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family of DNA transposons. Although it is an important tool in genetic appli- 249-Pos Board B29 cations and has been adapted for human gene therapy, its molecular mechanism Folding Rates from Thermodynamics Simulations: Apoazurin as an remains obscure. Here, we use a combination of NMR and CD spectroscopy Example and molecular modeling to show that the primary DNA recognition domain Dirar M. Homouz1, Margaret S. Cheung2,3. of the Sleeping Beauty transposase, the PAI subdomain, is well folded at low 1Khalifa University, Abu Dhabi, United Arab Emirates, 2Physics, University temperatures, but the presence of unfolded conformation gradually increases of Houston, Houston, TX, USA, 3Center for Theoretical Biological Physics - at temperatures above 15C. Furthermore, we show that only the folded confor- Rice University, Houston, TX, USA. mation of the PAI subdomain binds to the transposon DNA, suggesting that the In this work we employed the approach of the Mean First Passage Time choice of temperature may be important for the optimal transposase activity. To (MFPT) to calculate protein-folding rates from the thermodynamic simulations gain insight into the mechanism of hyperactive mutations K14R and K33A according to the Energy Landscape Theory developed by Bryngelson and located in the PAI subdomain of SB transposase, we determine the effects of Wolynes. The folding rates from MPFT depend on a single reaction coordinate mutations on its folding and DNA-binding properties. Overall, the results pro- on the energy landscape. Here we applied this technique with the aid of coarse- vide a molecular-level insight into the DNA recognition by the PAI subdomain grained molecular simulations for the computation of folding rates of protein of SB transposase. Apoazurin. Experimentally, apoazurin folds in a two-state manner that allows for the use of a structure-based model attributable to a smooth funnel-like en- 247-Pos Board B27 ergy landscape. Based on the fraction of native contacts as the choice of a single CPSF30, a Novel Non-Classical Zinc Finger Protein that Utilizes Iron & reaction coordinates, we computed the folding rates of Apoazurin at several Zinc Coordination for RNA Recognition cellular conditions that perturb the population shift of the energy landsape. Geoffrey Shimberg1, Dr. Jamie Michalek1, Andria Rodrigues2, Dr. Timothy Stemmler2, Dr. Sarah Michel1. 250-Pos Board B30 1Pharmaceutical Sciences, University of Maryland Baltimore School of Barrierless Transition Identified during Folding of Barstar by using Pharmacy, Baltimore, MD, USA, 2Biochemistry and Molecular Biology, Time-Resolved FRET from 5-Flurotryptophan 1 1 2 Wayne State University School of Pharmacy, Detroit, MI, USA. Guruswamy Krishnamoorthy , Anju Yadav , Jayant Udgaonkar . 1Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India, Zinc finger (ZF) proteins are eukaryotic proteins that are involved in transcrip- 2 tional and translational regulation. ZFs utilize cysteine and histidine residues to National Centre for Biological Sciences-TIFR, Bengaluru, India. coordinate zinc ions to fold and function properly. There are fourteen different Despite a large volume of theoretical support for barrierless (downhill) folding classes of zinc finger proteins, which are classified based upon the ligands of proteins, the extent of experimental observation is rather limited. This lim- involved in zinc coordination, the ZF domain folds, and the proteins’ biological itation is even more striking when we address the kinetic process of folding targets. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a rather than equilibrium titrations of folding-unfolding transition. In this work ‘non-classical’ ZF protein that contains multiple Cys3His zinc binding do- we use the fluorescence of a single 5-Fluorotryptophan (F-Trp) in barstar as mains and is involved in pre-mRNA regulation. CPSF30 contains 5 Cys3His the energy donor and study the evolution of intramolecular distances and dis- domains and a Cys2HisCys zinc knuckle domain. A construct of CPSF30 tance distributions during the kinetics of folding. Enhanced homogeneity of construct containing only CPSF30’s five Cys3His domains, denoted as fluorescence decay kinetics of F-Trp when compared to that of Trp enables un- CPSF305FE was prepared. This protein is predicted to have five zinc ions; equivocal interpretation of distribution of fluorescence lifetimes obtained from however, UV-visible and ICP-MS analysis of the isolated construct revealed the Maximum Entropy Method (MEM) as arising from folded and unfolded that CPSF305FE contains iron in addition to zinc in a 1:4:1 Fe:Zn:CPSF305FE conformations of the protein. Of the two intramolecular distances monitored stoichiometetry. XAS analysis identified the iron site as a 2Fe-2S cluster. during the folding process, W53-C82 distance showed continuous decrease CPSF30 is predicted to recognize AU-rich RNA sequences. To test this hypoth- while W53-C40 distance behaved in a two-state manner. Implication of these esis, RNA binding studies using both electrophoretic mobility shift assays observations to the folding process is discussed. a (EMSA) and fluorescence anisotropy (FA) of CPSF305FE with -synuclein 251-Pos Board B31 pre-mRNA (which contains an AU-rich sequence) along with a series of Development of the Line Confocal System for the Single Molecule mutated RNA sequences were performed. These studies revealed that Tracking of Fast Folding Dynamics of Proteins a CPSF30 binds to -synuclein pre-RNA with nanomolar affinity and requires Hiroyuki Oikawa1, Kiyoto Kamagata1, Munehito Arai2, the AU-rich sequence for binding. The binding interaction was best fit to a Atsuhito Fukasawa3,4, Hiroaki Yokota4, Toru Ide5, Satoshi Takahashi1. cooperative binding model, suggesting that the CPF30/RNA binding event is 1Institute of Multidisciplinary Research for Advanced Materials, Tohoku cooperative. University, Sendai, Japan, 2Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan, 3Hamamatsu Photonics K.K., Hamamatsu, Japan, 4The Graduate School for the Creation of Protein Folding and Chaperones New Photonics Industries, Hamamatsu, Japan, 5Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and 248-Pos Board B28 Technology, Okayama University, Okayama, Japan. Transition Path Times in Protein Folding Studied by Structure-Based Single-molecule fluorescence spectroscopy is a powerful method for the Simulation investigation of protein dynamics. However, the time resolution of single- Mashiho Ito, Shoji Takada. molecule fluorescence spectroscopy is usually limited to a few milliseconds. Graduate School of Science, Kyoto University, Kyoto, Japan. To improve the time resolution, we developed the line-confocal microscope In trajectories of protein folding and unfolding processes, the transition path combined with microfluidic chip. By using the device, we could trace the that connects denatured and native states is the most interesting part. Recently, time evolution of FRET efficiency from single molecules with the time reso- single-molecule FRET experiments together with a statistical inference theory lution of about 100 ms for the observation time of 5 ms. As the first example successfully identified the transition path times for folding of some small pro- of the developed method, we investigated the equilibrium unfolding transi- teins. Yet, its underlying physics is poorly understood. Here we conducted a tions of two mutants of the B domain of protein A (BdpA) doubly labeled comprehensive survey of transition path times for 29 small-to-medium two- with donor and acceptor fluorophores. In the case for the mutant with a shorter state-folding proteins using structure-based coarse-grained molecular dynamics donor-acceptor distance, the FRET traces revealed a conformational heteroge- simulations. Using the multi-canonical ensemble method, we first identified neity in the unfolded state. In the case for the mutant with a longer donor- folding transition temperature accurately. At the transition temperature, we acceptor distance, the presence of a minor conformation in the native state then performed relatively long simulations observing reversible folding and un- was detected. These results demonstrate that the developed method can reveal folding events, from which we identified and analyzed the transition path times. the complexity in the apparent two-state folding of BdpA. To track the confor- The distribution of transition path time for each protein can be explained as free mational fluctuations of denatured proteins and the fast protein folding transi- diffusion of particle in a reaction coordinate. We sought what reaction coordi- tion, we are improving the system. To obtain the better time resolution nates correlate with the transition path time. Among tested coordinates, we without sacrificing the observation time, we built a new system based on found that the average transition path time is most strongly correlated to the dif- hybrid photo detectors (HPD). HPD is a photon-counting detector which ference in the numbers of native contacts, i.e., contact energies, between native achieves a high sensitivity and a large effective area. By introducing HPD and denatured states. These results imply that the transition path time series can to the line-confocal microscope, we could obtain the single-molecule FRET be approximated as the nearly free diffusion in the reaction coordinate of native traces with the time resolution of 10 ms and the observation time of more contacts. than 10 ms. The traces of BdpA obtained by using the new system exhibited Sunday, February 8, 2015 51a the submillisecond dynamics in the unfolded state, the small fluctuations in N-cadherin is not promoted by attractive ionic interactions, but can be inhibited the native state and the folding transitions. by repulsive ionic interactions in the R14E mutant. 252-Pos Board B32 255-Pos Board B35 Coiled Coil Probes Capture the Mechanical Unfolding Pathway of a Large Effects of Crowding Agents and Volume Exclusion on Amyloid Beta Protein Fibrillation Qing Li, Zackary N. Scholl, Piotr E. Marszalek. Joe Hakim1, Santiago Schnell2. MEMS, Duke University, Durham, NC, USA. 1Department of Biomedical Engineering, Johns Hopkins University, Folding behaviors and mechanisms of large, multi-domain proteins remain Baltimore, MD, USA, 2Department of Molecular & Integrative Physiology, largely uncharacterized, primarily because of the lack of appropriate research University of Michigan Medical School, Ann Arbor, MI, USA. tools. To address these limitations, we develop novel mechanical folding Several common neurodegenerative disorders can be attributed to pathological probes that are based on antiparallel coiled-coil polypeptides. Such probes protein aggregation, including Alzheimer Disease, the most common form of can be conveniently inserted, at the DNA level, at different positions within dementia. It is known that macromolecular crowding promotes protein associ- the protein of interest where they minimally disturb the host protein structure. ations, including the formation of complexes and aggregates, both through During single-molecule force spectroscopy measurements, the forced unfold- theoretical works and in vitro. Therefore a possible contributing factor in the ing of the probes captures the progress of the unfolding front through the onset of Alzheimer Disease and other amyloidoses could be an increase in host protein structure. This novel approach allows unfolding pathways of large crowding content inside cells. In this work, we carried out computational sim- proteins to be directly identified. As an example we use this probe in a large ulations demonstrating the promotion of a generalized aggregation reaction multidomain protein with 10 identical ankyrin repeats and we determine, un- model for amyloid beta fibrillation by increasing crowding agent concentration equivocally and precisely, the unfolding pathway, its direction and order of within biologically relevant values. In our in silico experiment, we imple- sequential unfolding. This development facilitates examination of the folding mented simulations with emulating the biophysico chemical properties of the pathways of large proteins that predominate proteasomes of all organisms intracellular environment, including nonspecific affinity of reacting species but have since eluded study because of technical limitations using traditional to crowding agents, varying size and shape of crowding agents, glymphatic techniques. removal and new synthesis of reactants, and others, demonstrating conditions in which aggregation can be more highly dependent on crowding agent concen- 253-Pos Board B33 tration. Our work suggests loss of cellular crowding or volume control as a pre- Fast Closure of Long Loops at the Initiation of Folding of Globular cursor to pathological aggregation. Proteins Studied by FRET Based Methods Elisha Haas. 256-Pos Board B36 Life Sciences, Bar Ilan University, Ramat Gan, Israel. Enthalpy Mediated Protein Stabilization by Macromolecular Crowding The rate of protein folding is determined by the rate of passage through the tran- Michael Senske1, Lisa To¨rk1, Benjamin Born2, Martina Havenith1, sition state, however major structural transition precede the TSE formation. We Christian Herrmann1, Simon Ebbinghaus1. hypothesize that few non-local interactions are effective in the early phases of 1Ruhr-University Bochum, Bochum, Germany, 2Weizmann Institute of the folding transition prior to the cooperative transition. These interactions Science, Rehovot, Israel. loosely stabilize few closed loops which form the folding noncontiguous nu- Understanding the structure and stability of biomolecules in the cellular environ- cleus, reduce the chain entropy and determine the course of the folding pathway ment requires knowledge of their interplay with high concentrations of macro- (the ‘‘loop hypothesis’’). We study the order of formation of secondary structure molecules, osmolytes, salts and membranes. The macromolecular crowding elements and loop closure during the early phases of the folding of E. coli adeny- effect in a cell is usually described by the excluded volume theory based on hard- late kinase (AK) by combination of rapid mixing methods and time resolved core repulsions [1]. However, recent studies emphasize the role of further con- FRET spectroscopy. We find that at the initiation of folding of the AK molecule tributions aside from a pure volume effect including enthalpic and solvent effects the two N terminal closed loop structures in the CORE domain reach native end [2, 3]. We study cosolute effects at high molecular and macromolecular concen- to end distance within the first 300 microseconds of the transition. Three repre- trations via a thermodynamic analysis of the thermal unfolding of ubiquitin in sentative CORE domain b-strands have non-native end to end distance during the presence of different concentrations of cosolutes (glucose, dextran, poly(eth- the first 15 ms and undergo slow change (3 sec) to native distance. Not all helical ylene glycol), potassium chloride) [4]. In contrast to the excluded volume theory, segments in the CORE domain fold at the slow rate as the beta strands. We we observed enthalpic stabilization and entropic destabilization forces for all conclude that non local interactions are essential factor at the early phases of tested cosolutes. The enthalpic stabilization mechanism of ubiquitin in macro- the folding transition and that the folding of sub-domain elements is context molecular polysaccharide solutions of dextran was thereby similar to the effects dependent and should be studied in the whole molecule, in situ. observed in monomeric glucose. The more hydrophobic polymer polyethylene glycol destabilizes native ubiquitin, presumably via unspecific direct interactions 254-Pos Board B34 with the protein. Thereby, the macromolecular character of PEG and dextran - Basic Residue at Position 14 is not Required for Fast Assembly and and hence its excluded volume - is not to the determining factor which influences Disassembly Kinetics in Neural Cadherin the folding equilibrium of ubiquitin. Instead, our data suggest a key role for hy- Nagamani Vunnam1, Nathan I. Hammer2, Susan Pedigo2. 1 2 dration water molecules in the interaction of proteins and cosolutes. University of Minnesota, Minneapolis, MS, USA, Chemistry and References Biochemistry, University of Mississippi, University, MS, USA. [1]H.-X. Zhou, FEBS Letters, 2013, 587, 394-397 In spite of structural similarities, Epithelial- (E-) and Neural- (N-) cadherins are [2]Y. Wang et al., The Journal of the American Chemical Society, 2012, 134, expressed at different types of synapses and differ significantly in dimerization 16614-16618 kinetics. Recent studies proposed a transient intermediate in E-cadherin as the [3]R. Gilman-Politi and D. Harries, Journal of Chemical Theory and Computa- key requirement for rapid disassembly kinetics of the adhesive dimer. This in- tion, 2011, 7, 3816-3828 termediate in E-cadherin comprises four intermolecular ionic and H-bonding [4]M. Senske et al., J. Am. Chem. Soc., 2014, 136, 9036-9041 interactions between adhesive partners. These interaction are not preserved in N-cadherin except a basic residue at 14th position, which could stabilize 257-Pos Board B37 the intermediate through either H-bonding or ionic interactions with the partner Crowding and the Origin of Enthalpic Depletion Forces in Protein protomer. To investigate the origin of the rapid dimerization kinetics of N-cad- Interactions herin in presence of calcium, studies reported here systematically test the role Daniel Harries, Liel Sapir. of ionic and H-bonding interactions in dimerization kinetics using R14S, R14A Inst of Chemistry, The Hebrew Univesity, Jerusalem, Israel. and R14E mutants of N-cadherin. Analytical Size Exclusion Chromatographic In crowded environments, solutes preferentially excluded from macromole- and bead aggregation studies showed two primary results. First, N-cadherin/ cules can drive depletion attractions in important biomolecular association pro- R14S and N-cadherin/R14A mutants showed fast assembly and disassembly cesses and in protein folding. The established Asakura-Oosawa theory relates kinetics in the calcium-saturated state similar to wild-type N-cadherin. These these depletion forces to the excluded volume reduction and the ensuing en- results indicate that the fast disassembly of the calcium-saturated dimer of tropy gain upon macromolecular compaction. Accordingly, cosolute-induced N-cadherin occurs in the absence of ionic or H-bonding interactions between protein stabilization is often described in terms of entropically driven ‘‘crowd- the residue at position 14 and the partner protomer. Secondly, the dimerization ing’’. In agreement, many experiments of protein folding and other macromo- kinetics of N-cadherin/R14E were slow in the calcium-saturated state indi- lecular processes suggest that depletion forces are predominantly entropic for cating that electrostatic repulsion destabilizes the intermediate state. Taken some cosolutes, such as polyethylene glycol polymers. However, for other together these results indicate that the kinetics of dimerization in wild-type cosolutes, such as polyol osmolytes, the effect is enthalpically dominated,