, the motion of the molecules is Stabilized, Non-Fouling Transmission Electron Microscopy Grid Coatings constrained such that quadrupolar effects are displayed in spectral data. To for the Selective Capture of His-Tag T7 Virus and His-Tag Gro EL from prevent other effects such as bulk exchange of deuterons from impacting Cell Lysates 2 the signal, H5-labeled aniline is employed to study the supramolecular struc- Christopher J. Benjamin, Kyle J. Wright, Seok-Hee Hyun, ture of the liquid crystalline gels. This isotopomer of aniline has deuterons David H. Thompson. that are strongly bound to a rigid aromatic ring, allowing for study of solvent Department of Chemistry, Purdue University, West Lafayette, IN, USA. directed interactions without concern for conformation or bulk exchange Single-particle reconstruction has grown significantly with the improvements effects. This study determines the anisotropy and order parameter of the in various data collection and computational strategies including CTF fitting, binary liquid crystals to elucidate the structural and functional properties of the use of vitrified samples and the utilization of ultra-sensitive direct electron the system. detectors. Although these improvements have contributed significantly to the *This research is supported by the Photosynthetic Systems Program, Office recent evolution of 3D reconstruction analysis, the way samples are prepared of Basic Energy Sciences, United States Department of Energy (DE-FG02- for electron microscopy has remained largely unchanged. We report the devel- 07ER15903). opment of TEM grids that are modified with a stabilized, non-fouling coating 618a Wednesday, February 11, 2015 bearing nitrilotriacetic acid substituents at the end of a poly(ethylene glycol)- and mitochondria, and of single-membrane organelles such as the ER, lyso- grafted surface that promotes specific capture of protein targets for single some and peroxisome using ImageJ and Amira 3D software. Using UHVEM particle reconstruction analysis. The utilization of these grids for specific tomography, we also observed the 3D-structure of phycobilisomes, which are adsorption of the targeted protein onto the grid surface results in well- essential supramolecular complexes on the surface of the thylakoid membrane controlled surface concentration enhancements and a days-to-minutes reduc- in chloroplasts. Although many reports have provided structural models, we tion in time required for the preparation of a purified sample for cryoEM anal- offer the first 3D-structural model of the membrane surface from specimens ysis from an E. coli expression system. The selective and reversible capture that were not purified using specific detergents. of his-tag T7 bacteriophage and GroEL from crude lysates, as well as purified nanodisc-solubilized his-malFGK2, on these NTA-modified grids with an 3118-Pos Board B548 exceptionally low level of adsorption by non-target proteins has been observed. Regulation of Myosin VI Studied by Electron Microscopy Our data illustrates the utility of these grids for selective capture from complex Dario Saczko-Brack, Heike Ellrich, Christine Werner, Christopher Batters, mixtures, detergent-solubilized membrane protein isolates, and expression Claudia Veigel. systems yielding low copy numbers of the desired target in a manner that is Department of Cellular Physiology, Ludwig-Maximilians-Universita¨t well-suited for single particle reconstruction analysis. Mu¨nchen, Munich, Germany. Myosins are ATPase motor proteins that are activated by and traffic along actin 3116-Pos Board B546 filaments. This large protein family is divided into many classes with different Scanning Transmission Electron Tomography of Blood Platelets in Thick functional properties and specializations for various roles, including membrane Sections anchorage, longer range transport of cargo vesicles or cell signaling. Jake D. Hoyne1, Gina N. Calco1, Bryan C. Kuo1, Maria A. Aronova1, Myosin class VI is unique due to its reversed directionality along actin fila- Alioscka A. Sousa1, Qianping He1, Guofeng Zhang1, Irina D. Pokrovskaya2, ments, moving towards the pointed end, in contrast to almost all other classes, Laura MacDonald2, Andrew A. Prince2, Brian Storrie2, which move towards the barbed end of F-actin. Whilst the directionality is well Richard D. Leapman1. studied, other characteristics such as activation, cargo and lipid binding or 1NIBIB, National Institutes of Health, Bethesda, MD, USA, 2Department of dimerization are not fully understood. Using size exclusion chromatography, Physiology and Biophysics, University of Arkansas for Medical Sciences, titration studies and gliding filament assays we investigated myosin VI back- Little Rock, AR, USA. folding, cargo binding and mechanical activity. Electron tomography in the scanning transmission electron microscope (STEM) Furthermore, we applied electron microscopy and single particle image pro- can be performed on sections of stained plastic-embedded tissues or cells of 1 to cessing to determine the structural properties of myosin VI in different ionic 2 micrometer thickness without effects of chromatic aberration because there and nucleotide conditions. Two dimensional class averages based on various are no imaging lenses after the specimen. By using a small STEM probe conver- alignment and classification methods were made that allow for a detailed struc- gence angle of 1-2 mrad the geometrical broadening of the probe is restricted, tural analysis including a comparison with crystal structures. which enables a spatial resolution of a few nanometers. Furthermore, by using 3119-Pos Board B549 an axial bright-field detector instead of the standard high-angle annular dark- The Steric Fine Structure of Maurer’s Cleft in ‘‘Unroofed’’ Plasmodium field detector, image blurring due to multiple elastic scattering can be reduced Falciparum-Infected Erythrocytes in the lower part of the specimen. Here, we have applied STEM tomography to Eri H. Hayakawa1, Fuyuki Tokumasu2, Jiro Usukura3, Hiroyuki Matsuoka1, elucidate the 3D ultrastructure of human blood platelets, which are small anu- Takafumi Tsuboi4, Thomas E. Wellems5. cleate blood cells that aggregate to seal leaks at sites of vascular injury and are 1Lab of Medical Zoology and Parasitology, Department of Infection and important in the pathology of atherosclerosis and other diseases. Of particular Immunity, Jichi Medical University, Shimotsuke, Japan, 2Department of interest are the morphological changes that occur in alpha-granules, which Lipidomics, Grad school of Medicine, The University of Tokyo, Tokyo, contain important proteins released when platelets are activated. Axial bright- Japan, 3Division of Integrated Project, EcoTopia Science Institute, Nagoya field STEM electron tomographic tilt series were acquired at an accelerating University, Nagoya, Japan, 4Malaria Research Unit, Cell-Free Science and voltage of 300 kV from 1.5-micrometer thick sections of platelets that had Technology Research Center, Ehime University, Matsuyama, Japan, 5Lab of been prepared by rapid freezing and freeze-substitution; and the tomograms Malaria and Vector Research, National Institute of Allergy and Infectious were reconstructed from dual-axis tilt series. The tomographic reconstructions Diseases/NIH, Bethesda, MD, USA. revealed changes in ultrastructure that occurred on platelet activation including Plasmodium falciparum produces additional membrane systems, Maurer’s cleft release of alpha granules through channels connecting to the plasma membrane. (MC) and tubulovesicular network (TVN), in the host erythrocytes. The para- The research was supported by the intramural program of the National Institute sites use these membrane compartments to transport proteins to the surface of of Biomedical Imaging and Bioengineering, and the research in the Storrie lab- erythrocytes. Previous studies reported the structure of MCs by transmission oratory was supported in part by NIH grant R01 HL119393. electron microscopy (TEM) using ultra-thin layer specimen and suggested physical connections between MCs and erythrocyte membrane via an extension 3117-Pos Board B547 of MC membrane. However, fine structures of MC including filamentous exten- Three-Dimensional Microstructural Visualization of Mitosis using sions smaller than the thickness of diamond knives were likely missing in Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) and 3Mv the TEM images. To obtain intact structural information of MCs, we used Ultra-High Voltage Electron Microscope (UHVEM) Tomography with unroofing/rip-off technique for both normal- and parasitized-erythrocytes and Nanoscale Resolution at Whole Cell Level successfully captured accurate oval/global shape of MCs with elongated-fine Atsuko H. Iwane1,2, Keisuke Ohta2,3. < 1 2 3 filamentous extensions (diameters 10 nm). We also treated parasitized eryth- Osaka University, Suita, Japan, QBiC, RIKEN, Suita, Japan, Kurume rocyte with aluminum tetrafluoride, which are known to inhibit intracellular University, Kurume, Japan. vesicle transport, to clarify if the oval/global structures are MCs. In the pres- To better understand fundamental cellular properties, such as differentiation ence of aluminum tetrafluoride, the vesicle was no longer observed in parasit- and division, we are developing whole single cell 3D-structure analysis tech- ized erythrocytes. This result was in agreement with the previous study (Trelka nologies based on innovative electron microscopy. These new techniques are DP, et al., Mol Biochem Parasitol, 2000), demonstrating the oval/global struc- designed to reveal the dynamics and structure of intracellular material such tures are MCs which extends filaments to host erythrocyte membrane. Our EM as organelles and supramolecular proteins. Our main technologies include (1) images demonstrated that MCs in P. falciparum-infected erythrocyte involve Cryo-Tomography using Scanning-TEM and (2) FIB (Focused Ion Beam)- fine filaments reaching erythrocyte membrane which may provide a direct SEM and 3D-reconstruction. FIB-SEM is normally used to visualize metals transport pathway for their proteins to the surface of erythrocytes. and ceramics. We have modified it for the 3D reconstruction of an entire cell at a nanoscale resolution that lies between those of electron microscopy tomog- 3120-Pos Board B550 raphy and X-ray tomography. Towards Femtosecond Electron Diffraction of Proteins - Technical Last annual meeting, we described how FIB-SEM could visualize the basic 3D Challenges and Sample Preparation Strategies architecture of Cyanidioschyzon merolae (C. marolae). C. marolae is a primi- Henrike M. Mueller-Werkmeister1,2, Daniel Badali3, Oliver P. Ernst2, tive unicellular red algae whose cell division can be observed my manipulating R.J. Dwayne Miller1,3. the light/dark cycle. By synchronizing cells to a 6-h light/18-h dark cycle, we 1Chemistry, University of Toronto, Toronto, ON, Canada, 2Biochemistry, obtained > 75% S/M-phase cells at 89 hrs after synchronous culture start. University of Toronto, Toronto, ON, Canada, 3Max-Planck-Institute for Using these cells and FIB-SEM, we observed unique architectures of whole Structure and Dynamics of Matter, Hamburg, Germany. C. merolae cells during the mitotic cycle and successfully made 3D-models To study protein dynamics in real-time with atomic resolution is one of the of individual double-membrane organelles such as the nucleus, chloroplast dream experiments in biophysics. Up to now experimental tools with full Wednesday, February 11, 2015 619a atomic and ultrafast time resolution combined are lacking, yet will become [1] Lu, Z., et al. (2009). J. Struct. Biol. 168, 388-395. available. Femtosecond electron diffraction (FED) is a promising tabletop tech- [2] Shaikh, T.R., et al. (2014). Proc. Natl. Acad. Sci. USA 111, 9822-9827. nique with complementary features to XFEL measurements [Miller, R.J.D, Sci- ence, 2014, 343, 1108-1116]. It combines the high spatial resolution of 3123-Pos Board B553 diffraction methods with the high temporal resolution of femtosecond optical A Computational Modeling of Macromolecular Ensemble Conformation spectroscopy. The current electron brightness and time-resolution enabled first and Blurring in Cryo EM 1 2 2 studies of structural dynamics with atomic resolution on a time-scale of few Bijan Afsari , Jin Seob Kim , Gregory Chirikjian . 1Center for Imaging Science, Johns Hopkins University, baltimore, MD, hundred femtoseconds [Gao, M. et al., Nature, 2013, 496, 343-346] and even 2 gave insight into the structural dynamics during a chemical reaction [Jean- USA, Department of Mechanical Engineering, Johns Hopkins University, Ruel, H. et al., J. Phys. Chem. A, 2011, 115, 13158-13168]. The main challenge baltimore, MD, USA. for first applications of FED on proteins is sufficient sample preparation and It is well known that Cryo EM results in 3D construction in which subnuits of a development. Currently samples need to be single-crystalline, reversible and macromolecular complex may appear to be blurred and bloated. This issue af- match a size of about 100-150 nm thickness combined with a lateral size on fects the accuracy of positional and orientational information about the subnits the order of 100 mm x 100 mm. The presented work will outline and discuss cur- extracted from such reconstructions. The blurring can be due to classification or rent approaches for sample development on model systems Bacteriorhodopsin averaging steps in the analysis or due to mobility of the quaternary macromo- and give an overview on the femtosecond electron diffraction method including lecular structure. Such effects cannot be captured by traditional models of EM. the limitations for samples. Latest results on a crystalline organic spin- In this paper we propose a mathematical framework and algorithm to model crossover system [Jiang, Y., Ultrafast Phenomena XIX, 2014, in press] as this phenomenon. We use clustering methods such as the K-means algorithm well as on the model system Bacteriorhodopsin are reported to demonstrate to roughly resolve the different rigid subunits in the 3D density obtained the intrinsic capabilities of FED. from a macromolecular complex. Then we model the blurring effect in each (resolved) submit as an ensemble of rigid body motions (i.e., orientations 3121-Pos Board B551 and translations), specifically we use the Gaussian probability density (with un- Development of Cryo-Electron Microscopy Sample Preparation for the known covariance and mean) on the group of rigid body motions in 3D. We Examination of Nanobubble relate the shape of each resolved (blurred) subunit to the unknown parameters Xi Zhan, Lige Tonggu, Mo Li, Liguo Wang. of the Gaussian using the known structure of the subnit (from PDB). This gives Biological Structure, University of Washington, Seattle, WA, USA. a system of equations with unknowns being the parameters of the Gaussian. Cryo-electron microscopy (cryo-EM) has been widely used to characterize The system, in general, is underdetermined, however, we impose physically bio-macromolecules, such as DNA molecules, proteoliposomes, and protein meaningful regularization constraints to obtain unique solutions. Thereby we complexes. The targets of interests were effectively remained the original struc- are able to obtain more accurate orientation and positional information. We ture and morphology in amorphous ice. However, in cryo-EM, target of inter- show the performance of the algorithm on simulated data. We also discuss pros- ests in gas phase has not been studied yet. In this study, we utilized cryo-EM pect of this method in combination (or fusion) with SAXS data in order to rein- to examine the nanobubbles in frozen aqueous solution with a ‘‘sandwich’’ force information from both modalities. TEM grids assembly. With significantly increased surface area and lower 3124-Pos Board B554 buoyancy, nanobubble system greatly enhances the efficiency of oxygenation A Computational Modeling of Macromolecular Assemblies in SAXS and the time of retention in medical application. The substrate of ‘‘sandwich’’ Jin Seob Kim1, Bijan Afsari2, Gregory S. Chirikjian1. assembly provides sufficient nucleation site for the formation of bubbles. Addi- 1Mechanical Engineering, the Johns Hopkins University, Baltimore, MD, tionally, elimination of water-air interface increases the possibility to capture USA, 2Center for Imaging Science, the Johns Hopkins University, Baltimore, bubbles in nano scale. This method demonstrates a promising way to evaluate MD, USA. bubble system using cryo-EM and provides an insight on the study of nanobub- The determination of the structure of large macromolecular complexes is essen- ble in biomedical application. tial to reveal the function of the complexes in biological setting. For that pur- pose, several experimental methods for the structural determination have been 3122-Pos Board B552 developed and applied such as Electron Microscopy (EM) and Small-Angle X- Time-Resolved cryo-EM Study of Ribosome Subunit Association by ray Scattering (SAXS). In this study, we focus on SAXS. In particular, we are Mixing-Spraying interested in determining the spatial relationship between rigid subunits in a Bo Chen1, Sandip Kaledhonkar2, Ming Sun1, Bingxing Shen3, given macromolecular complex, which is especially important because confor- Ruben L. Gonzalez4,5, Joachim Frank1,2. 1 mational dynamics of rigid components in the given complex is directly related Dept. of Biological Sciences, Columbia University, New York, NY, USA, to its function. The present work is to develop a more efficient and effective 2Dept. of Biochemistry and Molecular Biophysics, Columbia University, 3 computational framework to reveal spatial relationship between complex sub- New York, NY, USA, Howard Hughes Medical Institute, Columbia units. One of important quantities in SAXS includes the so-called distance University, New York, NY, USA, 4Dept.of Biological Sciences, Columbia 5 distribution function (or pair distribution function), which is the distribution University, New York, NY, USA, Dept. of Chemistry, Columbia University, of distance between every pair of points in the complex. Given rigid sub- New York, NY, USA. components of the complex, this important quantity can be determined more Time-resolved cryogenic electron microscopy (cryo-EM) is a technique for efficiently when we apply the mathematical concept of the Fourier transform visualizing transient structures in a biological specimen in a pre-equilibrium sys- for rigid-body motion group. To this end, we develop a mathematical model tem. Capturing reactions in the sub-second range has been a practical challenge, to calculate the pair distribution function for a complex structure consisting due to the requirement of depositing the specimen rapidly on the grid without of several rigid sub-components. This new model is verified with several exam- blotting. To capture faster reactions in the sub-second range, Lu and coworkers ples. In the end, our modeling efforts will combine the current methodology [1] developed a mixing-spraying method, which allows a reaction involving two with similar ones for other experimental methods (e.g. EM) to reveal more macromolecular components to proceed for tens to hundreds of milliseconds. refined biological macromolecular structures. The first study of ribosome subunit association using time-resolved cryo-EM, by Shaikh et al. [2], were performed at 9.4 ms and 43 ms. In present work, 3125-Pos Board B555 we improved the mixing-spraying method, by designing an environmental Sub-Surface Serial Block Face Scanning Electron Microscopy chamber and optimizing the EM data yield, and applied the method to the Qianping He1, Maria A. Aronova1, David C. Joy2,3, Guofeng Zhang1, study of ribosome subunit association. We captured the subunit association re- Richard D. Leapman1. action in a pre-equilibrium state, by mixing the subunits and reacting for 60 ms 1NIBIB, National Institutes of Health, Bethesda, MD, USA, 2Department of and 140 ms. Our results showed that at 60 ms and 140 ms time points, 33% and Material Science and Engineering, University of Tennessee, Knoxville, TN, 42% of the large subunits have associated into 70S ribosomes, respectively, USA, 3Center for Nanophase Material Sciences, Oak Ridge National compared with 85% in a 15-min incubation control experiment. Three distinct Laboratory, Oak Ridge, TN, USA. conformations of the 70S ribosome were found: non-rotated, non-rotated with Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale 30S head swiveled and rotated. Our results demonstrate the capability of the 3D ultrastructure of tissue samples up to several hundred micrometers in size. mixing-spraying method of time-resolve cryo-EM to visualize multiple states In SBF-SEM, an ultramicrotome built into the SEM specimen stage succes- of macromolecules in a reaction within a sub-second time frame. In the future, sively removes thin sections from a plastic-embedded, heavy metal-stained the mixing-spraying method will be applied to study translation initiation, to specimen. After each cut, the freshly exposed block face is imaged at a low gain insights on the role of mRNA, initiator tRNA and initiation factors. incident electron energy using the backscattered electron signal, which is sen- Supported by HHMI and NIH R01 GM29169 and GM55440 (to J.F.) sitive to heavy atoms in the sample. Although the x-y resolution in the plane of 620a Wednesday, February 11, 2015 the block face is approximately 5 nm, the resolution along the z-axis in SBF- 3128-Pos Board B558 SEM is limited by the minimum slice thickness of around 25 nm. We have Accurate Determination of Tautomeric/Protonation States in Quantum- explored the feasibility of improving the z-resolution in SBF-SEM by recording Mechanic Driven Macromolecular Crystallographic Refinement images at more than one primary beam energy, thus sampling different depths Oleg Y. Borbulevych, Lance M. Westerhoff. below the block surface. We used Monte Carlo simulations of SEM images Research & Development, QuantumBio Inc., State College, PA, USA. from an epoxy block containing 5-nm diameter carbon spheres stained with Structure Based Drug Discovery (SBDD) is employed by virtually all pharma- 14% osmium positioned at different depths, as a model for small biological ceutical research and development organizations. Gaining an understanding of structures. A linear relationship was found between the depth of the spheres the protein:ligand complex structure along with the proper protonation and and the ratio of backscattered signals at primary beam energies of 1.4 keV explicit solvent effects is crucial for obtaining meaningful results from dock- and 6.8 keV, which allowed us to generate 3D tomograms with a depth resolu- ing, thermodynamic calculations, active site exploration, and ultimately lead tion of around 5 nm. Experiments are in progress to test this technique using a optimization. Recently, we incorporated our linear-scaling, quantum me- Zeiss Sigma-VP SEM equipped with a Gatan 3View SBF system. Sub-surface chanics (QM) DivCon tool with Phenix (e.g. Phenix/DivCon) in order to accu- SBF-SEM could potentially match focused ion beam (FIB) SEM in terms of rately elucidate the protein:ligand complex molecular structure. An intrinsic z-resolution, but with the added advantage of providing higher throughput problem of the X-ray crystallographic data is its inability to detect hydrogen and larger tissue volumes. The research was supported by the intramural pro- atoms - even at higher resolutions. It is generally extremely difficult to exper- gram of NIBIB. imentally determine the protonation/tautomeric state of the ligand and the surrounding active site. Traditionally, protonation can be established using 3126-Pos Board B556 the neutron diffraction; however, experimental requirements such as reliance Fixed Path Length Sample Holders Enable Robust Cryosaxs Measure- on very large crystals and on deuterium exchange limit the method’s suit- ments from Sub-Microliter Sample Volumes ability in SBDD. Andrea M. Katz, Jesse B. Hopkins, Steve P. Meisburger, In order to address this X-day data deficiency, we have challenged Phenix/ Matthew A. Warkentin, Robert E. Thorne, Lois Pollack. DivCon with various protonation candidates and applied rigorous statistical an- Cornell University, Ithaca, NY, USA. alyses to measure the agreement between the 3D structure of each candidate Small angle x-ray scattering (SAXS) gives structural information about bio- with electron density. While through the experiment we still cannot directly logical molecules in solution. However, large (~30 microliter) sample volumes observe hydrogen atoms, using the accurate QM functional we are able to are needed to mitigate radiation damage, limiting the use of SAXS in studying observe the presence/absence of hydrogen atoms by studying their influences rare molecules. By cryocooling SAXS samples, radiation damage and required on bound heavy atoms (Carbon, Nitrogen, Oxygen). To evaluate our protocol sample volumes are reduced by orders of magnitude [1], but challenges in we have chosen two protein:ligand structures 4N9S and 2JJJ for which both creating identically-sized frozen samples complicate background subtraction. neutron and X-ray structures and data are available in PDB. Ten probable pro- Here we present microfabricated silicon sample holders for cryoSAXS. These tonation states for the ligands in those structures have been generated, and each rigid sample holders have a fixed x-ray path length, simplifying background of the possible candidates has been refined against X-ray data with Phenix/ subtraction. Less than 800 nL of sample are required, facilitating measurements DivCon. We have found out that the top scored tautomer in each case coincides on expensive or hard-to-express molecules. These fixed path length, low vol- with the ligand structure revealed by the neutron diffraction. ume sample holders make cryoSAXS a more accessible technique capable of probing a wide range of biological molecules. 3129-Pos Board B559 1. S. P. Meisburger et al. Biophys. J. 104, 227 (2013). Transmission X-Ray Imaging Detector Captures the Last Light at NSLS Jen Bohon1, Erik M. Muller2, Wenxiang Ding3, Mengjia Gaowei4, 3127-Pos Board B557 Tianyi Zhou5, John Smedley4. 3D Dynamical Observations of Single Molecule Motions by X-Rays, 1Center for Synchrotron Biosciences, Case Western Reserve University, Electron and Neutron Upton, NY, USA, 2Department of Physics and Astronomy, Stony Brook Yuji C. Sasaki1, Keigo Ikezaki1, Kouhei Ichiyanagi2, Hiroshi Sekiguchi3, University, Stony Brook, NY, USA, 3Department of Electrical and Computer Naoto Yagi3. 1 2 Engineering, Stony Brook University, Stony Brook, NY, USA, The University og Tokyo, Kashiwa-city, Japan, KEK, Tsukuba, Japan, 4 3 Instrumentation Division, Brookhaven National Laboratory, Upton, NY, Research & Utilization Div., JASRI/SPring-8, Sayou-gun, Japan. USA, 5Department of Material Science and Engineering, Stony Brook We have proposed that single molecule techniques using shorten wavelengths, University, Stony Brook, NY, USA. for example, X-rays, electrons, and neutron [1]. Especially, Diffracted X-Ray Biological research constitutes a large and expanding scientific focus at syn- Tracking (DXT) using normal synchrotron orbital radiation (SR) source chrotron facilities. Structural biology researchers using x-ray facilities make (not XFEL) has been developed for obtaining the information of the 3D internal up the majority of this community, including use of techniques such as macro- motions of single proteins with both high time-resolution (micro-seconds) and molecular x-ray crystallography, small-angle x-ray solution scattering, x-ray high precision (nm/1000) [2, 3]. DXT can be monitored through trajectories of microscopy, x-ray absorption spectroscopy, x-ray fluorescence and x-ray foot- the Laue diffraction spots from the nanocrystal which was labeled on the indi- printing. Many of these technologies, as they are developed to take advantage vidual proteins. This concept can apply to utilize by using both electrons and of next-generation synchrotron sources, are trending toward use of high flux neutron. Instead of the Laue diffraction using white X-ray, the Electron beams and/or beams which require enhanced stability and precise understand- Back-Scattered Diffraction Pattern was adopted to monitor the 3D orientations ing of beam position and intensity from the front end of the beamline all the of the nanocrystals linked to the single protein molecules[4]. We called Dif- way to the sample. For high flux beams, major challenges include heat load fracted Electron Tracking (DET). Additionally, we call Diffracted Neutron management in optics (including the vacuum windows) and a mechanism of Tracking (DNT) for new single molecule measuring method in which the real-time volumetric measurement of beam properties such as flux, position, long time observation from the non-destructivity of a neutron is possible. and morphology. For beam stability in these environments, feedback from DXT, DET and DNT are assigned to labeling techniques through the nanocrys- such measurements directly to control systems for optical elements or to sample tals. The size effect between intramolecular motions of individual proteins and positioning stages would be invaluable. For x-ray footprinting, a focused the labeled nanocrystals becomes very important. We succeeded in the analysis ‘‘white beam’’ is used to maximize x-ray flux density over a practical sample of the quantitive size effects. As a result, we pointed out a possibility that size using a toroidal mirror. This intense beam can melt beryllium windows determinations of the intramolecular motions without labeled nanocrystals and is very complicated to measure, causing difficulties with properly focusing are carried out quantitatively. Additionally, by progressing of the automatic the mirror and with understanding where the beam is and exactly what is being DXT analysis corresponding to huge diffraction information, we obtained the delivered to the sample. To address these challenges, we are developing time-resolved dynamical information that statistical reliability is sufficiently diamond-based instrumented vacuum windows with integrated volumetric x- high. ray intensity, beam profile and beam-position monitoring capabilities. The pro- [1] Y. C. Sasaki, pp209-234 FUNDAMENTALS OF PICOSCIENCE, CRC totype device will be used as the exit window for the XFP beamline currently Press (2013). being developed at NSLS-II for x-ray footprinting. Current progress is pre- [2] H. Sekiguchi et al, PLOS ONE 8:e64176 (2013) sented, including successful demonstration of a >1kilopixel free-standing [3] H. Sekiguchi et al, Scientific Reports 4:6384 (2014) transmission imaging detector that was used to capture the last x-ray photons [4] N. Ogawa et al, Scientific Reports 3:2201 (2013) at the National Synchrotron Light Source. Wednesday, February 11, 2015 621a

Optical Spectroscopy: CD, UV-VIS, Vibrational, 3132-Pos Board B562 Detection and Identification of Amino Acids in Ficoll Solutions with Fluorescence Femtosecond Laser-Induced Breakdown Spectroscopy Poopalasingam Sivakumar, Yury Markushin, Elton Jhamba, Zakaria MRah, 3130-Pos Board B560 Leon A. Taleh, Angel Fernancdez, Noureddine Melikechi, Hacene Boukari. Molecular Mobility in Amorphous Sucrose Films Monitored by Riboflavin Physics and Engineering, Delaware State University, Dover, DE, USA. Phosphorescence - Potential Applications in Edible/Biodegradable Films We report femtosecond Laser-induced Breakdown Spectroscopy (fs-LIBS) Yan L. Wang, Maria G. Corradini, Richard D. Ludescher. measurements on several amino acids (Serine, Glutamine, and Cysteine) and Department of Food Science, Rutgers University, New Brunswick, NJ, USA. Albumin protein solutions mixed with ficoll at different concentrations. The The physical properties of amorphous biomolecules play a critical role in sta- goal is to assess the effects of a host matrix on the identification and spectral bility of food, pharmaceutical, and biotechnological products. Consequently, characterization of amino acids by fs-LIBS. fs-LIBS utilizes an intense short they have an impact on formulation design and quality control of amorphous laser pulse to obliterate a sample into basic constituents and to record the emis- products. sion spectrum of atoms, ions, and molecules in the cooling down of the plasma Molecular mobility, heterogeneity, and air permeability have been monitored plume. Several spectral peaks associated primarily with elemental composition in amorphous materials using Generally Recognized as Safe (GRAS) lumines- of a sample were observed in the fs-LIBS spectra in a range from 200 to 950 cent probes, i.e., tryptophan and erythrosine B. Given its GRAS status and nm. In addition, some molecular information associated with diatomic vibra- widespread availability, we hypothesize that riboflavin can be used as a conve- tional modes in certain molecules such as C-C and C-N were also obtained. nient and safe intrinsic luminescent probe to study the physical properties of The presence of ficoll affects the relative intensity and broadening of the C- amorphous films. Riboflavin phosphorescence has only been reported at tem- N band, which is a signature of the amino acids. The influence of ambient peratures below 77K and in solid matrices. gas on the fs-LIBS measurements was also analyzed under helium and argon The objectives of this study were: 1)to investigate riboflavin phosphorescence environments, and a different behavior of peaks associated with He and Ar sensitivity towards molecular mobility in a disaccharide model system, 2)to as a function of amino acids and ficoll mixture composition was observed. characterize the temperature dependence of riboflavin phosphorescence in The fs-LIBS data and their analysis compare favorably with those derived amorphous films. from Fourier Transform Infrared Spectroscopy (FTIR). Interpretation of the Sucrose was used as a model system to assess riboflavin’s applicability as spectral information enclosed in the emission of the diatomic molecules during a phosphorescent probe of the physical state of edible films. Steady-state laser ablation may lead to a better understanding of plume chemistry with a and time-resolved phosphorescence of riboflavin in amorphous sucrose direct consequence on chemical analysis of complex samples such as amino films were collected over the temperature range from 30 Cto60C. acids. Altogether, the results demonstrate the potential of fs-LIBS technique Emission spectra were fitted with a log-normal function. The performance as a detection method of biomolecules and for probing interactions of these bio- of a stretched exponential and a multi-exponential function were evaluated molecules with a host matrix. to characterize riboflavin lifetimes. The rate constant of non-radiative decay (kTS0), a measure of molecular mobility, was calculated from riboflavin’s average lifetime. Measures of the distribution of energetically 3133-Pos Board B563 distinct environments were obtained from bandwidth (G), stretching expo- Fluorescence Anisotropy Measurements of Fluoroscein Mixed with Ficoll nential factor (b), and fractional amplitudes (ai) parameters. The analysis Solutions of fitted parameters as a function of temperature revealed two temperature Elton Jhamba1, Zakaria M’Rah2, Yuriy Markushin2, regimes below Tg in sucrose films with a transition at 10C. Implications Noureddine Melikechi2, Hacene Boukari2. of these findings on riboflavin’s potential applications as a luminescent 1Physics and Engineering, Delaware State University, Dover, DE, USA, probe of physical properties of amorphous biomolecules and edible films 2Physics and Engineering, Delware State University, Dover, DE, USA. are discussed. We have conducted fluorescence anisotropy measurements of fluorescein fluo- rophores embedded in Ficoll solutions. Ficoll, a highly branched, hydrophilic polysaccharide, is used as a model crowding agent for studies of the effects 3131-Pos Board B561 of crowding on the behavior of nanoparticles. Fluorescein is a bright fluoro- Improving Far UV Circular Dichroism Calculations of Peptides and phore with well-known fluorescence spectrum. We have collected the measure- Proteins with the Dipole Interaction Model ments with an ISS K2 frequency-domain fluorometer (470-nm excitation Akongnwi C. Jungong1, Jenna Soukup1, Tsvetan Aleksandrov1, Rahul Nori1, wavelength and 1-100 MHz modulation frequency), and determined the fluo- Emma Miller1, Igor Uporov2, Kathryn A. Thomasson1. rescence lifetime, the fluorescence anisotropy, and the rotational correlation co- 1Chemistry, University of North Dakota, Grand Forks, ND, USA, efficient of the fluorescein as a function of ficoll concentration (up to 1200 mg/ 2Bioengineering and Bioinformatics, M. V. Lomonosov Moscow State ml.) As the ficoll concentration is increased, the lifetime of the fluorophores University, Moscow, Russian Federation. show little change from 4.08 in water to 4.23 in 1200 mg/ml ficoll. However, The dipole interaction model (assembled into the computer package the rotational correlation coefficient measurements show significant changes. DInaMo) uses a classical electromagnetic theory for calculating the far At low ficoll concentrations no measureable interactions was observed. As UV circular dichroism (CD) of peptides and proteins. DInaMo reduces the ficoll concentration is increased, two rotational modes emerge for better all amide chromophores to a single point with anisotropic polarizability fitting of the data, which can be associated with free fluorophores and and all nonchomophoric aliphatic atoms to points with isotropic polariz- fluorophore-ficoll interactions. Above 1000 mg/ml ficoll solutions the data ability. By determining interactions among the chomophoric and nonchro- can be fit with a single rotational mode due to slowing down of the rotation moporic parts of the molecule using empirically derived polarizabilities, of fluorophores. We compare the results from the fluorescence anisotropy the rotational and dipole strengths are determined leading to the calculation and lifetime measurements with those from viscosity measurements and fluo- of CD. Polarizabilities are largest for the chromophoric points and smaller rescence correlation spectroscopy measurements, the latter being related to for the nonchromophoric points with hydrogens having the smallest polar- the translation diffusion. izabilities. It is possible to collapse hydrogen polarizability onto the atom to which it is bound or ignore it in the CD calculation creating a united 3134-Pos Board B564 atom approach. Ignoring CH3 group hydrogens and treating only the p-p* transition reproduces experiment in the region of 180 to 210 nm Development of a Quantum-Mechanical Analysis of Stark Effects of well, showing bands with similar morphology and absorption maxima for Porphyrins Employed as Sensors of Internal Electric Fields in Biological the p-p* transition. Recent calculations on small peptides and proteins in Systems Hannah E. Wagie, Jorg C. Woehl, Peter Geissinger. which both CH3 and CH2 group hydrogens have been ignored seem to pro- Chemistry & Biochemistry, University of Wisconsin - Milwaukee, duce better CD results than ignoring only the CH3 group hydrogens. Also, Milwaukee, WI, USA. preliminary calculations using the mean polarizability values of the CH3 Chromophores in biological systems, such as heme, are exposed to strong and CH2 groups show some good prospects of further improving the CD calculations with the DinaMo package. In addition, initial calculations electric fields that are generated by charge distributions of the biosystems indicate that DInaMo has the potential of including the n-p* transition in themselves. These ‘‘internal electric fields’’ may be of magnitude of several the CD calculations. MV/cm, and, thus, may contribute to functional properties of a system. The 622a Wednesday, February 11, 2015 ability to measure such fields would help to elucidate the importance of ‘‘elec- UV-Vis scans show that the amount of change in absorbance at specific wave- trostatic structure’’ to a biosystem’s function. Our approach of measuring inter- lengths, coupled with characteristic wavelength shifts produced by different nal electric fields relies on changes to electronic transition energies of a solvents, can be used for detection of counterfeit drugs. The methods presented chromophore when exposed to an electric field, i.e., the ‘‘Stark effect’’. here could be used for quality control of medicines at or near the point of care in Analyzing the Stark shifts allows for extraction of magnitude and orientation parts of the world at higher risk of encountering defective pharmaceuticals. the internal electric field. Classically, the Stark shift is analyzed with a series of terms that are linear, quadratic, etc., in the electric field, which requires 3137-Pos Board B567 knowledge of the polarizability tensors for both ground and excited states to Hyperspectral Analysis of Laurdan Emission Spectra in Red Blood Cells extract the internal electric field. Usually the analysis is restricted to term linear and Giant Unilamellar Vesicles 1 2 2 in the external electric field, which for randomly oriented chromophores leads Catherine Leonard , Abdelmounaim Errachid , Julie Daubie , 3 2 1 to a systematic error. We pose a quantum-mechanical alternative to the clas- Didier Beghuin , Pierre-Jacques Courtois , Marie-Paule Mingeot-Leclercq , Donatienne Tyteca2. sical analysis based on infinite-order perturbation theory that overcomes the 1 neglect of non-linear terms. The challenge with the quantum-mechanical anal- Louvain Drug Research Institute (LDRI) - Cellular and Molecular ysis then becomes that, theoretically, it may require the description of an infin- Pharmacology Unit (FACM), Universite´ Catholique de Louvain (UCL), Brussels, Belgium, 2de Duve Institute (DDUV) - Cell Biology Unit (CELL), ite number of electronic transitions to account for the Stark shift. Here, we 3 present a method for determining the number and identity of electronic transi- Universite´ Catholique de Louvain (UCL), Brussels, Belgium, Lambda-X, tions that are required for quantum-mechanical Stark analysis, essentially open- Gosselies, Belgium. ing a practical path to using this more exact analytical option. As Lipid rafts are membrane microdomains enriched in cholesterol and sphingoli- demonstration, we apply this new protocol to a porphyrin sensor used to mea- pids that regulate the activity of several membrane proteins and their associated sure the internal electric field of myoglobin and show the accessibility of the pathways [1]. Laurdan, a fluorescent probe exhibiting a blue shift in emission method with reasonable computational resources. (from 440 to 490 nm) upon decreasing membrane order, has been extensively used to quantify and visualize membrane domains [2]. We here applied hyper- 3135-Pos Board B565 spectral microscopy to gain better insights on the variation of Laurdan emission MDDQM Investigations of the Length Scale and Forcefield Dependence of spectra in relation with membrane composition. As models, we used red blood the Time Dependent Fluorescent Stokes Shift of Wild Type Staphylococcal cells (RBCs) as the simplest and most characterized cell system, and giant uni- Nuclease and Charge Mutants lamellar vesicles (GUVs) of defined compositions (dioleoylphosphatidylcho- J. Nathan Scott1, Patrik R. Callis2. line (DOPC), dipalmitoylphosphatidylcholine (DPPC) and DOPC/DPPC/ 1Chemistry, Saint Francis University, Loretto, PA, USA, 2Chemistry and Cholesterol 1:1:1). Biochemistry, Montana State University, Bozeman, MT, USA. In preliminary studies, we validated an hyperspectral microscope prototype for Discussion persists as to the origin of the time dependent fluorescence spectral Laurdan imaging on GUVs and RBCs by comparison with spectrofluorimetry (Stokes) shift (TDFSS) in the range 100 fs to more than 100 ps for a number of and biphoton confocal microscopy for which reports are available [3]. This mi- tryptophan (Trp)-containing proteins. TDFSS reports on the dynamic reorgani- croscope yielded better spectral resolution (steps of 1 nm) as compared to zation of the local environment around the large dipole of Trp following exci- confocal microscopy (steps of 10 nm). This improved spectral resolution de- tation to the 1La state. Much of the discussion centers on the ubiquitous ‘‘slow’’ tected, besides well-known peaks at 440 and 490 nm, new peaks in the Laurdan (10 ps-5 ns) TDFSS component found only in proteins. Details of what deter- spectra of GUV (made of DOPC/DPPC/Cholesterol 1:1:1) and RBC mem- mines the fast (<2 ps) component in proteins are also of interest. Interest branes around 470 nm (and 540 nm). The biological significance of these focuses on two questions: (1) what are the relative contributions of protein new peaks needs to be identified and will be discuss according to experiments and water; and (2) what length scales characterize these contributions? We performed on GUV displaying different order and composition properties (gel have published extensive equilibrium MDþQM simulations on the single (DOPC), liquid-disordered (DPPC) and liquid-ordered (DOPC/DPPC/Choles- Trp of wild type Staph. nuclease, and four charge-changing mutants, all of terol 1:1:1) phases). which experimentally exhibit longer-decaying TDFSS (70-150 ps) than in To conclude, hyperspectral microscopy opens new perspectives for the use of other proteins, e.g., monellin and GB1 [D.P. Zhong, S.K. Pal, A.H. Zewail, environment-sensitive fluorescence probes, thus new possibilities of under- Chem. Phys,Lett. 503 (2011), p. 1-11.] In this work, we have performed non- standing membrane domains behavior. equilibrium, direct relaxation MDþQM molecular dynamics simulations for [1] Hryniewicz-Jankowska A et al. Biochim Biophys Acta, 2014, 1845:155-65. the wild type Staph. nuclease to investigate the origin of these long decay times, [2] Gaus K et al. Mol Membr Biol., 2006, 23:41-8. using both OPLS /TIP3P water and AMBER99sb-ildn/TIP4P water force fields. [3] Stott BM et al. J Lipid Res. 2008, 49:1202-15. Initial results suggest that while OPLS fails to capture these longer relaxation times, AMBER99sb-ildn shows much closer agreement with experiment. We 3138-Pos Board B568 will also report results for the four charge-changing mutants and comparisons Detection of Interactions of Inositol Phospholipids with Ion Channels 1 2 3 3 with other forcefields. Felix Chin , Anoop Saxena , Feng Qin , Ping-Chin Cheng . 1William East High School, East Amherst, NY, USA, 2SUNY Buffalo, 3136-Pos Board B566 Buffalo, NY, USA, 3SUNY-Buffalo, Buffalo, NY, USA. Detecting Counterfeit Pharmaceuticals through UV Spectrophotometry Inositol phospholipids account for only a small percentage of total lipids in cell Gabriela Figueroa1, Luis A. Palacio2, Bruce D. Ray2, Horia I. Petrache1, membranes. But they play crucial roles in diverse cellular processes such as Alfredo Lopez-Yunez3. signal transduction, cytoskeleton organization, membrane trafficking and 1Department of Biology, Indiana University-Purdue University Indianapolis, membrane permeability and transport. While some of the functions involve Indianapolis, IN, USA, 2Physics Department, Indiana University-Purdue regulated hydrolysis to generate second messengers, others are thought to University Indianapolis, Indianapolis, IN, USA, 3Alivio Medical Center, involve direct interactions with proteins. The repertoire of phosphoinositide- Indianapolis, IN, USA. binding proteins has been dramatically expanded in recent years. These include Abstract: According to the World Health Organization between 10%-30% many ion channels, from the first identified prototype inward rectifier potas- of medicines, in Africa, Asia and South America, are counterfeit or sub- sium channel (Kir) to the more recent transient receptor potential channels. standard, affecting the health of millions of people. Currently, there is no effec- However, in most cases the roles of the lipids are implicated from pharmaco- tive way to check the quality of a medicine at the point of care, leaving many logical maneuvering of signaling pathways. A direct observation of the inter- with treatable diseases at risk. The goal of this study is to identify UV-Vis action between lipids and proteins has rarely been possible. The recognition (240nm - 500nm) absorbance patterns that would indicate if a drug is sub- sites on the proteins are often interfered by structural-functional studies using standard or counterfeit. UV-Vis spectroscopy was selected as the method for site-directed mutagenesis. Owing to the complexity of gating mechanisms, the testing due to the maturity and availability of the technology. Pure Acetamin- molecule sites identified in this manner have been less reliable. In this work we ophen and Tylenol were used as controls for proof of concept. Samples were present a FRET approach for direct detection of interactions between phosphoi- prepared by dissolving different combinations of the pure active ingredient nositide lipids and ion channel proteins. Using an inward rectifier potassium and adulterants such as cement, rice flour, vitamin C and lactose in three channel and a transient receptor potential channel as prototype models, we different types of solvents (H2O, 0.1 M HCl, 0.1 NaOH). Various concentra- show the feasibility to generate recombinant proteins incorporating genetic do- tions (ranging from 0.01mg/ml to 0.04mg/ml) and mixing ratios were analyzed nors. We use fluorescent lipids as acceptors and show that the energy transfer using a UV-Vis Spectrophotometer. It was found that adulterants significantly is limited to only peripheral lipids around channel proteins. Our experiments decrease the absorption of acetaminophen at 245nm by interacting with its demonstrate that the assay has a resolution to probe specifically bound lipid benzene ring, while showing a slight increase in other parts of the spectrum. molecules. Wednesday, February 11, 2015 623a

3139-Pos Board B569 The cationic triangulenium dye, azadioxatriangulenium (ADOTA) was entrap- Food Colors as Intrinsic Luminescent Sensors in Edible Products ped in silica thin films obtained by the sol-gel process and in poly (vinyl) Sarah M. Waxman, Ariella Kashi, Adam Karami, Meera Patel, alcohol (PVA) thin films. ADOTA is a red emitting organic fluorophore with Maria G. Corradini, Richard Ludescher. a long fluorescence lifetime of ~20 ns. Silica thin films are ideal materials Rutgers University, New Brunswick, NJ, USA. for the entrapment of fluorescent molecules because they hold these molecules Limited use of luminescence spectroscopy in food and pharmaceutical applica- in a microporous support matrix. The smaller analyte species can easily diffuse tions can be related to two main causes: a)the inherent properties of most useful and interact with the fluorophore. For comparison, we also incorporated fluorophores (low availability, toxicity, high price and restricted solubility), ADOTA in PVA films, which serve as a model for a more rigid and isotropic and b)incomplete photophysical characterization of edible and safe-to-ingest matrix for the entrapment of fluorophores. The photophysical properties fluorophores. of ADOTA in silica thin films and PVA films were studied by means of To expand the use of luminescence spectroscopy to monitor quality and safety steady–state and time resolved fluorescence techniques. At 560 nm observa- of edible goods, photophysical properties of five generally-recognized-as- tion, the fluorescence lifetime of the ADOTA in silica matrix is 11.84 ns safe(GRAS) food colors that are routinely added to foods or pharmaceuticals compared to 19.95 ns in the PVA film. However, when observed at 620 nm, were assessed. The sensitivity of the food dyes’ fluorescence emission intensity the fluorescence lifetimes of ADOTA entrapped in both the matrices are almost to the surrounding medium’s rigidity and polarity were also determined. 20 ns. We have found that the ADOTA incorporated in silica thin film has a Environmental polarity moderately impacted the location of the fluorescence in- wider lifetime distribution (Lorentzian distribution) compared to ADOTA in tensity peaks and bathochromic shifts were observed for all dyes as the polarity PVA film. The local environment of ADOTA molecules in silica thin film is of the solvents increased. The Stokes shifts,(lem-lexc), were estimated to be 45- rich in water and ethanol, which creates the possibility of forming aggregates 90nm depending on the medium and molecular structure of the synthetic color. due to high concentration of dye within a small confined area. In contrast to All these food dyes were practically non emissive in common fluid solvents, the PVA matrices, the porous silica films allow restricted rotations of ADOTA which can explain the limited information on their photophysical properties. molecules, which result in faster and complex fluorescence anisotropy decays. Excited state tautomerization and/or internal twisting, already reported in These types of fluorescent hybrid materials are ideal for developing fluores- synthetic non-edible dyes in low viscosity solutions, can also constitute the pre- cence based biosensors, highly luminescent materials in medicine, functional dominant non- radiative relaxation pathway of the studied food colors in fluid materials in optoelectronic devices and optical components like solid state environments. The medium’s rigidity was altered by changing temperature and tunable lasers. composition. As the viscosity and consistency index of the surrounding me- dium increased, the dyes’ fluorescent emission intensity also increased, which 3142-Pos Board B572 suggests the molecular rotor character of these dyes. The maximum fluores- A Comparison of Photophysical Characteristics of rHDL Encapsulated cence intensity of each dye vs. viscosity relationship was characterized by a Anti-Cancer Drug Valrubicin and Free Valrubicin power law model and the sensitivity of each dye to changes in viscosity was Sunil Ajit Shah1, Rahul Chib1, Sangram Raut1,2, Jaclyn Bermudez3, evaluated in terms of its parameters. Nirupama Sabnis4, Divya Duggal1, Andras Lacko4, Zygmunt Gryczynski1,2, Ignacy Gryczynski1. In principle, the large Stokes shift and sensitivity to viscosity supports the 1 potential use of these food colors as probes of microviscosity or molecular Department of Cell Biology and Immunology, Center for Fluorescence crowding in edible goods. Technologies and Nanomedicine, UNT Health Science Center, Fort Worth, TX, USA, 2Department of Physics and Astronomy, Texas Christian 3140-Pos Board B570 University, Fort Worth, TX, USA, 3Cell Biology and Immunology, North Probing the Internal and External Structure of Carbon Nanodots through Texas Eye Research Institute, UNT Health Science Center, Fort Worth, TX, Fluorescence Quenching USA, 4Department of Integrative Physiology and Anatomy, UNT Health Rachel Taylor, Jan Karolin, Chris Geddes. Science Center, Fort Worth, TX, USA. UMBC Chemistry, The Institute of Fluorescence, Baltimore, MD, USA. Drug delivery via nanotechnology is one of the rapidly developing fields in In past several years, there has been significant investigation into the various cancer therapeutics. Targeted drug delivery has the advantage of having synthetic routes of carbon nanodots along with their associated photophysical minimal interaction with healthy tissue, thereby reducing the toxicity of the properties [1-3]. Carbon nanodots are naturally fluorescing nanometer-sized drug to the rest of the body. rHDL nanoparticles are an efficient method of particles with interesting and unique photophysical properties, which make drug delivery for highly lipophilic anti-cancer drugs. Scavenger receptors them highly applicable for various applications in the life sciences [2-3]. Our class B type I (SR-BI), which are highly expressed on cancer cells interact lab has been investigating these particles produced by various combustion routes with rHDL nanoparticles for effective drug delivery to the cancer cell and for many years, studying both the photophysical and plasmon-enhanced photo- tumor. Valrubicin is an anti-cancer drug, with intrinsic fluorescence. In this physical properties [1]. In order to fully understand the photophysical properties experiment, we compared the photophysical properties of free valrubicin of carbon nanodots, in this poster we have examined the both the internal and and rHDL valrubicin via steady state and time resolved fluorescence measure- external structure of these particles in an attempt to ascertain the origins of ments. The steady-state anisotropy of rHDL valrubicin is higher as compared the fluorescence signature/s, using a combination of differently charged ions; to free valrubicin, suggesting its encapsulation in rHDL nanoparticles. A which ultimately results in both static and dynamic quenching processes being longer rotational correlation time was observed for rHDL valrubicin in time observed. Our results reveal significant vibronic structure of the nanodots’ chro- resolved anisotropy measurements compared to free valrubicin, further mophore, which can readily be quenched by non-charged ions (acrylamide), supporting steady state anisotropy data.. We also studied the cellular internal- suggesting a buried fluorescent chromophore center. ization of free valrubicin and rHDL valrubicin using confocal microscopy. [1] Y. Zhang, H. Gonc¸alves, J. C. G. Esteves Da Silva, and C. D. Geddes, This could help track the movement of rHDL nanoparticles within the cancer ‘‘Metal-enhanced photoluminescence from carbon nanodots,’’ Chem. Com- cells. mun. 47, 5313-5315 (2011). [2] S. Baker and G. Baker, ‘‘Luminescent Carbon Nanodots: Emergent Nano- 3143-Pos Board B573 lights,’’ Angew, Chem. Int. Ed. 49, 6726-6744 (2010). Spectral Distortions in Metal-Enhanced Fluorescence [3] H. Li, Z. Kang, Y. Liu, and S-T. Lee, ‘‘Carbon nanodots synthesis, proper- Jan O. Karolin, Hilla Ben Hamo, Chris D. Geddes. ties and applications,’’ J. Mater. Chem, 22, 24230-24253 (2010). Institute of Fluorescence, Baltimore, MD, USA. In recent years our laboratory and others have demonstrated many examples 3141-Pos Board B571 and concepts in Metal-Enhanced Fluorescence1 (MEF), a surface plasmon phe- Fluorescence Studies of a Long Lifetime Fluorophore, ADOTA in Silica nomenon, which amplifies both fluorescence and luminescence signatures in and PVA Thin Films the near-field, i.e. less than one wavelength of light away from a metallic ob- Rahul Chib1, Sangram Raut1,2, Sunil Shah1, Beata Grobelna3, ject1. In all of these examples of MEF, and for over a decade, the fluorescence Irina Akopova1, Ryan Rich1, Thomas Just Sørensen4, Bo W. Laursen4, spectra has simply been reported as being enhanced, i.e. the emission is greater Zygmunt Gryczynski1,2, Ignacy Gryczynski1. from a plasmonic substrate as compared to a suitable control sample. 1Department of Cell Biology and Immunology, Center for Fluorescence However, in this paper we will show that Metal-Enhanced Fluorescence from Technologies and Nanomedicine,, UNT Health Science Center, Fort Worth, both a variety of plasmonic substrates and using a range of different fluoro- TX, USA, 2Department of Physics and Astronomy, Texas Christian phores, often results in fluorophore spectral distortion2. More often than not, University, Fort Worth, TX, USA, 3Faculty of Chemistry, University of the red edge of the fluorescence spectra is observed to be distorted, as compared Gdansk, Gdansk, Poland, 4Nano-Science Center and Department of to the emission spectra of a fluorophore observed in the far-field and distal Chemistry, University of Copenhagen,, København, Denmark. from plasmonic interactions. In addition, a significant MEF effect often results 624a Wednesday, February 11, 2015 in notable changes in the spectrum full width at half maximum (FWHM). 3146-Pos Board B576 We discuss these new effects in terms of the mechanism of plasmonic Sedimentation Velocity Analysis of the EGFPs in E. coli Whole Cell enhancement. Extracts using Fluorescence Detection System 1Metal-Enhanced Fluorescence, Edited by Geddes, C.D., John Wiley and Sons, Jia Ma, Huaying Zhao, Peter Schuck. New Jersey, June 2010, 625 pgs, ISBN: 978-0-470-22838-8. NIBIB, NIH, Bethesda, MD, USA. 2 Spectral Shifts in Metal-Enhanced Fluorescence, Karolin, J. and Geddes, The analytical ultracentrifugation (AUC) is a classic biophysical instrument to C.D., (2014), Applied Physics Letters, 105, 063102. analyze protein interactions in solutions. A recently introduced fluorescence detection system (FDS) improves the specificity and sensitivity of the AUC, 3144-Pos Board B574 and offers the potential to analyze the protein interactions in biological fluids. Ultrasensitive Detection Allows for Singlet Oxygen Phosphorescence We explore challenges posed by the application of FDS-AUC to the study of Detection, an Important Prerequisite for Photodynamic Therapy protein interactions in E.coli whole cell extracts, using fluorescent proteins Marcelle Koenig1, Manoel Veiga1, Sebastian Tannert1, Felix Koberling1, (EGFPs) as model system. At experimentally feasible concentrations of cell Volker Buschmann1, Matthias Patting1, Marcus Sackrow1, Michael Wahl1, extracts, we find no discernable effects of hydrodynamic nonideality on the Rainer Erdmann1, Peter Kapusta2, Christian Wolf3, Christian Kaufmann3, sedimentation of EGFPs. However, at high concentrations E.coli whole cell ex- Humberto Rodriguez3. tracts produce significant signals from auto fluorescence with complicated 1PicoQuant GmbH, Berlin, Germany, 2J. Heyrovsky Institute of Physical quenching patterns. Goal of this work is to establish detection limits and Chemistry, Prague, Czech Republic, 3PVcomB/Helmholtz-Zentrum Berlin develop procedures to improve specificity. fu¨r Materialien und Energie GmbH, Berlin, Germany. Detection sensitivity from the ultraviolet to the near infrared spectral region 3147-Pos Board B577 is a key parameter to meet today’s demand for handling smallest analyte Two-Color Imaging using Spectral Variants of iRFP670 and iRFP682 amounts and short measurement times in the optical evaluation of miscella- Near-Infrared Fluorescent Proteins neous samples. The introduction of single photon counting based data acqui- Mikhail Baloban, Daria M. Shcherbakova, Vladislav V. Verkhusha. sition has proven to yield a major sensitivity increase and very high dynamic Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, range - it is the ideal method for measuring weak luminescence. Albert Einstein College of Medicine, Bronx, NY, USA. We present the hardware and handling optimization of a state of the art spec- The new class of fluorescent proteins (FPs) engineered from bacterial phyto- trometer for steady-state and time-resolved fluorescence measurements. The chromes (BphPs) attracts much attention for in vivo imaging due to their high sensitivity of the spectrometer was shown by measurements of popular near-infrared (NIR) fluorescence spectra. These FPs utilize widely available fluorescent dyes as well as the Raman spectrum of water under well defined in mammalian cells biliverdin (BV), a product of heme degradation, as a chro- and reproducible conditions. The achieved sensitivity allows us to quantify mophore and, therefore, are as easy to use as common GFP-like proteins. We singlet oxygen generation and to characterize the singlet oxygen phosphores- recently reported five NIR FPs, called iRFPs, with different fluorescence and cence decay, a prerequisite when studying photosensitisers like porphyrins biochemical properties. Interestingly, two of them, iRFP670 and iRFP682, ex- and phthalocyanines used for example in photodynamic therapy (PDT). More- hibited the twice higher molecular brightness, as well as the blue-shifted absor- over, with the help of an integrating sphere fluorescence quantum yields of low bance (643 nm and 663 nm) and fluorescence (670 nm and 682 nm) compared fluorescent samples like Ru(bpy)3 in water can be determined very precisely. to other iRFPs. Here we characterized the unusual properties of these NIR FPs The fibre connection of the spectrometer to a time-resolved fluorescence micro- in detail. Our biochemical and biophysical analysis showed that iRFP670 and scope (MicroTime100/200) was also realized. The combination of the advan- iRFP682 incorporate the BV chromophore in two distinct confirmations. A sin- tages of both setups makes it e.g. possible to perform 2D-lifetime imaging gle amino acid mutation resulted in a depletion of one BV confirmations in the with a freely tunable detection window for low luminescent samples even far protein binding pocket and, consequently, in 30 nm red-shifts of both absor- into the near infrared region. The measurements with such a combination bance and fluorescence. The point mutation also caused a slight decrease in give not only the spectral and lifetime information of a luminescent sample the molecular brightness and an increase in the pH stability of the obtained but also the spatial information which is especially important for hetergeneous red-shifted variants, which we named iRFP670-red and iRFP682-red. The samples. effective brightness of the iRFP670-red and iRFP682-red in live mammalian cells was comparable to that of the parental proteins, suggesting that the high 3145-Pos Board B575 efficiency and high specificity of the incorporation of endogenous BV chromo- Accounting for Photophysical Processes and Specific Signal Intensity phore was not affected. Spectrally resolvable fluorescence of the iRFP670 and Change in Fluorescence-Detected Sedimentation Velocity Analytical iRFP670-red pair, as well as of the iRFP682 and iRFP682-red pair, allowed Ultracentrifugation easy separation of two cellular populations using FACS cytometry and straight- Huaying Zhao1, Jia Ma2, Maria Ingaramo2, Eric Andrade2, Jeff MacDonald3, forward two-color fluorescence microscopy of live cells, thus making them the Glen Ramsay3, Grzegorz Piszczek4, George Patterson2, Peter Schuck2. probes of choice for cell labeling in the NIR region. 1 2 NIBIB, National Insitutes of Health, Bethesda, MD, USA, NIBIB, National 3148-Pos Board B578 Institutes of Health, Bethesda, MD, USA, 3Aviv Biomedical Inc, Lakewood, 4 Tuning the Photophysical Properties of the Green Fluorescent Protein NJ, USA, NHLBI, National Institutes of Health, Bethesda, MD, USA. with Unnatural Amino Acids Fluorescence detected sedimentation velocity (FDS-SV) analytical ultracen- Gregory M. Olenginski, Christine M. Phillips-Piro, Scott H. Brewer. trifugation has emerged as a powerful technique for the study of macromo- Chemistry, Franklin and Marshall College, Lancaster, PA, USA. lecular interactions, particularly high-affinity protein interactions, with Green fluorescent protein (GFP) is a widely utilized protein in imaging due to hydrodynamic resolution exceeding that of diffusion-based techniques, and its favorable optical properties. These photophysical properties have previously with sufficient sensitivity for binding studies at low picomolar concentra- been modified through site-directed mutagenesis utilizing naturally occurring tions. In order to fit the FDS data structure, in the quantitative analysis it amino acids. Here, we have further modulated the photophysical properties is essential to adjust the conventional sedimentation models for detailed of GFP in a systematic fashion by the site-specific incorporation of unnatural description of the sedimentation boundaries. A key consideration is the amino acids (UAAs) into this protein. UAAs greatly enhance the ability to change in the macromolecular fluorescence intensity during the course of modulate the optical properties of this protein in a controlled manner. Optical the experiment, caused by slow drifts of the excitation laser power, and/or data on these new GFP constructs containing UAAs will be presented along by photophysical processes. In the present work we demonstrate that FDS- with initial crystallographic data relating structural changes in the protein to SV data have inherently a reference for the time-dependent macromolecular changes in the optical properties. signal intensity, resting on a geometric link between boundary migration and plateau signal. We show how this new time-domain can be exploited to 3149-Pos Board B579 study molecules exhibiting photobleaching and photoactivation. This ex- Use of the Methyl Ester of a Fluorescent Unnatural Amino Acid to Facil- pands the application of FDS-SV to proteins tagged with photoswitchable itate Site-Specific Incorporation of Fluorescent Probes in Proteins fluorescent proteins, organic dyes, or nanoparticles, such as those recently Joshua R. Berlin1, William Lopez1, Mohit R. Jain2, Jorge E. Contreras1. introduced for sub-diffraction microscopy. At the same time, we find con- 1Pharmacology and Physiology, Rutgers-New Jersey Medical School, ventional fluorophores undergo minimal photobleaching under standard illu- Newark, NJ, USA, 2Center for Advanced Proteomic Research, Rutgers-New mination in the FDS. These findings support the application of a high laser Jersey Medical School, Newark, NJ, USA. power density for the detection, which we demonstrate can further increase Fluorescent unnatural amino acids (UAAs) can be efficiently incorporated the data quality. in target proteins in vivo by expressing suppressor tRNA/aminoacyl-tRNA Wednesday, February 11, 2015 625a synthetase (tRNA/aaRS) pairs in the presence of target protein mRNA that 3152-Pos Board B582 contains an orthogonal stop codon in the coding sequence and adequate Investigation of E. coli Heptosyltransferase I Dynamics cellular levels of UAA. While conceptually straightforward, many technical Joy M. Cote. barriers impede facile application of this technology in a broad array of eu- Chemistry, Wesleyan University, Middletown, CT, USA. karyotic expression systems. Cell loading of UAAs is one such obstacle since Interest in new methods to treat gram-negative bacterial infections has emerged amino acids usually require specific transporters for cellular uptake. To due to a significant increase in antibiotic resistance amongst bacteria. Bacterial improve UAA cell loading, the methyl ester of one fluorescent UAA, L- biofilms are a major contributor to this immunity. Heptosyltransferase-I (HepI) Anap (L-Anap-AM) was tested in eukaryotic expression systems. L-Anap- is an essential enzyme for the biosynthesis of lipopolysaccharides (LPS), an AM is soluble in ethanol and DMSO, and readily diluted into aqueous solu- important component to bacterial biofilms. Cells deficient in HepI have tions. Full length GFP (containing a stop codon mutation at Y39) and decreased intestinal colonization and are more susceptible to hydrophobic an- AHA2 Hþ-ATPase (stop codon mutation at W71) were produced in yeast tibacterials, which makes HepI a good target for developing inhibitors. HepI is strains expressing a tRNA/aaRS pair for L-Anap after growth in L-Anap- a member of the GT-B structural subclass of glycosyltransferases. Crystal AM containing media. Both expressed proteins were fluorescent and GFP structures of GT-B enzymes have been observed to interconvert between showed efficient FRET between L-Anap and the protein fluorochrome. LC/ open and closed conformations based up the ligation state of the proteins; we MS/MS studies also showed that L-Anap was located at residue 39 in GFP. therefore hypothesize that HepI will also interconvert between open and closed These studies demonstrated that L-Anap-AM is correctly incorporated into conformations to enable catalysis. In HepI, there are eight tryptophan residues, peptide chains during translation. Studies were also carried out in Xenopus oo- which enable us to observe changes in the intrinsic tryptophan fluorescence cytes in which nuclear injection of the tRNA/aaRS pair for L-Anap was fol- upon substrate binding. Using wild-type and mutant forms of HepI we are at- lowed by injection of cRNA for Connexin 26 (Cx26) or the Shaker KV tempting to discern which regions are undergoing conformational changes channel containing a stop codon mutation at specific locations. Both Cx26 upon binding of the sugar acceptor substrate (associated with an observed and Kþ currents were measured in injected oocytes, using a two- blue shift in the fluorescence). Individual HepI tryptophan residues have microelectrode voltage clamp, only after incubation in an L-Anap-AM con- been mutated to phenylalanine. Arginine residues that we hypothesize to taining storage buffer. These studies demonstrate that L-Anap-AM can be have an important role in substrate induced conformational changes have used effectively to generate UAA-containing proteins in a variety of eukary- also been mutated. Fluorescence circular dichroism have been used to deter- otic expression systems. mine the impact of these residues upon binding. Enzyme kinetics were also per- formed on all mutants to ensure that the mutagenesis was not impacting catalysis. Data thus far suggest that a conformational change is indeed needed 3150-Pos Board B580 for chemistry to occur. Monitoring whether a large dynamic closing occurs, Dithioamide Peptides and Proteins: Synthesis and Application to Tracking is also being explored using mutagenesis and site specific fluorophore incor- Protein Conformational Changes by Fluorescence Spectroscopy poration. Ultimately, an enhanced understanding of HepI’s protein dynamics Yun Huang. and mechanism may lead to the design of more effective gram-negative Department of Chemistry, University of Pennsylvania, Philadelphia, PA, therapeutics. USA. Tracking protein conformational change is important to understand the folding and function of proteins. Fo¨rster resonant energy transfer (FRET) and photo- 3153-Pos Board B583 induced electron transfer (PET) are widely used to get time-resolved structural The Role of Chaperone Proteins in Cataract Aggregation: A Two- information on protein motions. However, the relatively large size of fluoro- Dimensional Infrared Study phores and quenchers may introduce significant perturbations to protein struc- Tianqi O. Zhang, Martin T. Zanni. ture. The thioamide bond, a single atom substitution of the peptide bond, has Dept. of Chemistry, University of Wisconsin-Madison, Madison, WI, USA. recently been shown to be a minimalist fluorescent quencher of various fluoro- Crystallin proteins need to maintain their native structures at high concentration phores by either FRET- or PET- based mechanisms. Unlike commonly used in the lens to enable vision. When these proteins aggregate into opaque de- fluorescence probes, thioamides are sufficiently small that they can be placed posits, cataracts are formed. Most kinds of isolated crystallin proteins form am- at nearly any position in the protein sequence without significant alteration yloid fibril structures when treated with denaturants in vitro, but these of the secondary structure. However, moderate quenching efficiency may limit structures have not been confirmed from examination of a cataractous lens. It its sensitivity for some applications. Here, we show that two consecutive thio- is hypothesized that alpha crystallins form molecular chaperones that bind to amide bonds can be incorporated into peptide and protein backbones, and the unfolded proteins and interrupt the aggregation pathways. We study the aggre- quenching effect is strengthened compared with a mono-thioamide. Thus gation of gammaD-crystallin in the presence of alphaB-crystallin with two- dithioamide bonds provide increased sensitivity to detect protein conforma- dimensional infrared (2D IR) spectroscopy that is sensitive to the secondary tional changes and may be used for advanced spectroscopy applications like structure of proteins. Using 13C isotope labeling of either the gammaD- Fluorescence correlation spectroscopy (FCS) and Fluorescence lifetime imag- crystallin or the alphaB-crystallin, we can independently watch structural ing microscopy (FLIM). changes in both proteins simultaneously. In addition, cross-peaks in the 2D IR spectra reveal coupling between the different proteins. Using these tools, we have observed interaction between alphaB-Crystallin and aggregated 3151-Pos Board B581 gammD-Crystallin. Our study provides a new way of monitoring protein- Parallels between Enzyme Action and Tryptophan Fluorescence Bright- protein interactions and will be valuable in the further studies of molecular ness in Proteins chaperone interactions with disease-related protein aggregates and their 1 2 Pedro L. Muı´n˜o , Patrik R. Callis . intermediates. 1Chemistry, Saint Francis University, Loretto, PA, USA, 2Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA. Thousands of scientists studying proteins rely on the intensity, wavelength, 3154-Pos Board B584 and lifetimes of emitted light (fluorescence) from the amino acid tryptophan Development of a Vibrational Hydration Ruler (Trp) because it is extremely sensitive to its ‘‘environment’’ in a protein. But, Elise Tookmanian, Edward Fenlon, Scott Brewer. what exactly does ‘‘environment’’ mean? In the last several years, we have Chemistry, Franklin & Marshall College, Lancaster, PA, USA. combined classical molecular dynamics with simplified quantum mechanics Amino acids covalently modified with spectroscopic reporters offer the and electrostatics to gain considerable insight into what environments pro- potential to probe local protein hydration with high spatial and temporal mote and quench Trp fluorescence. Close parallels can be drawn between resolution when coupled with the appropriate spectroscopic technique. Three our simulations of Trp fluorescence brightness and simulations of enzyme azidophenylalanine residues have been synthesized and, in combination with effectiveness, especially for the ‘‘single electron transfer’’ mechanism. We the commercially available 4-azido-L-phenylalanine, form a series of unnatural have carried out MD simulations of Staphylococcal nuclease and ribonuclease amino acids (UAAs) containing the azide vibrational reporter at varying T1 and determined the electric potential difference between the phosphorus distances from the aromatic ring of phenylalanine. The azide vibrational subject to nucleophilic attack and the putative electrophile. These are charac- reporter was selected due to the position, sensitivity and extinction coeffi- terized by potential differences of 2-3 volts, with fluctuations spanning cient of the azide asymmetric stretch vibration. The sensitivity of the azide 1.5 volts, quite similar to the energy gaps between the fluorescing state of reporters for these UAAs was investigated in solvents that mimic distinct Trp and charge transfer states that result in fluorescence quenching. We local protein environments. Three of the four azido modified phenylalanine shall report results for a variety of enzymes representing six major classes residues were successfully genetically incorporated into a surface site in super- of enzymes. folder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal 626a Wednesday, February 11, 2015 aminoacyl-tRNA synthetase in response to an amber codon with high efficiency the protein region (1900-1485 cm-1 and 3500-3010 cm-1) by PCA of serum and fidelity. The site-specific incorporation of these UAAs was verified by samples. SDS-PAGE and ESI-Q-TOF mass analysis. IR spectroscopy was then utilized to probe the protein hydration state for the azide group in these sfGFP constructs. 3157-Pos Board B587 Investigation of Gender Effect on Obesity using a Model of Inbreed Obese Mouse Lines by Fourier Transform Infrared Imaging 3155-Pos Board B585 Fatma Kucuk Baloglu1, Gudrun Brockmann2, Sebastian Heise2, The Effect of Selenium Treatment On-Diabetic-Induced Structural Varia- Sebnem Garip1, Feride Severcan1. tions in the Molecules of Rat Kidney Plasma Membrane 1Biological Sciences, Middle East Technical University, Ankara, Turkey, Rafig Gurbanov1, Sherif Abbas2, Mehmet Bilgin3, Feride Severcan2. 2Department of Breeding Biology, Humboldt Universitatzu, Berlin, 1Biochemistry, Middle East Technical University, Ankara, Turkey, 2Biology, Germany. Middle East Technical University, Ankara, Turkey, 3Biophysics, Adnan Lipid accumulation and storage of lipids in adipocytes during obesity cause Menderes University, Aydın, Turkey. structural and functional changes in adipose tissue conformation. The expan- Diabetes Mellitus is a metabolic disorder affecting the great amount of sion of visceral (VAT) and subcutaneous (SCAT) adipose tissue mass in the world’s population, in which fat, protein and carbohydrate metabolism is body is the main reason of obesity and many times it results in disturbed lipid severely affected by deficient insulin secretion or function. In this study, and glucose metabolism. Gender is an important factor for the research in the Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) obesity and other metabolic diseases because it leads to different fat distribu- Spectroscopy was used to study diabetic kidney disease-induced structural tion and the pathophysiology. This study aims to determine gender effect on changes, which encountered as a complication of diabetes. Furthermore, the the structural and functional parameters on VAT and SCAT. To achieve this, protecting and possible therapeutic role of selenium in the course of diabetic FTIR microspectroscopic imaging technique and UCP1 immunohistological kidney disease disclosed. The detailed spectral analysis of ATR-FTIR spec- staining have been used. FTIR microspectroscopy is a rapid and effective troscopy revealed that, protein and saturated lipid content of diabetic kidney technique to monitor molecular alterations in biological tissues induced by plasma membrane prominently diminished. The decrease in the unsaturated different conditions such as disease, chemical treatment and variations in lipid content indicates diabetes-induced lipid peroxidation. Nevertheless, the the environmental factors. UCP1 protein content gives information about administration of selenium at low and medium concentrations improved the the amount of brown adipose tissue (BAT), and therefore about the transdif- condition by changing the lipid and protein content to the normal values. ferentiation of BAT to the white adipose tissue (WAT). The results of FTIR The ordered structure of plasma membrane lipids due to diabetes turned imaging study revealed a decrease in unsaturation level of lipids and an in- back to healthy structure with the selenium treatment. The diabetes caused crease in the amount of triglycerides in adipose tissue samples. Furhermore, the decrease of membrane dynamics however; selenium treatment increased the longer hydrocarbon acyl chain length was obtained in the lipids of obese the dynamics of membrane. Hierarchical Cluster Analysis (HCA) and Prin- samples. All of these spectral paramaters could be used as biomarkers in cipal Component Analysis (PCA) applied to the control, diabetic and selenium obesity. The results of the present study showed that, these obesity indicators treated groups revealed clear separation of the groups with high heterogeneity are more significant in SAT of female mice, whilst, they are more significant in the lipid and protein spectral regions. These chemometric methods show inVAT of male mice. The amount of UCP1 protein which is a marker of the that, low and medium dose selenium treated groups successfully segregated transdifferentiation, showed a decrease in male samples rather than females. from diabetic group and clustered close to the control group which indicates Consequently, obesity has adverse effects on health of both genders but espe- recovery effect of selenium at these concentrations in diabetic animals. To cially on men. conclude, lipid and protein structure and content of the diabetic kidney plasma membranes deteriorated, which restored after selenium administration, more preferentially at low dose. The results of the study suggest selenium treatment 3158-Pos Board B588 at appropriate dose may be related to insulin mimetic and antioxidant proper- Measuring the Distribution of Taurine Molecule Inside Biological Tissue ties of selenium. via Intrinsic Molecular Vibrations using Nonlinear Raman Spectroscopy Masahiko Kawagishi1, Yuki Obara2, Takayuki Suzuki2, Masumi Hayashi3, Kazuhiko Misawa2, Sumio Terada1. 3156-Pos Board B586 1Department of Neuroanatomy and Cellular Neurobiology, Tokyo Medical A Novel Method for Early Diagnosis of Malignant Pleural Mesothelioma and Dental University, Tokyo, Japan, 2Department of Applied Physics, from Human Serum Samples: ATR-FTIR Spectroscopy Tokyo University of Agriculture and Technology, Koganei, Japan, 3Wired Dilek Yonar1, Abdulsamet Sandal2, Salih Emri2, Feride Severcan1. Co., Ltd., Komae, Japan. 1Biological Sciences, Middle East Technical University, Ankara, Turkey, Distributions of small molecular weight (less than 300 Da) organic com- 2Faculty of Medicine, Dept. of Chest Diseases, Hacettepe University, Ankara, pounds inside biological tissue have been obscure because of the lack of Turkey. appropriate methods to measure them. Although fluorescence techniques Malignant pleural mesothelioma (MPM) is an aggressive and rare form of are widely used to characterize the localization of large molecules such as cancer which arises from environmental fibrous minerals (tremolite asbestos proteins and nucleic acids, they cannot be easily applied to the cases with or erionite) exposures. Since it is difficult to differentiate the benign pleural small molecule compounds: Fluorescent labels are relatively large compared thickenings from carcinomas, MPM can only be diagnosed in the advanced to the target compounds and can interfere with the chemical properties of stage. Therefore, it is important to develop a new method with high speci- them. Raman spectroscopy is a technique to study vibrational information ficity and sensitivity for the early diagnosis of MPM. Fourier Transform intrinsic to and characteristic of the chemical species of compounds. We Infrared (FTIR) spectroscopy is a novel and non-invasive method that pro- used coherent anti-Stokes Raman scattering (CARS) spectroscopy to detect vides high specificity and sensitivity in the diagnosis of cancer. Moreover, and identify a small molecule compound, taurine, in aqueous environment FTIR with its attenuated total reflectance (ATR) tool is eminent technique without labeling. Molecular species could be uniquely identified from the because of its rapidity and ease to put into clinical practice. Hence, we spectral shape of the broadband vibrational spectra of target compound. used ATR-FTIR spectroscopy coupled with chemometric analysis methods The local distribution of the compound could be determined from the spectral to characterize the molecular alterations as well as to differentiate the intensity. We have developed a phase-sensitive CARS spectroscopy capable experimental groups from each other. FTIR spectra of the samples collected of measuring the broadband spectrum simultaneously without losing high fre- from patients diagnosed with malignant pleural mesothelioma (MPM), lung quency resolution. We also utilized a time-resolved technique to remove non- cancer (MLC), benign, and healthy control (C) were recorded in the 4000- resonant noise signals over a wide spectral range produced by water mole- 650 cm-1spectral region.. Recording the spectra and analysis of the spectral cules. We combined these techniques to selectively detect resonant vibra- data were obtained with Perkin Elmer Spectrum One Program. Spectral tional CARS signals from a target compound. We measured taurine inside analysis indicated a significant decrease in the lipid, protein, carbohydrates, mouse cornea tissue soaked in solution as an initial model experiment. We and nucleic acid contents in MLC and MPM with respect to the healthy detected a Raman peak of taurine near 1000 wavenumber / cm inside cornea, samples. Hierarchical Cluster Analysis (HCA) and Principal Component and successfully characterized its depth profile in the tissue. Our CARS Analysis (PCA) were performed to differentiate the studied groups based spectra measurement can be a promising method to measure and visualize on the spectral differences. HCA of the samples demonstrated that all stud- the distribution of small bio-related compounds in biological background ied groups successfully differentiated from the control. Moreover, success- without using any labeling, paving the way for new cell biological analysis ful clustering of the all groups (control, MLC, MOC, MPM) was obtained in in various disciplines. Wednesday, February 11, 2015 627a

Bioengineering as a molecular identifier may open new possibilities for more sophisticated sensor design while OmpG’s monomeric structure greatly simplifies nanopore 3159-Pos Board B589 production. The Alpha Beta Rearrangement of the Asp-Gly Sequence 3162-Pos Board B592 Kazuki Koda. Multicolor Monomeric Near-Infrared Fluorescent Proteins Science and Engineering, Kinki University, Higashi-osaka, Japan. Daria M. Shcherbakova, Mikhail Baloban, Vladislav V. Verkhusha. The protein proopiomelanocortin (POMC) is a precursor several nerve peptide Albert Einstein College of Medicine, Bronx, NY, USA. hormones, including MSHs, ACTH, CLIP, LPH, b-endorphin, and the Joining The genetically encoded near-infrared fluorescent probes are preferable for peptide (JP). The biological activity of POMC derived peptides have been well non-invasive in vivo imaging. In the near-infrared spectral region (650-900 studied except for JP. To investigate the biological function of JP, we chemi- nm) mammalian tissues are relatively transparent to light because the combined cally synthesized JP by an Fmoc solid phase method. However, the yield of absorption by hemoglobin and water is minimal. the synthesized JP was poor and a highly efficient a/b-rearrangement in the Previously, we have developed five spectrally distinct fluorescent proteins, Asp-Gly sequence was observed. Therefore, we evaluated the rearrangement iRFP670, iRFP682, iRFP702, iRFP713 (aka iRFP) and iRFP720, from bacterial of the Asp-Gly sequence under several conditions to estimate the biological phytochromes. As a chromophore, iRFPs use a heme derivative, called bili- activity and the stability of the correct configuration of JP. verdin, abundant in mammalian cells. All iRFPs incorporate endogenous bili- For this purpose, JP was chemically synthesized by ordinary Fmoc and Boc verdin efficiently and autocatalytically, do not require its exogenous supply solid phase methods. After deprotection, JP was separated and identified by and, therefore, can be used as easily as GFP-like proteins. iRFPs are dimers reverse-phase HPLC and MALDI-TOF/MS, respectively. The rearrangement and can mainly serve for labeling of organelles and whole cells. iRFPs have of the Asp-Gly moiety was observed in the case of the Fmoc method but enabled multicolor imaging of deep tissues in living animals. was not significant in the case of the Boc method. Here we report a palette of monomeric iRFPs suitable for protein tagging, To estimate the stability of the Asp-Gly moiety, JP was treated with several which also do not require external biliverdin. To engineer these proteins we first buffers in the pH range of 1-8. The a/b-rearrangement was gradually increased used rational design to monomerize the proteins and engineer spectral shifts. in a pH-dependent manner and was significantly observed under strongly acidic Then we applied directed molecular evolution with the high-throughput conditions. In addition, salt effects for the rearrangements were also estimated. screening for selection of mutants, which incorporate biliverdin efficiently The results will be discussed in this paper. and specifically. As the result, we have engineered three spectrally different monomeric iRFPs, named miRFP670, miRFP703 and miRFP709. miRFPs 3160-Pos Board B590 are characterized by high effective brightness in mammalian cells, high pH sta- Photo-Regulation of Small G Protein Normal and Oncogenic K-Ras using bility and high photostability. We demonstrated that miRFPs perform well as Photochromic Molecules fusion tags for cellular proteins. Seigo Iwata1, Kaori Masuhara2, Nobuhisa Umeki3, Kazunori Kondo2, The set of miRFPs should enable imaging of several tagged proteins in living Shinsaku Maruta1,2. mammals, and thus will be useful in cell and developmental biology and 1Div. Bioinfo., Grad. Sch. Eng., Soka Univ., Tokyo, Japan, 2Dept. BioInfo., biomedicine. The developed molecular evolution approaches could be used Fac. Eng., Soka Univ., Tokyo, Japan, 3Cellular Informatics Lab., RIKEN, for optimization of genetically encoded tools derived from other photoreceptors Wako, Japan. including flavoproteins and opsins. Ras is one of the small G-proteins known as a molecular switch mediating cellular signalling. Switching ON state of Ras is induced by exchange of bound 3163-Pos Board B593 GDP for GTP and OFF state is by hydrolysis of GTP to GDP. Interestingly, the Design and Characterization of Force-Sensitive DNA Origami Components core nucleotide-binding motif of Ras is considerably conserved with the ATP Yi Luo1, Michael W. Hoduba2, Michael G. Poirier1,3, Carlos E. Castro1,2. driven motor proteins, myosin and kinesin. Therefore, it is believed that these 1Biophysics Graduate Program, The Ohio State University, Columbus, OH, nucleotide requiring proteins share common molecular mechanism utilizing USA, 2Department of Mechanical and Aerospace Engineering, The Ohio nucleotide hydrolysis cycle. Previously, we have incorporated photochromic State University, Columbus, OH, USA, 3Deparment of Physics, The Ohio molecules, 4-phenylazophenyl maleimide (PAM), into the functional site of State University, Columbus, OH, USA. kinesin as a photo-switching nano device and succeeded to regulate kinesin Scaffolded DNA origami is powerful design and fabrication tool for the cre- ATPase activities reversibly upon visible light (VIS) and ultra-violet (UV) light ation of nanoscale objects via bottom up self-assembly. These objects have irradiation. Therefore, it is expected that Ras can be also regulated using photo- ~nm level geometric complexity and spatial accuracy, which is comparable chromic molecules. to biological machinery. DNA origami has been used to create different a In this study, we performed basic study to control the function of Ras using wide range of objects such as drug delivery containers or platforms to guide photochromic molecules upon VIS and UV light irradiations. We prepared molecular robots. Current applications of DNA origami exploit the large stiff- normal and oncogenic Ras mutants which have a single cysteine at functional ness of bundles of dsDNA to create structures that maintain a well-defined sites and modified with photochromic molecules of azobenzene and spiropyran and static geometry. However, DNA origami nanostructures with mechani- derivatives stoichiometrically. The GTPase activities of PAM-Ras were revers- cally functional components, such as springs or actuators have remained ibly altered upon VIS and UV light irradiations. In order to monitor the effect largely unexplored. We aim to make DNA origami devices that are respon- on GTPase kinetic pathway by the photoisomerization of PAM, we synthesized sive to force magnitudes typically seen in biomolecular system (~picoNew- fluorescent GTP analogue, NBD-GTP. The Kinetic studies suggested that the tons). We have currently developed a binary approach to make force- initial binding step of NBD-GTP to Ras and the dissociation step of NBD- sensitive DNA origami components and demonstrated this approach through GDP from Ras were regulated by the photoisomerization of PAM. the design of a binary force sensor. This force sensor incorporates structures similar to DNA hairpins into DNA origami designs. The hairpin-like struc- 3161-Pos Board B591 tures undergo a conformational change at a specific force threshold. We Harnessing the Dynamical Movement of OmpG Loops for Protein Sensing have characterized the conformational change dynamics of this force sensor Monifa Fahie, Christina Chisholm, Min Chen. using different experimental methods including single-molecule total internal Department of Chemistry, University of Massachusetts Amherst, Amherst, reflection fluorescence microscopy, transmission electron microscopy and MA, USA. magnetic tweezers. We have shown that such dynamics can be tuned accord- Oligomeric protein nanopores with rigid structures have been engineered for ing to the design to meet the requirement of a wide range of applications. the purpose of sensing a wide range of analytes including small molecules An analog force sensor is also in development using similar approaches. and biological species such as proteins and DNA. We chose a monomeric b- Ultimately we aim to use these devices to measure forces of molecular inter- barrel porin, OmpG, as the platform from which to derive the nanopore sensor. actions in cellular systems, for example cellular traction forces applied during OmpG is decorated with several flexible loops that move dynamically to create cell migration. a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating charac- 3164-Pos Board B594 teristic of the loop’s movement in and out of the porin was substantially altered Remodeling Protein Interfaces to Regulate Recognition by analyte protein binding. The gating characteristics of the pore with bound James R. Horn, Megan L. Murtaugh, Sean W. Fanning, targets were remarkably sensitive to molecular identity - even providing the Christopher A. Smith, Dionne H. Griffin. ability to distinguish between homologues within an antibody mixture. A total Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL, USA. of five gating parameters were analyzed for each analyte to create a unique Protein affinity reagents play an important role across a wide-range of life fingerprint for each biotin binding protein. Our exploitation of gating noise science applications. Efforts to enhance affinity reagents through protein 628a Wednesday, February 11, 2015 engineering typically focus on improved affinity or stability. Here, we of translating genetic based cellular behavior into macro-scale robotic examine methods to engineer coupled equilibria that are linked to the protein locomotion. binding event, which result in protein variants that can be regulated revers- ibly, based on environmental conditions. Using single domain (VHH) anti- bodies as model affinity reagents, along with combinatorial libraries, which 3167-Pos Board B597 provide a route to screen new and wild-type residues across a significant Regulation of Cell Function via Extracellular Biophysical Environment: A amount of interface surface area, we explore the creation of either pH- or Theoretical- Experimental Approach metal ion-dependent protein recognition. The resulting protein variants Toloo Taghian1, Abdul Sheikh2, Daria Narmoneva1, Andrei Kogan1. 1 2 have been analyzed to evaluate the structural and thermodynamic conse- University of Cincinnati, Cincinnati, OH, USA, Yale University, New quences of the remodeled interfaces on protein regulation and stability. Haven, CT, USA. The results suggest the ability to introduce new function, such as a reversible Regulatory promise of electric field (EF) as a non-pharmacological, non- linked binding event, is likely to scale with the complexity/size of the invasive tool to control cellular functions is of great therapeutic interest. protein-protein (ligand) interface. Furthermore, the combinatorial approach However, biophysical mechanisms for the cell-EF interactions are not under- to introduce new function should be generally transferable to other protein stood. We developed a theoretical-experimental approach to investigate EF affinity reagent scaffolds. effects on cells in electrode-free physiologically-relevant configuration, i.e. with cells attached to a substrate. Cell is modeled as a membrane-enclosed hemisphere with realistic parameters. Our numerical results demonstrated 3165-Pos Board B595 that EF frequency is the major parameter that controls the mechanism of A Continuous-Flow C. elegans Sorting System with Integrated Optical EF interactions with cells in realistic environment. Non-oscillating or low- Fiber Detection and Laminar Flow Switching frequency EF leads to charge accumulation on the cell surface membrane Nitish Thakor1, Yuanjun Yan1, Li Fang Ng2, Li Theng Ng2, and results in field screening from the cytoplasm, suggesting that in this Kwan Bum Choi3, Jan Gruber2, Andrew Bettiol3. regime, cell responses are regulated by EF interactions with the surface 1Singapore Institute for Neurotechnology, Singapore, Singapore, 2Yale-NUS membrane receptors. In contrast, high-frequency EF penetrates the cell mem- College, Singapore, Singapore, 3Department of Physics, National University brane and reaches cell cytoplasm, where it may directly activate intracellular of Singapore, Singapore, Singapore. responses. Theoretical simulation predicts the non-uniform distribution of the Sorting of C.elegans genotypes is a routine task in most C.elegans research induced field within the cell membrane, which depends on the applied EF labs. Conventionally, the worms are sorted manually which is both labor frequency. Importantly, substrate properties significantly affect both magni- intensive and slow. In recent years microfluidics has become a useful and tude and distribution of the induced field on the cell membrane, underscoring important tool for biologists to study C.elegans, including sorting. Although the need for a comprehensive, physiologically-relevant modeling approach they have shown successful sorting results, the immobilization of worms which for EF-cell interactions. These theoretical predictions were confirmed in was adopted in most of these works causes aversive stimulation to the worms our experimental studies of the effects of applied EF on responses of and their behavior will potentially be altered when carrying out post-sorting vascular cells. Results show that non-oscillating EF increases vascular endo- characterizations. thelial growth factor (VEGF) expression while field polarity controls cell In this work, both worm detection and switching are achieved without any adhesion rate. High-frequency, but not low-frequency, EF provides differen- intervention of the continuous worm flow. The genotypes of the worms are tial regulation of cytoplasmic focal adhesion kinase and VEGF expression detected by integrated optical fibers based on their fluorescence, without depending on the substrate, with increased expression in cells cultured on the need for immobilization. Switching is based on the steering of laminar RGD-rich synthetic hydrogels, and decreased expression for basement mem- flow boundaries. A novel design that integrates two control inlets dynami- brane (matrigel) culture. These results advance our understanding of complex cally switches the fluidic flow to desired outlets by changing the relative mechanisms underlying cell-EF interactions and may contribute to future EF- pressure in the control inlets, which cause the two laminar flow boundaries based therapies. to steer. Compared to previously reported microfluidic C. elegans sorting devices, sorting in this system is conducted in a continuous flow environment 3168-Pos Board B598 without any immobilization technique or need for multilayer mechanical Mechanobiology of mRNA Localization in Breast Cancer Cells valves to open and close the outlets. The continuous flow sorter not Susan M. Hamilla1, Stavroula Mili2, Helim Aranda-Espinoza1. 1 2 only increases the throughput but also avoids any kind of invasive or University of Maryland, College Park, MD, USA, National Institutes of possibly damaging mechanical or chemical stimulus. We have characterized Health, Bethesda, MD, USA. both the detection and the switching accuracy of the sorting device at Introduction: Metastasis occurs when cancer cells form secondary tumors in different flow rates, and efficiencies approaching 100% can be achieved distant areas of the body. Localization of RNAs in lamellipodial regions has with a high throughput of about 1 worm/s. To confirm that there was no been proposed to play an important role during metastatic progression. In significant damage to C. elegans following sorting, we recovered the sorted one pathway, the tumor suppressor protein, adenomatous polyposis coli worms, finding no differences in behavior and propagation compared to (APC) targets RNAs to cell protrusions. APC -associated mRNAs have been control. implicated in cellular migration and metastatic progression. Therefore, locali- zation or not of these mRNAs has functional significance in cellular migration and metastasis. Additionally, It has been shown that cancer cells modulate their 3166-Pos Board B596 gene expression in response to the mechanical properties of the substrate. Discovering Emergent Behavior of Host-Microbiome Interactions with Furthermore, mRNA localization at adhesion sites is influenced by mechanical Biomimetic Robotics tension, which is adjusted by cells as a function of the mechanical properties of Keith C. Heyde, Warren C. Ruder. the cell environment. Therefore, mechanical properties of tissues may play a Virginia Tech, Blacksburg, VA, USA. role during metastasis by modulating localization of mRNAs. As a result, Although underlying dynamics governing host-microbiome interactions this study investigates APC- associated mRNA localization as a function of are poorly understood, recent studies have shown that commensal bacteria substrate stiffness. play an important role in regulating the health and behavior of their host. Methods: We used the MCF10A cell series, a breast cancer progression model In order to better elucidate this interaction, we designed a biomimetic composed of cell lines representing pre-malignant to invasive transformation robotic host platform comprised of an onboard synthetically engineered to investigate mRNA localization. By using in situ hybridization to fluores- microbiome. By computationally simulating engineered gene networks in cently label mRNAs, and micropatterned polyacrylamide gels of varying these commensal communities, we discovered complex emergent behaviors stiffness, we observed APC associated mRNA localization. Glu-tubulin in the host, such as stalk-and-strike hunting patterns, dependent exclusively and vinculin were immunostained to study their relationship to mRNA on biochemical network dynamics. This simulation models behavior at localization. multiple scales from the molecular kinetics of genetic transcription to Results: On stiffer substrates (280kPa), we observed increased mRNA locali- the physical actuation of robotic components. Taken as a whole, this study zation compared to softer substrates (0.87kPa). Staining of cytoskeletal provides both a computational tool for understanding inter-kingdom elements such as Glu-tubulin and vinculin showed a correlation between the communication while presenting a design for a biomimetic system capable abundance and location of the proteins and APC-associated mRNAs. These Wednesday, February 11, 2015 629a results reveal biophysical roles of adhesion and cytoskeletal tension in regu- a problematic duration in vivo. Our objective was to design, collect and test lating mRNAs to protrusions. the avidity of an antibody with a bivalent Fab display. In addition to the Fab elements, mutations were made in the constant region (Fc) of the antibody to include an anti-CD3 small chain variable fragment, enabling binding to the 3169-Pos Board B599 CD3 glycoprotein found on the surface of T cells, and a 6-histidine, to be Mechanics of Optic Vesicle Morphogenesis in the Chick Embryo used during purification. Two Escherichia Coli expression methods were Seyedhadi Hosseini, Larry Taber. attempted; experimenting with phosphate depletion and ITPG induced pro- BME, Washington university in saint louis, Saint Louis, MO, USA. moters. Neither provided significant yield, so a mammalian expression model In the vertebrate embryo, the eyes are initially bulges that grow outward from was chosen. The mammalian model produced a higher yield using transiently the sides of the primitive forebrain. As these optic vesicles (OVs) elongate, they transfected kidney cells. The protein was purified using fast protein liquid chro- come into contact with the surrounding surface ectoderm (SE), and both layers matography, running through two columns. A Protein A column selected for Ig then invaginate to create the optic cup (prospective retina) and lens vesicle. The folds, isolating all formed antibodies. A HisTalon column selected for histidine, initial shaping of the OVs sets the stage for these later events. To explore the removing formed protein with double CD3 reactive Fc elements, which cause mechanical factors involved in shaping the OVs, we used experiments on chick systemic immune response. Unfortunately errors in our MHC receptor expres- embryos along with computational models. First, mechanical dissections were sion prevented the project from completing our avidity studies. used to remove the SE. Our analysis of OV shapes suggests that the SE exerts asymmetric loads that cause the OVs to flatten and shear caudally during the earliest stages of eye development and later to bend in the caudal and dorsal 3172-Pos Board B602 directions. These deformations cause the initially spherical OVs to become Exploring Biologically based Malnourishment through a Gut-On-A-Chip pear-shaped. Exposure to the myosin II inhibitor blebbistatin reduced these Approach effects, suggesting that cytoskeletal contraction controls OV shape by regu- Eric S. Parigoris, Kyle B. Justus. lating tension in the SE. To test the physical plausibility of these interpretations, Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA. we developed finite-element models of the forebrain, including frictionless Malnourishment results in a cycle that degrades the ability to effectively contact between the SE and OVs. With differential growth included in the absorb nutrients even after adequate food has been consumed due to a disrup- OVs, these models were used to simulate each experiment (control, SE tion of gut homeostasis. We aim to better understand impaired nutrient ab- removed, no contraction). For each case, the predicted shape of the OV agrees sorption in malnourished individuals through a microfluidic based lab-on-a- reasonably well with experiments. Our results support this idea that a combina- chip approach. We are developing a malnourished model of an intestine tion of differential growth in the OV and external pressure exerted by the SE into existing gut-on-a-chip device design fabricated through soft lithography are sufficient to cause the global changes in OV shape observed during the techniques. In order to develop a more physiologically relevant model, we earliest stages of eye development. have studied the effects of different components of bacteria on the mechanical properties of the gut cells. Whole bacteria, spheroplasts, and the endotoxin 3170-Pos Board B600 lipopolysaccharide (LPS) were all introduced to the gut cell layer to determine Cell-Free Expression Systems: From Gene Circuits to Self-Assembly their adverse effects on the stability of the cells. We characterized this Processes in a Test Tube malnourished model by studying the tight junction integrity along with cyto- Ryan Marshall, Mark Rustad, Jonathan Garamella, Vincent Noireaux. skeletal organization of the microvilli in both healthy and malnourished University of Minnesota, Minneapolis, MN, USA. models. Additionally, we have looked at the permeability of the monolayer Cell-free transcription-translation (TX-TL) systems are becoming powerful as an indicator of epithelial integrity. Our work would be useful in many areas platforms to construct complex biological systems in vitro through the expres- including evaluating malnourished nutritional absorption in vitro as well as sion of DNA programs. Considerable efforts have been made to improve those demonstrating the response of malnourished cells in a more physiologically systems in the past decade. Our laboratory has developed the most efficient relevant assay. and versatile in vitro E. coli TX-TL system to express synthetic or natural DNA programs encoding for self-assembly processes. Either in test tube reactions, in emulsion droplets or in liposomes, cell-free TX-TL reactions 3173-Pos Board B603 Developing a Microfluidic Device for Adenoviral Transfection of are now used for research studies ranging from elementary gene circuits to Pancreatic Islets minimal cells. I will present our custom-made cell-free TX-TL system, its current capabilities Pamuditha N. Silva, Zaid Atto, Uilki Tufa, Dawn M. Kilkenny, and limitations. Recently we have shown that entire phages can be synthesized Jonathan V. Rocheleau. in cell-free TX-TL reactions from their genomes. The bacteriophage T7, Institute of Biomaterials and Biomedical Engineering, University of Toronto, composed of about 60 genes, is entirely synthesized in a single test tube reac- Toronto, ON, Canada. tion from its 40 kbp genomic DNA. Replication of the T7 DNA instructions Diabetes is a global epidemic that is associated with the deterioration of pancre- atic beta-cell mass and function. Beta-cell function within the islet has pre- occurs concurrently with phage expression and self-assembly. The phage viously been studied ex vivo and the role of specific proteins is commonly phiX174 composed of a dozen of genes can also be synthesized from its genome. Encapsulated inside cell-sized phospholipid liposomes, the TX-TL investigated with overexpression models using viral transfection. However, system is used to construct a prototype of minimal cell. I will present poor penetration and reduced transfection efficiency limits the application of this cell-free synthetic biology platform, our last experiments and how this this technology in whole ex vivo islets. To improve transfection efficiency system can be used to study the relationship between information and self- and maintain islet structure, we aim to develop a method to bring viral particles deeper into the tissue. Improving upon a previous microfluidic device design organization. featuring hydrodynamic traps (nozzles) in series, a spiral-shaped microfluidic device with parallel nozzles was created to trap, transfect and deliver nutrients 3171-Pos Board B601 to islets. The device was engineered to provide sufficient resistance and pres- Increasing Targeting and Efficacy of Anti-Tumor Antibody sure drop along the nozzles, allowing for homogeneous transfection throughout Justin McKetney1, Rebecca Kerr2, Edward J. Collins2. captured islets. Computer-generated models were used to optimize our design 1Biology, San Diego State University, San Diego, CA, USA, 2Microbiology parameters to minimize pressure drop variations along with shear induced dam- and Immunology, University of North Carolina Chapel Hill, Chapel Hill, age of islets. Through quantitative fluorescence microscopy, we have measured NC, USA. the media exchange rate through islets and have determined a range of oper- In cancer research one of the most difficult aspects is finding a treatment that ating flow-rates. We have also shown successful culturing of the loaded islets selectively targets diseased cells without harming healthy cells in their prox- in our microfluidic device. Beta-cell-specific transfection in whole islets with imity. This lead to inquiry about the utilization of a patients own immune sys- desired genes will facilitate the investigation of islets in a more physiologically tem. It has been found that cytotoxic immune cells infiltrate tumor masses but relevant environment. Therefore, we have developed a beta-cell specific shuttle are inactivated by intercellular signals. Our lab sought to construct an antibody vector in order to deliver fluorescent protein-tagged genes via adenoviral par- that would bind specifically tumor cells with a high affinity, as well as acti- ticles. Currently viral penetration efficiency is being tested in this device vating infiltrating T lymphocytes. Previously, our lab identified a antigen bind- through fluorescence microscopy and western immunoblotting. We aim to ing fragment (Fab) that localized to tumors and bound an E75-MHC receptor create a multi-purpose platform that is not limited to the study of beta-cell found on their cell surface with nanomolar affinity. Using SPR studies the biology in islets, but may be translated to the investigation of other tissue half-life of this binding was found to be less than a 2 minutes and exhibited models and diseases. 630a Wednesday, February 11, 2015

3174-Pos Board B604 In the field of gene delivery systems, it is still unknown that virus vector Polypeptides for Bio-Tethering and Self-Assembly of Lithium Ion Battery showed much higher transportation efficiency of plasmid DNA from cytoplasm Electrodes to nucleus in contrast to non-virus vectors. To improve gene transportation Alex Winton. efficiency, which directly lead to increase transfection efficiency, gene trans- Chemistry, UMBC, Catonsville, MD, USA. portation mechanism through nucleus membrane should be cleared. But The goal of this research is to isolate multifunctional polypeptide sequences for unfortunately there are no experimental protocol to quantitatively evaluate bio-tethering electrode material and carbon nanotubes in order to decrease in- the transportation efficiency through the nucleus membrane. So, in this study, ternal resistance in lithium ion batteries thereby improving their performance. nuclei were extracted from cells by gentle detergent treatments and captured Phage display is the key technique performed in our research. This involves the into a PDMS or PMMA microchamber at a single nucleus level. After the use of an M13 bacteriophage library which has been genetically engineered to confirmation of capturing a single nucleus into a microchamber, nucleotides express a randomized polypeptide sequence of 12 amino acids on the pIII minor and nuclear membrane stained nuclei, transportation of mRNA by molecular coat protein. To perform phage display a library of bacteriophage expressing beacon were observed. these randomized polypeptide sequences were mixed with electroactive mate- rials lithium manganese oxide(LMO) and lithium titanate oxide(LTO). Sam- 3177-Pos Board B607 ples were then shaken and elution of bound phage was performed by adding Decoding Long Nanopore Reads of Bacteriophage Phi X 174 increasing concentrations of surfactant in order to isolate phage that stick Andrew H. Laszlo, Ian M. Derrington, Brian C. Ross, Henry Brinkerhoff, tightly to the electroactive materials. The supernatants from each of the succes- Andrew C. Adey, Ian C. Nova, Jon M. Craig, Kyle W. Langford, sive wash rounds were saved and titered. The strongest elutions represent Jenny Mae Samson, Riza Daza, Kenji Doering, Jay Shendure, bacteriophage expressing a peptide sequence with high binding affinity for Jens H. Gundlach. the tested materials. Isolated colonies produced from single bacteriophage Physics, University of Washington, Seattle, WA, USA. were then sequenced. Results have currently been obtained for LTO and Nanopore DNA sequencing is a single molecule sequencing technique with LMO binding peptides. As research progresses we will refine and isolate other promise for long reads, low cost, and minimal sample preparation. We have peptides that bind specifically to electroactive materials as well as conductive built on our progress in nucleotide sensitivity and DNA control to interpret carbon nanotubes, current collectors, and separator materials. Binding studies the procession of ion current levels observed as DNA translocates through will be performed to characterize the amino acid sequence as well as to improve the nanopore MspA. Approximately 4 nucleotides affect the ion current of peptide-material interactions, such that electrode binders may be replaced each level, therefore, we measured current corresponding to all 256 4-nucleo- by these peptides. These peptides will then be used to generate an aqueous, tide combinations (quadromers). This quadromer map proves to be highly self-assembling, lithium ion battery electrode. predictive of ion current levels for nanopore reads of bacteriophage phi X This work was funded by the UMBC Department of Chemistry and 174. We show nanopore sequencing reads of up to 4,500 bases in length, which Biochemistry. can be unambiguously aligned the phi X 174 reference genome. We also demonstrate proof of concept hybrid genome assembly and polymorphism 3175-Pos Board B605 detection. Shocking the World of Batteries: A Bio-Inspired Approach to Electrode 3178-Pos Board B608 Construction A Systematic Method for Designing DNA Nanostructure Assembly Scott J. Riley1,2. 1 2 Processes Chemistry, Frostburg, Brooklyn Park, MD, USA, Chemistry, UMBC, John Zenk, Chanon Tuntivate, Rebecca Schulman. Cantonsville, MD, USA. Johns Hopkins University, Baltimore, MD, USA. In the modern world there exists an ever increasing demand for more efficient Inspired by the complexity and diversity of biological structures that forms of energy storage. Electrochemical cells are the most widely used form self-assemble in vivo, a fundamental goal of bionanotechnology is to self- of portable energy storage and are possible candidates for large scale energy assemble biomolecular complexes with defined architecture from a set of storage. From mobile devices to cars, batteries are integrated into many facets starting components. To design such structures, many standard methods of daily life, yet little improvement in their performance is being recognized. involve constructing components that strongly bind to their neighbors through While batteries have been improving over the years, the growth is slow and orientation-specific interactions. While these design methods ensure the target staggered. An improvement similar to the discovery of lithium ion batteries structure is favored thermodynamically, in practice the yields of the resulting needs to be made in order to keep up with demand. This presentation is focused assembly processes vary widely and do not conform to equilibrium predictions. on maximizing battery performance by interfacing biology and batteries. To investigate why yields might be limited, we have developed a simple model My research focuses on using a bio-inspired approach to address battery perfor- of biomolecular complex self-assembly using reported rate constants and ther- mance. We use the technique of phage display to isolate Solid Binding Peptides modynamic parameters typical for biomolecular components. Such a coarse- (SBP) that bind specifically to Lithium Cobalt Phosphate (LCP). This technique grained model is particularly useful in cases where we consider the assembly has yielded two peptides that bind specifically to LCP. These specific SBPs of engineered components and purposely design the process to include simple are then combined with SBPs that are specific to carbon nanotubes (CNT). interactions. With these kinetic simulations, we show that even though com- This creates a multifunctional peptide that will be used to bio-tether conducting plexes are small and equilibrium is well defined, rarely is thermodynamic CNTs with LCP and thus generate better conducting hybrid electrodes. equilibrium achieved over timescales commensurate with experiments. We This new model for the electrode would enhance multiple aspects of battery find that rates of complex nucleation and component rearrangement together performance, including capacity, cyclabilty, and power. determine assembly yields. Our goal is to use this model in conjunction with experimental information 3176-Pos Board B606 to build a computer-guided, scalable design process for DNA origami com- Micro- and Nano-Chamber Array Chips for a Single Molecule Analysis ponents. We have used fluorescence-quenching assays to measure origami- Noritada Kaji. origami reaction rates and equilibrium constants that are driven by specific Nagoya University, Nagoya, Japan. Watson-Crick complementary (i.e., ‘‘sticky end’’) interactions. A feedback This paper reports the design, fabrication, and application of micro- and nano- loop between experimental measurements of a self-assembly process and a scale chamber array chips toward a single nucleus assay as a new experimental coarse-grained, computationally tractable model can guide the design of the format for research in biology. assembly process by making it possible to focus our assembly efforts on Micro- and nanoscale chamber array on a chip becomes a powerful new tool for specific interactions that make a strong difference in yields, or to redesign bioanalysis since it could stochastically capture and measure biomolecules at a the process to include qualitatively new types of component interactions. single molecule level. Our fundamental interests are how reaction-space size affects an enzyme activity and how small space we could extrapolate enzyme 3179-Pos Board B609 kinetics in small space from old enzyme kinetic data in bulk. So we have Microfluidics for Rare Cell Capture measured a single b-galactosidase activity in various sizes of micro and nano- Chwee Teck Lim. chambers, from 5 mm to 500 nm cubic, and found that the activity gradually Mechanobiology Institute & Dept of Biomedical Engineering, National decreased according to its chamber size. Because the specific surface area in- University of Singapore, Singapore, Singapore. creases according to the decrease of the chamber volume, non-specific adsorp- The presence and frequency of rare cells such as Circulating Tumor Cells tion might be a factor to suppress the activity. However, several experiments (CTCs) in bloodstream of patients with epithelial cancers is an important including surface coating and repetition of the enzyme capturing elucidated intermediate step in cancer metastasis and can provide valuable insights that non-specific adsorption is not a major factor affecting the activity. into disease stage and treatment evaluations. In fact, the presence and number Wednesday, February 11, 2015 631a of CTCs in peripheral blood has been associated with the severity of the dis- Engineered Biosurfaces ease and have potential use for early detection, diagnosis, prognosis and treat- ment monitoring. Nevertheless, their numbers are rare, typically less than one 3182-Pos Board B612 to about a hundred cells in 1ml of blood. The isolation of CTCs using micro- Pushing Micropatterning to the Nanoscale fluidics is attractive as the flow conditions can be accurately manipulated to Martin Fo¨lser1, Marco Lindner2, Eva Sevcsik1, Iris Bergmair2, achieve an efficient separation. Here, we will demonstrate several effective Gerhard Schu¨tz1. microfluidic separation methods by utilizing the unique biophysical property 1Vienna UT, Wien, Austria, 2Profactor GmbH, Steyr, Austria. differences such as size and deformability of cancer cells from that of blood Protein-protein interaction in live cell membranes can be shown by labelling cells as well as exploiting the fluid dynamics in specially designed microflui- the proteins of interest and mapping the distribution of the respective fluores- dic channels to isolate the generally stiffer and larger CTCs from the more cent signal in the membrane. Colocalization analysis of the images can indicate deformable and smaller blood cells. Using this approach, we are able to affinity of whatever kind, however background fluorescence, the excess of one retrieve viable CTCs with high isolation efficiency and purity. With blood interaction partner, coincidental overlaps and the difficulties of multicolor- specimens from cancer patients, we confirmed the successful detection, isola- imaging limit applicability and sensitivity of the approach. We presented a tion and retrieval of CTCs. Identification of CTCs will aid in the detection of method to circumvent many of the difficulties by arranging one of the proteins malignancy and disease as well as allow further insights into the metastatic as bait in a well-defined pattern in the cell membrane and measuring and process. comparing the abundance and diffusion characteristics of the second protein in bait-rich and bait-depleted areas and tested it on proteins of the immunolog- 3180-Pos Board B610 ical synapse as we showed the recruitment of Lck to CD4-enriched areas. To Effect of Magnetic Nanoparticles on Improving Doxorubicin Treatment of create those patterns the bait protein is immobilized by antibodies that are T47D Breast Cancer Cells 1 1 2 1 linked to Streptavidin, which is deposited in an underlying structure using Sarah A. Alobaid , Yuan You , Hasanain D. Al-Saadi , Michael J. Rossi , soft lithography. The standard protocols for this technique allow only the cre- Saion K. Sinha3. 1 ation of feature sizes which are at least one order of magnitude larger than the Biology & Environmental Science, University of New Haven, West Haven, supposed size of functional domains in the cell membrane. To miniaturize the CT, USA, 2Electrical and Computer Engineering, University of New Haven, 3 size of patterns to widths of 200nm and below great efforts in lithography are West Haven, CT, USA, Physics & Electrical and Computer Engineering, required as substrates have to be chosen more carefully and novel stamp mate- University of New Haven, West Haven, CT, USA. rials such as polyhedral oligomeric silsesquioxane, which contain custom Chemotherapeutic and anticancer therapeutics face restricted usage at higher tailored functional groups, have to be used to achieve optimal stiffness control. concentrations, because of potential serious side effects on healthy cells. There- The characterization of the imprints using combined AFM and fluorescence fore, finding more effective delivery mechanisms may enhance the efficacy of microscopy reveals a large environmental influence (humidity, temperature) the compounds and decrease side effects. Recently, Magnetic Nanoparticles during deposition of proteins and storage on the quality of the imprint and (MNP) have been demonstrated to increase the performance of some anticancer the substrate -protein binding affinity. We further investigate the micro- and drugs. In the current study the effect of MNPs used in tandem with an anti- nanoscale homogeneity of deposited streptavidin and demonstrate the usage cancer drug was studied on breast cancer cell lines with and without the effect of the miniaturized platform as a protein-protein interaction assay. of external magnetic field. MNP (functionalized and biocompatible Fe3O4 NPs 160 nm diameter) was 3183-Pos Board B613 coupled with Doxorubicin (DOX), a commonly used anti-breast cancer drug, Structural and Functional Study of Midbody during Cytokinesis and magnetically delivered to T47D breast cancer cells. Control studies were Rongqin Li, Weiwei Zhang, Q. Peter Su, Boxin Xue, Yujie Sun. performed with the intrinsic MNP and it was observed that they are not toxic Biodynamic Optical Imaging Center, Peking University, Beijing, China. to the cells. 50% cell viability was observed with a 1 mg/ml concentration of Midbody, an organelle forming during cytokinesis, plays a pivotal role in the DOX and this concentration was further used for MNP studies. The MNPs com- final step of mitosis via recruitment of proteins and regulation of vesicle fusion. bined with DOX were magnetically delivered to the cancer cells, both as a func- Recently, many studies are emerging to reveal the molecular mechanism for the tion of time and magnetic field. Optical and Atomic Force Microscope (AFM) functions of midbody-located proteins in cellular bridge abscission. Here, we were used to study the effect of these external parameters on the MNP penetra- aim to resolve the morphology and infrastructure of midbody to understand tion into the cell. From these microscopy studies. It was observed that more its role in cytokinesis. To achieve this goal, we apply super-resolution fluores- MNP entered the cells with increasing time and magnetic field until a saturation cence imaging and atomic force microscopy (AFM) to uncover the relationship point was reached. Also it was observed that there was a 20% increase in cell between midbody structure and functions. We focus our attention on central- mortality for the cells treated with DOX l MNPs. This study was then modelled spindlin, a tetramer composed of two MKLP1 and two MgcRacGAP, which with variable permeability medium and thus an optimum value of the time and is critical for midbody formation. We found that microtubule-associated pro- magnetic field was obtained. tein MKLP1 is a ring-like structure, the length and width of which is about 1.5 micron and 1 micron, respectively. Midbody height acquired by AFM is 3181-Pos Board B611 about 700 nanometer. The petal-like protrusions on the top of midbody exhibit Novel ‘Theranostic’ Magnetic Nanoparticles for Therapy and Imaging large rigidity as high as 500kPa. Interestingly, AFM results show that midbody Farah Benyettou1, Rachid Rezgui2, Ali Trabolsi1, Mazin Magzoub2. is an asymmetric, palm-wrapping-fist structure. We speculate that the asym- 1 2 Chemistry, NYU Abu Dhabi, Abu Dhabi, United Arab Emirates, Biology, metric structure may be responsible for the asymmetric abscission during NYU Abu Dhabi, Abu Dhabi, United Arab Emirates. cytokinesis. We report the development of a novel therapeutic nanoplatform, Targeted Chemotherapeutic Nanoparticles (T-CNPs), for the diagnosis and treatment 3184-Pos Board B614 of cancer. The T-CNPs are composed of iron oxide NPs that are functionalized Probing the Minimum Geometric Requirements for T-Cell Stimulation with water-soluble organic nanocontainers - for the encapsulation of a Haogang Cai1, David Depoil2,3, Michael P. Sheetz4, Michael L. Dustin2,3, combination of hydrophobic and/or hydrophilic drugs that can act synergisti- Shalom J. Wind5. cally - and conjugated to cell-penetrating peptides (CPPs) to enhance specific 1Department of Mechanical Engineering, Columbia University, New York, targeting of tumors and internalization into cancer cells. The nanoplatform is NY, USA, 2Department of Pathology, New York University, New York, NY, designed to simultaneously target a tumor, monitor it by magnetic resonance USA, 3Kennedy Institute of Rheumatology, University of Oxford, Oxford, imaging (MRI), and treat the disease by controlling the gradual release of the United Kingdom, 4Department of Biological Science, Columbia University, delivered anti-cancer drugs using a non-invasive external stimulus, which New York, NY, USA, 5Department of Applied Physics and Applied will concurrently induce hyperthermia. We will highlight new ways for the Mathematics, Columbia University, New York, NY, USA. active release of encapsulated drugs, particularly by an external magnetic field, The immune recognition process involves an elaborate arrangement of adhe- and promote the understanding of complex in vivo phenomenon when the sion, costimulatory and signaling molecules organized into a stereotypic molecules are optimally delivered. The nanoplatform combines stability, geometric structure known as the immunological synapse (IS). We have biocompatibility, and dual-purpose functionality (tumor treatment and imag- developed a versatile engineered platform to probe the minimum geometric ing), with multiple therapeutic strategies (targeted drug delivery and hyperther- requirements (in terms of spacing and stoichiometry) for T-cell stimulation. mia). Our system therefore consolidates diagnosis and targeted therapy into Arrays of metallic nanodots, ~ 2-10 nm in size, to which a UCHT1 Fab anti- a single, centralized system of treatment. This novel ‘theranostic’ strategy body was bound, were created by nanolithography. These served as individual has the potential to pave the way for treatment of cancer in a highly selective T-cell receptor (TCR) binding sites. The adhesion molecule ICAM-1 was and effective, yet relatively sensitive, manner. either statically bound to a PEG-silane brush surrounding the nanodots, or 632a Wednesday, February 11, 2015 it was allowed to move freely within the arrays in a supported lipid bilayer. lipids, the penetration of the liquid crystal into the lipid region, the propagation T-cells were plated on arrays comprising individual TCR binding sites, small of the interfacial orientation to the bulk phase behavior of the liquid crystal clusters and extended hexagonal close packed arrays, with spacings ranging film, and the effects of liquid crystal orientation on the phase transitions of from 40 nm and below to 1 mm. TCR signaling strength was monitored the lipids themselves. by measuring phosphorylated tyrosine (pY) intensity. T-cell adhesion was assessed by the number of cells bound to the arrays, as well as by the area 3187-Pos Board B617 of spread cells. Surface Modification of Solid-State Nanopores for Sticky-Free Transloca- Systematic variation of the spacing and cluster size of TCR binding sites in the tion of Single-Stranded DNA arrays enabled determination of the minimum conditions that support T-cell Zhipeng Tang. signaling and the formation of the IS. TCR signaling increased with decreasing Peking University, Beijing, China. spacing below a threshold spacing ~ 60 nm. For clusters with these spacings Nanopore technology is one of the most promising approaches for fast and the formation of the stereotypic ‘‘bullseye’’ geometry that characterizes the im- low-cost DNA sequencing application. Single-stranded DNA detection is pri- mune synapse became evident, with ICAM-1 excluded to the periphery. In mary objective in such device, while solid-state nanopores remain less explored terms of stoichiometry, at least 4 TCR-binding sites (within ~ 60 nm) were than their biological counterparts due to bio-molecule clogging issue caused required for T-cell adhesion and spreading. Further, in the absence of ICAM- by surface interaction between DNA and nanopore wall. By surface coating 1 (or at low concentrations), a threshold density of agonist sites was found, a layer of polyethylene glycol (PEG), solid-state nanopore can achieve long above which the TCR apparently plays a dual role of immune activity and lifetime for single-stranded DNA sticky-free translocation at pH 11.5. Associ- adhesion. ating with elimination of non-specific binding of molecule, PEG coated nano- pore presents new surface characteristic as less hydrophility, lower 1/f noise 3185-Pos Board B615 and passivated surface charge responsiveness on pH. Meanwhile, conductance Voltage Gating in Nanopores Containing Anthraquinone Mimics Biolog- blockage of single-stranded DNA is found to be deeper than double-stranded ical Membrane Proteins DNA, which can be well described by a string of blobs model for a quasi- Matthew Pevarnik, Weibin Cui, Luke Theogarajan. equilibrium state polymer in constraint space. Electrical & Computer Engineering, University of California, Santa Barbara, Biosurface Interactions Santa Barbara, CA, USA. Ion channels formed by membrane bound proteins play a key role in the proper 3188-Pos Board B618 functioning of the human body. These membrane proteins are able to regulate Predicting Adhesion of Functionalized Nanocarriers for Specific Peptide the transport of water, ions, and larger molecules through small openings in the Sequences using Atomistic Potentials of Mean Force protein under a transmembrane potential. Much study has gone into illumi- Matt McKenzie1, Aravind Rammohan1, Jacob Miner2, nating how these membrane proteins sense voltage. Some mechanisms include Natesan Ramakrishnan3, Ravi Radhakrishnan3. charged residues in proteins that can reorient in an electric field or side chains 1Corning Inc, Corning, NY, USA, 2RPI, Troy, NY, USA, 3UPENN, that have an intrinsic dipole moment. Here we report the first synthetic nano- Philadelphia, PA, USA. pore to mimic and explore these possible mechanisms. In this work we use atomistically computed potentials of mean force (PMF) We chemically attached a 9, 10 Anthraquinone (AQ) to the interior of an between peptide sequences and cell surface receptors as inputs to a Monte Alumina nanopore. In the presence of an applied voltage we found the AQ Carlo based code for predicting adhesion of nanocarriers onto surfaces func- responds in a manner similar to biological pores exhibiting voltage gating. tionalized with peptides. Here we demonstrate the validity of this approach The conductivity verse voltage of the AQ modified nanopore followed a classic to capture adhesion equilibrium constants by implementing experimentally sigmoidal gating curve, identical to biological membrane proteins. Through measured interaction potentials for the ICAM-ICAM1 system as a look-up this plot, it is possible determine the exact mechanism of the gating effect. In table. This methodology was then propagated to predict adhesion for two our system, we determined that the AQ was gaining one electron when the different peptide sequences: KGGEPRGDTYR and GERGDSFFAFRSPF. applied voltage reached a certain level. At this voltage level the AQ was From ligand-docking studies, we derived the binding free energy, and estimated reduced to form a radical semiquinone. The potential required to form the semi- the PMFs and obtained the differences in adhesion equilibrium constants. This quinone state can be determined from the conductivity plot. This technique work showcases a method for incorporating sequence specificity in predicting can also be applied to other molecules where the chemical reaction changes adhesion equilibrium constants for nanocarriers onto functionalized surfaces. it response to an electric field, such as deprotonation, cation incorporation Ó 2014 Corning Incorporated. All Rights Reserved. and radical formation. Corning Restricted 3186-Pos Board B616 3189-Pos Board B619 Modeling of the Liquid Crystal/Lipid Interface for Bio-Sensing Appli- Activated Membrane Surfaces by Functionalized Peptides cations Daniel R. Scott1, Vitalii Silin2, David Vanderah3, John P. Marino3, Donya Ohadi, Mark Uline. Susan Krueger1, Hirsh Nanda1. Chemical Engineering, University of South Carolina, Columbia, SC, USA. 1National Institute of Standards and Technology, Gaithersburg, MD, USA, The control of the orientation of liquid crystal thin films at surfactant-decorated 2Institute for Bioscience and Biotechnology Research, UMD, Rockville, MD, interfaces is of primary importance in the development of liquid crystalline USA, 3Institute for Bioscience and Biotechnology Research, NIST, devices as well as many applications in biological systems. Highly detailed Rockville, MD, USA. molecular level modeling of these interfaces is needed to help us to create For integral membrane proteins (IMP), an assessment of their structures and in- liquid crystal-based sensors that respond to specific chemical and biological teractions with other membrane proteins within a bio-mimetic lipid bilayer signals. These liquid crystal/surfactant systems have been studied extensively environment is critical for determining their cellular function. Hydrophobic due, in part, to both their birefringence optical properties and orientational sequences prevalent within the transmembrane domain(s) of IMPs, however, sensitivity to surface interactions. It is therefore critical to understand the make these proteins susceptible to aggregation, and thus create difficulties in interplay between the conformational entropy of the surfactants, the rotational examining their structural and functional properties via canonical techniques. entropy of the liquid crystals, the intermolecular and molecule-surface interac- Working exclusively with transmembrane (TM) segments of polytopic mem- tions, and the packing at the interfaces to be able to create design platforms for brane proteins - in the form of soluble peptides - bypasses many of the pitfalls sensing applications using these systems. of full-length protein preparations, while allowing for the opportunity to In the absence of liquid crystal molecules, binary DPPC/DOPC lipid mono- examine the properties of TM domains within bio-mimetic membrane environ- layers undergo phase transitions from liquid-expanded to liquid-condensed ments. In this study, peptides mimicking the TM domains of the epidermal phases as the lipid areal density decreases. DPPC has two fully saturated fatty growth factor (EGFR) and CD4 receptors, both cell-signaling membrane pro- acid tails, and DOPC has two monounsaturated fatty acid tails. The area per teins, have been reconstituted into POPC lipid bilayers. The formation of their molecule of this phase transition is highly dependent on the temperature and native alpha-helical structure within vesicle membranes was observed from CD the composition of the monolayer. In this work, we present the effect of liquid spectra, and proper orientation of the peptides passing through the membrane crystal nematic elasticity on liquid-expanded/liquid-condensed phase diagram. was demonstrated by tryptophan fluorescence using brominated lipids. Func- We use theoretical model predictions for the phase behavior of liquid crystal tionalized with an N-terminal biotin tag, and utilizing an engineered planar thin films in the presence of various lipid monolayer mixtures to better under- lipid bilayer system ideally set up for surface plasmon resonance measure- stand the fundamental interactions that control molecular reorganization. These ments, the TM peptides demonstrated capabilities of ‘‘activating’’ a membrane results shed light on the interplay between the conformational entropy of the surface by the capture of streptavidin. Prospectively, these peptides Wednesday, February 11, 2015 633a reconstituted as near-native membrane proteins within artificial bilayer systems we present adhesion equilibrium behavior for 3 different receptor-peptide could be utilized in formulating bioactive surfaces for powerful biomedical and sequences across a range of length scales, from 50 nm, to 500 nm. We believe biosensor applications. Novel techniques for re-forming the full-length IMP this approach offers a clear path to scaling up to mammalian cells (5-20 construct will be discussed. microns). 3190-Pos Board B620 3193-Pos Board B623 Characterization of Peptides Designed to Control Crystal Nucleation and Mapping Interactions between Silver Nanoparticles and Biomolecules at Growth the Atomic Level Shourya Sonkar Roy Burman1, Michael S. Pacella2, James J. De Yoreo3, Jeffrey Comer1, Horacio Poblete2, Emilio I. Alarcon3. Jeffrey J. Gray1,4. 1Nanotechnology Innovation Center of Kansas State and Institute of 1Chemical and Biomolecular Engineering, Johns Hopkins University, Computational Comparative Medicine, Kansas State University, Manhattan, Baltimore, MD, USA, 2Biomedical Engineering, Johns Hopkins University, KS, USA, 2Center for Bioinformatics and Molecular Simulations, Baltimore, MD, USA, 3Physical Sciences Division, Pacific Northwest Universidad de Talca, Talca, Chile, 3Bio-nanomaterials Chemistry and National Laboratory, Richland, WA, USA, 4Program In Molecular Engineering Laboratory, University of Ottawa Heart Institute, Ottawa, ON, Biophysics, Johns Hopkins University, Baltimore, MD, USA. Canada. Organisms, from algae to humans are known to mold complex, hierarchical The association of biomolecules with silver nanoparticles (AgNPs) has been hard tissues from minerals using biomolecular templates and additives. shown to modify the nanoparticles’ stability as well as their behavior in the Molecular-level mechanistic understanding of how these biomolecules, partic- physiological environment. However, the details of how silver nanoparticle ularly proteins, participate in the nucleation and growth of these inorganic surfaces – replete with heterogeneities – interact with the equally heteroge- crystals has been a longstanding goal. We design peptides with transformative neous surfaces of biomolecules remain elusive, yet essential to understanding abilities over calcite crystals using Rosetta. Based on the theory of how addi- the origin of the biological activity of AgNPs. Leveraging molecular dynamics tives alter crystal nucleation and growth, we employ four modification strate- simulation and free-energy/kinetics calculations, we have constructed maps de- gies to modify the morphology of the crystal, viz. a peptide binding to a tailing interactions of bare and functionalized AgNPs with peptides, proteins, face, an array of peptides binding to a face, peptides pinning steps and peptides and lipid bilayer membranes. blocking kinks. To test the designs, we employ a variety of techniques ranging from measurements at the atomic scale to full crystal observations. We also 3194-Pos Board B624 investigate alternative mechanisms of modification by comparing the interac- Protein Corona and Secondary Structure in Response to Nanoparticle tions predicted by Rosetta in other select states to those in the target state. Pegylation Sabiha Runa1, Alexandra Hill1, Victoria Cochran2, Christine Payne1. For each design, we obtain the solution-state structure of the peptide by circular 1 dichroism. To test peptides designed against a non-native face of calcite, we School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA, artificially stabilize the face for binding measurements. The overall crystal 2 morphology change is then tested by incubating supersaturated precursor Department of Chemistry and Chemical Biology, Harvard College, solutions with the design peptides. To confirm the predicted mechanism of Cambridge, MA, USA. growth alternation, we observe the change in kinetics of calcite step growth Nanoparticles are versatile tools for biophysical applications. Using these par- with peptide doping using in situ AFM, and report calcite step velocities. ticles requires a close examination of the protein corona: the layer of proteins Finally, by nucleating calcite on a monolayer of the designed peptides, we that adsorbs onto the particle surface. Modifying nanoparticle surfaces with examine the face on which calcite nucleated and compare it to our target polyethylene glycol (PEG) has been shown to reduce corona formation. face. These experimental results provide a feedback loop to the next generation Because tightly bound ‘hard’ corona proteins can block surface ligands that of designs and enable the rational design of bio-surface interactions. could be used in targeting applications, a reduction in corona is desirable and can enhance our ability to effectively utilize nanoparticle surface modifications. 3191-Pos Board B621 First, gold nanoparticles were PEGylated and characterized with dynamic light Examining Bacterial Cell Interactions using Atomic Force Microscopy scattering. Using gel electrophoresis, a three-fold decrease in corona formation Ronald Aucapina, Nadia Ouedraogo, Megan A. Ferguson. was found for PEGylated nanoparticles compared to bare nanoparticles. With a SUNY New Paltz, New Paltz, NY, USA. reduction of corona confirmed, we next investigated the secondary structure of Given the prevalence of bacterial biofilms in both native and engineered envi- the corona proteins. PEGylated and bare nanoparticles were incubated with ronments, our understanding of their interactions with both other bacteria and bovine serum albumin, the most prevalent serum protein. Using CD spectros- abiotic surfaces is quite limited. In this research we use an AFM to analyze the copy, we probed the secondary structure of the adsorbed albumin. Significant interactions of bacteria such as E. coli and a saprophytic, biofilm forming structural changes were not detected. In addition, bovine serum albumin, a2- variant of B. bacteriovorus with other bacteria and chemically characterized macroglobulin, and transferrin were each incubated with free PEG. Once again, surfaces. Tipless AFM cantilevers were left unmodified (Si3N4), or coated no alteration of protein secondary structures were found, even in the presence with a monolayer of E. coli. These cantilevers were then used to collect of a one hundred molar excess of free PEG. These results conclude that PEG force curves on biofilms of B. bacteriovorus and E.coli as well as chemically can quantitatively reduce corona formation without altering structural aspects characterized surfaces such as mica, silicon, and poly-L-lysine-coated glass. of corona proteins. The greater the cantilever’s contact time with the surface, the more force and energy was required to retract from the surface. E. coli-coated cantilevers 3195-Pos Board B625 had more adhesion to B. bacteriovorus biofilms than to E. coli biofilms, but A Theoretical Study of Polymer-Based Drug Delivery Systems E. coli B. bacteriovorus Ebtisam A. Aldaais1,2, Mark J. Uline3. even - interaction paled in comparison to adhesion 1 between E. coli biofilms and abiotic surfaces. Further results probing biofilms Biomedical Engineering, University of South Carolina, Columbia, SC, USA, 2Biomedical Engineering, University of Dammam, Dammam, Saudi Arabia, with cantilevers that have been chemically modified with acid or amine groups 3 will be presented. Chemical Engineering, University of South Carolina, Columbia, SC, USA. A variety of interactions between drug delivery devices and local cells and tis- 3192-Pos Board B622 sues impact clinical outcomes in terms of both therapeutic action and biological A Self-Consistent Multiscale Methodology for Predicting Adhesion of response. Understanding the competition of interactions in highly inhomo- Mammalian Cells onto Functionalized Surfaces geneous environments such as those relevant in tissue engineering, nanotech- Aravind R. Rammohan1, matthew mckenzie1, Jacob Miner2, nology, and those responsible for biological cell function is critical to the Natesan ramakrishnan3, Ravi Radhakrishnan4. further development of design platforms for delivery systems. We use a three 1Corning Inc., Corning, NY, USA, 2RPI, Troy, NY, USA, 3UPENN, dimensional mean-field theory to study the competition between electrostatic, Philadelphia, PA, USA, 4UPENN, philadelphia, PA, USA. van der Waals and steric interactions in determining the molecular organization Predicting cell adhesion onto surfaces functionalized with peptides is inher- of micelles made of ampliphilic diblock polybases designed to carry doxoru- ently a multiscale problem since the adhesion interface is mediated largely bicin to cancer cells. The micelles are assumed to target cancer cells primarily by interactions of specific peptides with surface receptors. This interaction oc- through electrostatic binding as several cancers are known to flip negatively curs over length scales on the order of nanometers, while typical mammalian charged lipids to the outer-leaflet. The polyelectrolyte micelles spontaneously cells are on the order of microns. In this work, we showcase a self-consistent form self-assembled aggregates whose physical properties are manipulated approach for obtaining specifics of interactions between peptide sequences by the composition of the solution in contact with the polymer system. These and receptors, and then applying this chemical information to describe these in- theoretical calculations show that chemical equilibrium and the relevant teractions for cells that are decorated with these receptors. Using this approach physical interactions present in responsive polymer micelless couple in such 634a Wednesday, February 11, 2015 a highly non-additive manner that the qualitative physics can only be accurately affinity between the peptide and the active material was determined through determined through a highly detailed molecular theory. site-directed mutagenesis of specific amino acids in the peptide sequence. We find that charge regulation stabilizes micellar domains over a wide range of Binding peptides for LMNO and multiwalled carbon nanotubes (MWCNTs) pH by reducing the local charge in the aggregate at the cost of chemical free are combined to form bifunctional peptide that serve as a nanobridge to con- energy and gaining in the van der Waals attractive interactions. The balance nect two materials with synergistic properties. In this presentation I will of interactions in this highly inhomogeneous environment determines the discuss research on determining how SBPs bind to electroactive materials, boundaries between different carrier and release morphologies. We predict and I will also show the impact that multifunctional SBPs have on improving the formation of polymer micelle phases based on the proper choice of solution battery electrode performance. pH and salt concentration, and one can use these predictions to provide design guidelines for the creation of responsive polymer delivery systems presenting 3198-Pos Board B628 self-organized patterns with the desired functional properties. Current Fluctuation Analysis in a Protein Nanopore Marı´a Queralt-Martı´n, M. Lido´n Lo´pez, Vicente M. Aguilella, 3196-Pos Board B626 Antonio Alcaraz. Red Blood Cell Behavior within the Exclusion Zone Laboratory of Molecular Biophysics, Department of Physics, Universitat Istva´n Husza´r1, Andra´s Laki2, Kristo´f Iva´n2, Miklo´s S. Kellermayer1. Jaume I, Castello´n de la Plana, Spain. 1Department of Biophysics and Radiation Biology, Semmelweis University, Current fluctuation analysis has been widely used over the recent years to Budapest, Hungary, 2Faculty of Information Technology and Bionics, study the kinetic effects of different biological systems like neuronal networks Pa´zma´ny Pe´ter Catholic University, Budapest, Hungary. or biomembranes. In particular, noise analysis has been successfully em- Various hydrophilic polymers have been reported to induce the formation of ployed in protein ion channels to investigate the transport mechanisms that an exclusion zone (EZ) at their surface, which is devoid of particles and may control the channel function. In this work, current fluctuations are analyzed extend to several hundred microns. It has previously been suggested that even in a protein nanopore, OmpF from E. coli. The study is performed for cells may be excluded from the vicinity of the gel, thus raising the possibility different electrolytes, including KCl, LiCl, MgCl2, and CaCl2, over a wide of developing microscale cell-separation technologies. Here we explored the range of concentrations and voltages. Previous studies addressing current fluc- behavior of red blood cells (RBCs) in the vicinity of Nafion with the aim tuations in OmpF investigated the pH titration of the channel residues by of devising a cell-separation method and of understanding the microscale analyzing the Lorentzian-like shape of the power spectral densities [1]. A mechanisms of EZ formation. We assembled a PDMS-based microfluidic complementary approach is followed here, based on the noise studies of device housing a 1-mm diameter, 50-micron thick cylinder of Nafion. A Hoogerheide and colleagues in synthetic nanopores [2]. Special attention is suspension of RBCs, prepared from anti-coagulated whole human blood by paid to the additional white noise seen in the low frequency range of the repeated washing/resuspension in PBS, was infused in the device. The posi- power spectral density. The average noise scales with the square of the dc cur- tion, shape and spectral properties of RBCs were followed with brightfield rent, showing that this frequencyindependent excess noise originates from video microscopy. Contrary to expectations, RBCs were not excluded from conductance fluctuations. These fluctuations are analyzed here in terms of Nafion surface. Rather, a three-phase process of aggregation, lysis and discol- the ionic concentration to disclose the different transport mechanisms occur- oration propagated gradually across the stationary RBC suspension from ring in OmpF channel. the Nafion surface towards distal regions. During the discoloration phase [1] E.M. Nestorovich et al. (2003) Biophys. J. 85:37183729 RBCs turned brown, pointing at the possibility of acid-hematin formation. [2] D.P. Hoogerheide et al. (2009) Phys. Rev. Lett. 102:256804 Microscpectroscopy measurements supported this hypothesis. Thus, the vicin- ity of fresh Nafion surface is a highly acidic environment. The spatial and 3199-Pos Board B629 temporal propagation of the process suggests that protons diffuse out of the Naturally Synthetic: Using Biology to Improve Technology polymer. Soaking Nafion extensively in PBS resulted in the disappearance Mark A. Allen, Evgenia Barannikova, Scott Riley, Alexander Winton. of the RBC-associated phenomenon, indicating that the thermodynamic Chemistry and Biochemistry, University of Maryland Baltimore County, driving force of particle exclusion is most likely the presence of a steep proton Baltimore, MD, USA. gradient between Nafion and the surrounding buffer solution. Due to its high Nature has a diverse toolkit that can be utilized to address a broad array of prob- acidity, unequilibrated Nafion has limited applications in cell-separation lems. Among these tools are DNA, lipids, polysaccharides, and proteins, each methodologies. of which has been used for direct applications from sensors to electronics. Among these macromolecules, proteins represent nature’s most diverse poly- 3197-Pos Board B627 mer with a range of functionality determined by 20 different natural building Solid-Binding Peptides as a Biotemplate for Li-Ion Battery Electrodes blocks. The functionality of proteins is determined by their amino-acid content Evgenia Barannikova, Mark Allen. and structure. For instance proteins that bind to metal ions for biomineralization Chemistry and Biochemistry, UMBC, Baltimore, MD, USA. typically express higher levels of amino acid residues containing carboxylate Li-ion battery electrodes composed of electroactive materials at the nanoscale side groups or histidines and cysteines. The amino acid makeup of a protein level show higher capacity and energy density over macroscale structures. or polypeptide determines its properties (whether it can bind to metal ions, However, nanoscale battery materials are prone to aggregation upon cell protein surfaces or nearly any other functional material because proteins can cycling, which reduces the specific capacity and coulombic efficiency, thus, be designed to bind to a large number of materials). leading to poor cycling stability. Using a biotemplating approach for electrode The focus of my group is to identify functional polypeptides and use these to fabrication presents opportunities that may overcome aggregation and improve the properties of technologically relevant materials. Our lab uses a improve conductivity through introduction of biological nanoscale templates technique called phage display in order to identify solid binding polypeptides that would precisely control the position of electroactive nanoparticles in inti- that are specific for binding to and the mineralization of electroactive materials mate proximity with conductive material and provide structural support upon and use these materials to prepare new lithium ion batteries. Once peptides are cycling. Engineering of nanoscale bridges between electroactive and conduc- identified, they will be synthesized and combined with other peptide chains that tive material is done using solid-binding peptides (SBP) that have specific have already been isolated that bind to carbon nanotubes (CNTs) to make binding affinity for the materials of interest. In our study, SBP for cathode ma- multifunctional polypeptides. My research is multidisciplinary and involves terial Li2Mn3NiO8 (LMNO) was isolated using M13 bacteriophage through the integration of biology, biochemistry, synthesis and nanomaterials science Phage Display procedure (New England BiolabsÒ). The nature of binding in order to address significant technological problems.