Nu Biophysical Society presents From Molecules to Behaviour

MBU In-house Symposium 2014

Abstract Booklet

Molecular Biophysics Unit

Indian Institute of Science

Bangalore 560012

India

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Table of Contents

1. Molecular Biophysics …………………………………… 5

2. Molecular Biophysics Unit at IISc ………………………. 5

3. Areas of research at the Unit ……………………………. 6

4. Program schedule for the One day Symposium ………..... 7

5. Plenary Lectures abstracts ……………………………….. 9

6. Student talk abstracts …………………………………… 13

7. Student poster abstracts ………………………………… 30

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Molecular Biophysics

Molecular Biophysics is an interdisciplinary science encompassing Biology, Physics, Chemistry, Engineering, Math and Computation. Integrating theory and experiments, it aims at understanding biological systems at various levels. Understanding the physical and chemical principles underlying life forms has been the impetus for many scientists and this has propelled this field over the decades.

Molecular Biophysics Unit at the Indian Institute of Science

The Molecular Biophysics Unit (MBU) at the Indian Institute of Science was founded in 1971 by G.N. Ramachandran. The Unit is currently engaged in frontline research in contemporary molecular Biophysics and Structural Biology. The research activities in the Unit focus on the structure, conformation and interactions in biomolecules, with the main objective of explaining biological activity in molecular terms. The unit has grown over a quarter of century, during this period, more than 200 young scientists have obtained their Ph.D. degrees and the number of research publications exceeds 1000. Seven faculty members have received prestigious S.S. Bhatnagar prize, eight members are fellows of the Indian Academy of Sciences and seven members are fellows of the Indian National Science Academy.

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Areas of Research at the Unit

 Protein folding and dynamics

 Computer modeling and dynamics of biological molecules

 Unusual DNA structure and control of transcription

 Genome organization

 Ion channels and electrophysiology

 Lectins and lectin-carbohydrate interactions

 X-ray crystallography of proteins and viruses

 Theoretical studies on peptide and protein conformation

 DNA-protein interactions

 Ionophores, drugs and their interaction with membranes

 Synthetic protein design and protein engineering

 Membrane channel forming peptides

 NMR studies of proteins and peptides

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MOLECULAR BIOPHYSICS UNIT In-House Symposium

Life Sciences at the intersection of Physics, Mathematics and Chemistry

Program Date: 14 June, 2014 Venue: Faculty Hall 8:45 – 9:00 AM Chairman’s Address Prof. RaghavanVardarajan (MBU, IISc) 9:00 – 10:00 AM Plenary Lecture 1 Dr. Vatsala Thirumalai, NCBS

Title: Mind the gap: Gap junctions and neural circuit assembly in larval zebrafish

Student Talks (Session 1) 10:00 AM – 10:45 AM Chairpersons: Nilesh Aghera & Sufyan Ashhad

10:00 – 10:15 Krishnayan Basuroy Prof. P. Balaram's group 10:15 – 10:30 Karuna Dixit Prof. Siddhartha P. Sarma's group 10:30 – 10:45 M. Saranya Dr. Rahul Roy group

Tea Break and Poster Session 1 (10:45 AM - 11:30 AM)

Student Talks (Session 2) 11:30 Noon – 12:45 PM Chairpersons: Anurag Pandey & Thyageshwar C

11:30 – 11:45 S M Arif Prof. M. Vijayan's group 11:45 - 12:00 Debanjan Dasgupta Prof. Sujit K. Sikdar's group 12:00 – 12:15 Kumar Perinbam Prof. B. Gopal's group 12:15 – 12:30 Geeta Deka Prof. M. R. N. Murthy's group 12:30 – 12:45 Deivanayaga Barathy V Prof. K. Suguna's group

Lunch Break: Main Guest House (12:45 PM – 2:00 PM) Student Talks (Session 3) 2:00 PM - 3:15 PM Chairpersons: Mamta Bangera & Ashutosh Gulati

2:00 - 2:15 Asmita Gupta Prof. Manju Bansal's group 2:15 - 2:30 Anindita Das Dr. Rishikesh Narayanan's group 2:30 - 2:45 Chetana Baliga Prof. R. Varadarajan's group 2:45 - 3:00 Richa Mudgal Prof. N. Srinivasan's group 3:00 - 3:15 Saurabh Yadav Prof. Surolia

Tea Break and Poster Session 2 (3:15 PM – 4:00 PM)

Student Talks (Session 4) 4:00 PM – 5:00 PM Chairpersons: Rustam Ali & Indra Mani Sharma

4:00 – 4:15 Soma Ghosh Prof. Saraswathi Vishveshwara's group 4:15 – 4:30 Hitesh Verma Dr. Jayanta Chatterjee 4:30 – 4:45 Mahavir Singh Mahavir Singh 4:45 – 5:00 Sunanda Margrett Prof. Dipankar Chatterji

5:00 – 6:00 PM Plenary Lecture 2 Prof. Rohini Balakrishnan, CES, IISc

Title: Acoustic communication in crickets: Behavioural ecology to biophysics Awards and Vote of Thanks (6:00 PM– 6:15 PM)

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Plenary Lecture Abstracts

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Plenary Lecture 1

Mind the gap: Gap junctions and neural circuit assembly in larval zebrafish

Dr. Vatsala Thirumalai

National Centre for Biological Sciences NCBS-TIFR, GKVK, Bangalore.

Gap junctions are cytoplasmic and localized in fiber tracts, neuropilar continuities between cells, through which areas and on somata. In the cerebellum, ions, second messengers and small we localized Cx35 puncta on Purkinje molecules can be exchanged. They are neurons using cell-specific markers. In formed by assemblies of connexin the hindbrain, the Mauthner neuron was proteins in vertebrates. Of the several labeled at club endings and in the axonal known isoforms of connexins, Connexin cap region. To test the function of Cx35 36 (Cx36) in mammals and Cx35 in during development, we designed and teleosts are predominantly expressed in microinjected splice-blocking morpholino the nervous system. Using antisense oligonucleotides specific for immunohistochemical methods in Cx35 and validated that Cx35 mRNA was zebrafish, we show that Cx35 is first seen mis-spliced. I will discuss results of our in the commissures and the optic tectum recent experiments in the cerebellum and at 2dpf. Other regions like the cerebellum hindbrain, which implicate Cx35 in acquire it later at 4-5dpf. glutamatergic synaptogenesis and Immunoreactivity was mostly punctate neuronal activity.

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Plenary Lecture 2

Acoustic communication in crickets: Behavioural ecology to biophysics

Prof. Rohini Balakrishnan

Centre for Ecological Sciences Indian Institute of Science Bangalore 560 012

Many animal groups such as crickets, of Kudremukh National Park in the frogs and birds use long-distance acoustic Western Ghats. The aim is to examine signals for mate attraction. The senders sender and receiver strategies for are typically males and the „intended‟ communication in the complex, noisy receivers are females of the same species. acoustic environment of the dusk chorus. Each species typically has a unique In this context, signal structure, signal acoustic signal by which it may be degradation and signaller behaviour have identified at a distance. Females use been examined for evidence of sender spectral and temporal properties of the strategies to avoid masking interference acoustic signal to recognize and then and maximize high-fidelity information locate calling males of their species, who transfer. Receiver strategies are being are potential mates. In most natural examined in terms of auditory mechanics, environments, a large number of physiology and behaviour. Another acoustically communicating species co- important evolutionary force determining exist and signal simultaneously, giving signal structure and signaller behaviour is rise to the conspicuous dawn chorus of predation: I will describe some recent birds and the dusk chorus of crickets and work examining the role of bat predation frogs. This raises the question of how in shaping cricket communication males and females of each species systems. I will also illustrate the manage to communicate with each other importance of biophysical measurements in the face of high levels of masking of structures involved in sound interference and signal degradation. Over production, transmission and reception the past ten years, my research group has for an understanding of the ecology and worked on an assemblage of acoustically evolution of acoustic communication communicating species of crickets and systems. katydids in the tropical evergreen forests

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Student Talk Abstracts

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Session 1: 10:00 AM – 10:45 AM

The C11/C9 Mixed Helices with Alternate Directionality Hydrogen Bond in the Crystal Structures  of Hybrid Peptides Krishnayan Basuroy, Vasantham Karuppiah, Narayanaswamy Shamala and Padmanabhan Balaram

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Helices are most abundant secondary aminomethylcyclohexanecarboxylic acid), structures in proteins and α-polypeptides. we observed formation of the mixed The two major helix types, 310 and α- C11/C9 structures, where αβ segments helices, in α-polypeptides, are form forward direction C=Oi…HNi+3 characterized by intramolecular hydrogen intramolecular hydrogen bonds and the bonds that run in the same direction and βα segments form reverse direction have hydrogen bonds of the type C=Oi…HNi-1 hydrogen bonds. In these C=Oi…HNi+n (n = 3 for the 310 helix and examples α-residues adopted semi- n = 4 for the α-helix) which require extended poly-proline (PII :   -60º,   orientation of peptide units in the same 120º / PIIˊ:   60º,   -120º) like direction. Helices with mixed hydrogen conformations and -residues adopted the bond directionality in which alternating backbone conformation   90º, θ  60º, C=O…HN hydrogen bonds run in   -90º. opposite directions are unprecedented in We will present the the structural chemistry of α-polypeptides. conformational analysis of mixed helical While studies in solution provide strong structures observed in some of the crystal evidence for the occurrence of mixed structures of (αβ)n hybrid peptides and helices in oligo-β-peptides and hybrid  discuss the Interconversion between sequences, limited information is C11(unidirectional helix) and C11/C9 currently available in the crystalline state. (alternate directionality helix) structures. During the course of studies of 2,2 (αβ)n hybrid peptides containing the β - 2,2 disubstituted residue, β Ac6c (1-

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Session 1: 10:00 AM – 10:45 AM

Structural studies of Sesbania Mosaic Virus (SeMV) VPg (Viral Protein Genome linked) in the free and bound state

Karuna Dixit, H. S. Savithri and Siddhartha P. sarma

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Sesbania mosaic virus (SeMV) is a plant (RdRP). The uridylated VPg (Viral virus that belongs to the genus Protein Genome linked) is attached to the sobemovirus and infects Sesbania 5‟ end of the RNA and serves as template grandiflora. The length of the genus is  for RNA synthesis. Polyprotein 4 Kb. The genome has four open reading processing requires the protease domain frames of which ORF2 codes for two to be fused with VPg protein. In isolation, polyproteins. Polyprotein processing is a the VPg proteins are known to be major strategy employed by both animal intrinsically disordered. Here we have and plant viruses to generate more than used NMR spectroscopic methods to one functional protein. In SeMV, the N- study the state of the VPg protein in the terminal protease domain coded by ORF2 free state as well as in an intermolecular mediates the processing. On processing, it complex with the protease domain. The releases protease, VPg, P10, P8 and a results of our studies will be presented. RNA dependent RNA polymerase

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Session 1: 10:00 AM – 10:45 AM

Probing viral genome evolution by single RNA sequencing M. Saranya1, Vaseef Razvi2, Katsu Shiroguchi3, Guru Prasad4, Rahul Roy1, 2

1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 2Department of Chemical Engineering, Indian Institute of Science, Bangalore 560 012 3RIKEN Center for Integrative Medical Sciences, Yokohama, Japan 4Translational Health Science and Technology Institute (THSTI), Gurgoan, India

E-mail: [email protected]

RNA viruses propagate as quasispecies of we are developing a method that would closely related genotypes in the host. This give us the full-length sequence of each helps the virus in rapid adaptation, drug individual viral RNA molecules and resistance and expansion. Sequence thereby provide the tools to track the diversity and virulence of a viral species sequence variations in a viral population. is regulated by the host selection A complete sequence landscape of the pressures at different levels of its life viral pool will help explain the cycle. Ability of a particular variant in a contribution of various mechanisms like genotype to propagate rapidly compared recombination and mutation to viral to others, is due to its inherent genetic genome evolution. In addition, full-length modifications. Our main objective is to viral sequences will allow us to correlate define such genetic contributions of the the inherited elements such as co- full-length virus genome to its infectivity, evolving residues to functional changes in which we term as “infectivity fitness”. To viral constituents. probe the sequence-infection relationship,

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Session 2: 11:30 AM – 12:45 PM

Conformational selection and ligand binding in Mycobacterium tuberculosis uracil-DNA glycosylase (MtUNG) Arif S M,1 Geethanandan K,1 Mishra P,1 Surolia A1 , Varshney U2 and Vijayan M1

1Molecular Biophysics Unit, Indian Institute of Science, Bangalore. 2Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore.

E-mail: [email protected]

Uracil-DNA glycosylase excises uracil closed conformation while others remain from a single or double stranded DNA in the open conformation. Variability in arising either from deamination of the C-terminal region in the second cytosine or incorporation of dUMP during domain is also observed among the replication. We report here the structures structures. Thus, MtUNG appears to of the complexes of M. tuberculosis present an interesting case of uracil-DNA glycosylase (MtUNG) with conformational selection. Uracil and all uracil and its fluoro, chloro, nitro, amino the analogues bind to the protein with and thio analogues. The protein is known reasonable but varying affinity. The to exist in a closed conformation when compounds exhibit two modes of binding. bound to DNA and in an open The locations of the uracil moiety in the conformation in the absence of DNA. two cases are related by a two-fold DNA binding is also associated with symmetry passing through N3 and C6. movements in the C-terminal region in The existence of the two modes can be the second domain. Although DNA is largely explained in terms of steric bound to the protein in none of the interactions. Further examination of the present structures, some of them exhibit structures is in progress.

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Session 2: 11:30 AM – 12:45 PM

Calcium permeable AMPA receptor dependent long lasting plasticity of intrinsic excitability in fast spiking interneurons of the dentate gyrus decreases inhibition in the granule cell layer

Debanjan Dasgupta, and Sujit Kumar Sikdar

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012. E-mail: [email protected]

The local fast-spiking interneurons with membrane hyperpolarization and (FSINs) are considered to be crucial for depolarization respectively. Including a the generation, maintenance and selective blocker of calcium-permeable modulation of neuronal network AMPA receptors (CP-AMPARs) in the oscillations especially in the gamma bath significantly attenuated the calcium frequency band. Gamma frequency rise and blocked the membrane potential oscillations have been associated with dependence of the calcium rise in the different aspects of behaviour. But the FSINs, suggesting their involvement in prolonged effects of gamma frequency the observed phenomenon. Chelation of synaptic activity on the FSINs have not intracellular calcium blocked the been explored previously. Using whole expression of plasticity. Blocking HCN cell current clamp patch recordings, we channel conductance or CP-AMPARs observed a sustained decrease of intrinsic during the experiment also forbade the excitability in the FSINs of the dentate long lasting expression of the plasticity. gyrus (DG) following repetitive synaptic Simultaneous dual patch recordings from stimulations of the mossy fibers at 30 Hz FSINs and synaptically connected (gamma bursts). Interestingly, when the putative granule cells (GCs) confirmed gamma bursts were paired with that the decreased intrinsic excitability in membrane hyperpolarization, the decrease the FSINs accompanied decreased in excitability following the induction strengths of inhibitory post-synaptic protocol accentuated further, while it was potentials in the GCs. Experimentally attenuated when the gamma bursts were constrained network simulations using paired with membrane depolarization. NEURON predicted increased spiking in Paired pulse ratio measurement of the the GC owing to decreased input synaptic responses did not show resistance in the FSIN. We hypothesize significant changes during the that the plasticity in the FSINs induced by experiments. However, we observed post- local network activity may serve to synaptic calcium rise associated with the increase information throughput into the induction protocols. Interestingly, the downstream hippocampal subfields maximum and the minimum increase besides providing neuroprotection to the occurred during pairing of gamma bursts FSINs.

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Session 2: 11:30 AM – 12:45 PM

Structural insights into the role of the Bacillus subtilis reductase YwfH (BacG) in tetrahydrotyrosine synthesis

P Kumar and Gopal B

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

The synthesis of the di-peptide antibiotic structurally similar to other characterized bacilysin involves the sequential action of short-chain dehydrogenase/reductases multiple enzymes in the bac operon. despite marginal sequence similarity. YwfH (also referred to as BacG) The structures of YwfH in different catalyzes the stereo-selective reduction of conformational states provide a rationale H2HPP (dihydro-hydroxyphenylpyruvate) for the ping-pong reaction mechanism. to H4HPP (tetrahydro- The identification and role of the residues hydroxyphenylpyruvate) in this in the catalytic tetrad (K113-Y117-S155- biosynthetic pathway. YwfH is a N158) in proton transfer was examined NADPH dependent reductase that by mutational analysis. Together, the facilitates the conjugate addition of a structures and biochemical features reveal hydride at the C4 olefin terminus of synchronized conformational changes that H2HPP. Here we describe the structure of facilitate co-factor specificity and YwfH in three conformational steps- the catalysis of H4HPP formation en-route to apo form, an apo-like conformation and tetrahydrotyrosine synthesis. the NADPH complex. YwfH is

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Session 2: 11:30 AM – 12:45 PM

Structural studies to elucidate the catalytic mechanism of Diaminopropionate ammonia lyase Geeta Deka1, Shveta Bisht1, M. R. N. Murthy1, H.S. Savithri2

1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 2Department of Biochemistry, Indian Institute of Science, Bangalore 560 012

E-mail: [email protected]

Diaminopropionate ammonia lyase Reduction of catalytic efficiency (DAPAL) is a non-stereo specific fold- (Kcat/Km) of D120N EcDAPAL for D- type II pyridoxal 5‟ phosphate (PLP) DAP by 140 folds and presence of the dependent enzyme that catalyzes the uncatalyzed ligand in external aldimine conversion of both D/L isoforms of the form at the active site in the crystal nonstandard amino acid structure suggested that Asp120 indeed Diaminopropionate (DAP) to pyruvate abstracts proton from D-DAP. Lys77, and ammonia. DAP is important for the which was speculated to be important for synthesis of nonribosomal peptide proton abstraction from L DAP; however antibiotics such as viomycin and seemed to be crucial for PLP binding only. capreomycin. Structural studies on Presence of non-covalently bound PLP in EcDAPAL bound to a reaction the L77W mutant and occurrence of both intermediate (aminoacrylate) suggested the ketoenamine, enolimine forms of that the enzyme follows a two base internal aldimine in L77R mutant mechanism, where Asp120 and Lys77 provided an in depth insight into the function as general bases to abstract unique chemistry of internal aldimine proton from D-DAP and L-DAP formation in PLP dependent enzymes. respectively. To understand the reaction Thus, these studies provide deeper mechanism structural and biochemical insights into the reaction mechanism of characterization of active site mutants EcDAPAL, representing the overall Asp120 (Asp120Asn/Ser/Thr/Cys) and reaction mechanism followed by several Lys77 (Lys77His/ Thr/Ala/Val) of other fold-type II PLP pendent enzymes. EcDAPAL has been carried out.

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Session 2: 11:30 AM – 12:45 PM

Crystal structure of an aspartic proteinase-like domain from Mycobacterium tuberculosis

Deivanayaga barathy V, Sushanth Kumar, Muraleedhara Reddy T, Kaza Suguna

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

A search in the genome of M. host. The crystal structure of one of the tuberculosis showed a weak similarity to domains showed that it has a pepsin-like the HIV aspartic proteinase sequence. fold and the catalytic site architecture of This region corresponds to the C-terminal known aspartic proteinases. However, no domain of a PE family protein in M. proteolytic activity was detected. The size tuberculosis (Rv0977). A search with the of the molecule is intermediate to sequence of this domain revealed the eukaryotic pepsins and the homodimeric presence of similar domains in two other retroviral pepsins. A close examination of proteins of M. tuberculosis, Rv1983 and the binding site revealed subtle Rv2519. The presence of two signature differences when compared to the active motifs, DTG and DSG, of aspartic enzyme structures. Appropriate mutations proteinases in the full sequence of this of some of the residues in this region to domain encouraged us to take up further convert it to an active enzyme did not studies on this domain. Previous reports make it active. Multiple sequence identifying the protein as a surface alignment of M. tuberculosis aspartic antigen and our findings on the proteinases shows that the critical occurrence of similar domains in two substrate binding residues of Rv2519 are other PE proteins of M. tuberculosis similar in nature to those in pepsins. indicated that these domains probably Hence, it is likely that Rv2519 could be play an important role in infecting the an active aspartic proteinase.

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Session 3: 2:00 PM - 3:15 PM Molecular basis for nucleocytoplasmic transport of tRNA by Exportin-t

Asmita Gupta, Senthilkumar Kailasam and Manju Bansal

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Export of mature, fully processed tRNA structural motions in Xpot after cargo molecules from nucleus to cytoplasm is release and identified various molecular carried out by Exportin-t (Xpot) protein determinants responsible for cargo in mammals (Los1p homologue in binding. We employed classical all atom S.cerevisiae). It belongs to a family of molecular dynamics simulation methods nuclear import/export proteins called to study all the molecular complexes Karyopherins, which are formed of a involving Xpot. The overall repeating motif of 35-40 amino acids conformational change in Xpot due to called HEAT repeats. The export and cargo release was attributed to a highly subsequent release of tRNA cargo by fluctuating C-terminal region. The Xpot involves Ran protein and GTP identity of residues in GTP and GDP hydrolysis in the cytoplasm which is bound states of transporter protein was associated with Ran. Despite the also established, besides analyzing the availability of crystal structures of nuclear change in the molecular electrostatic and cytosolic forms of Xpot, the surface potential of Xpot during molecular details regarding the sequential conformational change after tRNA release. events leading to tRNA release and The energetics of complete and conformational change by Xpot remains intermediate complexes were also studied unclear. We studied a range of molecular in order to illustrate differential binding complexes including free Xpot protein affinities of Xpot in presence or absence and intermediate state complexes bound of its specific cargo and directionality of either to Ran or tRNA, to illustrate gross the overall transport process.

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Session 3: 2:00 PM - 3:15 PM Neuronal ion channels and their spatial localization regulate feature selectivity in single neurons Anindita Das and Rishikesh Narayanan

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

An outstanding question in the quest to transformation. In this talk, I will present understand information encoding in our a quantitative approach to addressing this brain is what features neurons extract lacuna employing what is known as the from the multi-dimensional sensory input spike triggered average (STA), a measure that they receive from the external world. of the specific input features that a neuron Memory encoding requires processing selectively responds to with an action and integration of information from potential. The approach involves the multiple sensory cues, a feat achieved by quantification of several STA-derived the well-organized circuitry of the measures to assess the role of VGICs in hippocampus, a brain region critically altering the features that neurons respond involved in learning and memory. to. Through the course of the talk, I will Pyramidal neurons in the hippocampus present several lines of evidence that are prodigious computational devices that demonstrate that VGICs and their specific transform tens of thousands of afferent localization along the neuronal membrane synaptic inputs into action potentials that can radically alter the specific input encode critical features about these inputs. features that neurons respond to. These However, little is understood on how the results demonstrate that the biophysical biophysical properties of single neurons properties of a neuron play a critical role and the voltage-gated ion-channels in information encoding by regulating the (VGIC) that reside on their plasma specific features that a neuron is selective membrane aid in this information to.

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Session 3: 2:00 PM - 3:15 PM Rational design of cold sensitive phenotypes Chetana Baliga1, Kajari Mondal1, Antara Bhattacharjee1, Sandipan Majhi1, K. VijayRaghavan2 and Raghavan Varadarajan1

1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 2National Centre for Biological Sciences, TIF, Bangalore.

E-mail: [email protected]

Cold sensitive (cs) mutants behave like phenotype, with activity at 37 oC and no loss-of-function mutants at temperatures activity at 30oC. In addition, previously below the restrictive temperature but isolated partial loss-of-function mutants display wild type phenotypes at higher of yeast Gal4 were expressed in S. temperatures and are useful tools to probe cerevisiae under the control of a heat gene function in vivo. They are rarer than inducible promoter. Three of the four temperature sensitive (ts) mutants and the mutants tested showed cs phenotypes. We molecular mechanisms responsible for cs also designed buried site mutants in Yeast phenotypes are poorly understood. Herein, Trp1 and Ura3 proteins and screened we propose a method for rational design them for cs behavior. Finally, the four of cs mutants. It is known that loss of Gal4 mutants were individually cloned function mutants can often be rescued by into a Drosophila P element vector under overexpression [Bajaj et al, 2008]. We control of an eye specific GMR promoter have previously shown that it is possible containing Heat Shock Elements (HSEs). to accurately predict buried residues Transgenic Drosophila lines were solely from amino acid sequence generated, crossed to UAS reporter lines [Varadarajan et al, 1996] and to engineer and progeny were screened for reporter destabilizing mutants at predicted, buried gene expression at multiple temperatures. positions [Chakshusmathi et al, 2004]. In three cases, cs phenotypes were We hypothesize that destabilized and/or observable. These data demonstrate that partial loss-of-function mutants can be cs phenotypes need not result from converted to cs ones by increasing their complex, temperature-dependent expression level selectively at high mutational effects. Instead, many partial temperature. In order to test this loss-of-function mutants can be converted hypothesis, 45 known ts mutants of the to cs ones by either increasing their E.coli toxin CcdB were overexpressed at expression level or activity at high 37oC while maintaining basal levels at temperatures; or by decreasing the same 30oC. In all cases, this resulted in a cs at low temperatures.

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Session 3: 2:00 PM - 3:15 PM Design of artificial proteins to aid detection of related natural proteins Richa Mudgal1, Ramanathan Sowdhamini2, Nagasuma Chandra3, Narayanswamy Srinivasan4* and Sankaran Sandhya3

1 IISc. Mathematics Initiative , Indian Institute of Science, Bangalore. 2 National Center for Biological Sciences, Bangalore. 3 Department of Biochemistry, Indian Institute of Science, Bangalore. 4 Molecular Biophysics Unit, Indian Institute of Science, Bangalore.

E-mail: [email protected]

With the advent of high-throughput evolutionary relationships, we methods for genome and proteome demonstrate that such designed sequences sequencing, the need for efficient and when integrated with natural protein sensitive tools for functional annotation sequences in databases and employed in of proteins is -increasing. In the absence sequence-based remote homology protein structures, sequence similarity is a searches, show improved fold coverage key factor in detecting remote and up to 66% in the number of correct relationships. However sequence-based evolutionary relationships discovered, search methods are rendered less effective with an average increase of 15.6% at due to the non-uniform dispersion of the significantly low error rates (1.7%). protein sequence space. Here, we describe Although sequences could not be a computational approach to purposefully designed between some families, fill-in the void and sparse regions designed sequence between other families between two related protein families within the fold aided in detecting 373 through directed design of protein-like difficult relationships. Additionally, „linker‟ sequences. Protein families are artificial sequences when plugged-into represented as multiple profiles and other generic sequence databases such as related protein domain families are Pfam also empowers remote homology aligned using a profile-profile alignment detection and fold recognition. method, AlignHUSH. Where reliable alignments were achieved, a Roulette Reference: wheel-based method was used to design 3,611,010 artificial sequences R. Mudgal, R. Sowdhamini, N. Chandra, corresponding to 374 SCOP folds. Using N. Srinivasan & S. Sandhya (2014) J. 3024 queries with known distant Mol. Biol., 426, 962-979.

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Session 3: 2:00 PM - 3:15 PM Investigating the modulators of Neuropathic Pain Saurabh Yadav and Avadhesha Surolia Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Nociception has evolved as a mechanism channels), intracellular signaling to avoid a potentially hazardous stimulus molecules (like MAPK, CaMK, ERK, and thus helpful in the survival of the PKA, cAMP, ERK, CREBP , NFkB etc) organism. In general, pain enhances the etc. have been reported to play a role in sensitivity of the injured tissue, against its pathogenesis. NFkB is one of the key noxious stimulus, thereby protecting the regulators of cellular transcription, and injured area from further injuries and thus homeostasis. It was known to play a very helpful for the healing process. important role in inflammatory pain, Physiological pain, hence is generally however its role in neuropathic pain has transient (acute pain) in nature. Chronic been discovered recently. In the past few pain may occur as a result of pathological years there has been many reports chronic inflammation (inflammatory pain) showing that siRNA mediated as well as or lesions to the nervous system pharmacological inhibition of p65 subunit (Neuropathic pain). Neuropathic pain of NFkB leads to alleviation of arises due to nerve lesions of primary neuropathic pain. However the sensory neurons of the central or mechanism of regulation of the levels of peripheral nervous system and is p65 subunit itself is not known. We tried associated with dysesthesia , hyperalgesia, to study the cellular pathways involved in and mechanical allodynia being one of regulation of p65 expression. Our data the most distinctive feature of neuropathic shows that a whole new pathway pain. involving ILK1 as an intermediate plays a Molecular mechanism behind role in the regulation of p65 expression in neuropathic pain has not been fully neuronal cells and modulate their firing understood, although various properties which may contribute to the inflammatory mediators, ion channels pathophysiology of neuropathic pain. (like voltage gated Na, K, and Ca

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Session 4: 4:00 PM – 5:00 PM Investigating the mechanism of iron dependent Repressor (ideR) activation and DNA binding Soma Ghosh, Nagasuma Chandra, Saraswathi Vishveshwara Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 E-mail: [email protected]

Metalloproteins form a major class of systems were identified and compared. enzymes in the living system and are Details at the atomistic levels were involved in critical biological functions investigated using hydrogen bonds and such as catalysis, redox reactions and as protein structure networks [PSN/PDG] “switches” in signal transductions. and further quantified using free energy Irondependent repressor (ideR) is a metal [MMPBSA] calculations. sensing transcription factor that regulates The results show significant free iron concentration in Mycobacterium variation between the metallated and the tuberculosis. Crystal structures of ideR nonmetallated systems. At the level of bound to DNA are available and indicates monomers, iron strongly influences the the formation of a 'dimer-of-dimer' flexibility of the structure and complex, with the two dimers binding to intradomain movements, which in turn opposite sides of the DNA double helix. promotes dimerization and DNA binding. Apart from regulating iron homeostasis, Mode of dimerization in the absence and ideR is also known to promote bacterial presence of iron and how that influences virulence. Various experiments have DNA binding has also been thoroughly suggested that ideR exists as a discussed. monomerdimer equilibrium, with the Perhaps, the most striking results equilibrium shifting towards the dimer in are obtained from the simulations of the the presence of the metal. The two ideR-DNA complex in the absence of metallated dimer, in-turn binds to DNA metals. As compared to the metallated and facilitates its biological function. In systems, the protein molecules are seen to this study, we seek to understand the role move away from the DNA in the absence of iron in ideR activation and DNA of metals. Such drastic changes in the binding at the atomistic level. This is ideR-DNA interactions not only provides performed at two levels, a) shift in molecular insights about the role of iron, monomer-dimer equilibrium, and b) but also about the mechanism of DNA interaction of ideR with DNA. Molecular binding and unbinding. These results dynamics (MD) was employed to obtain would be presented in details in the talk. the ensemble structures in the presence Further, the possible role of iron as an and absence of metals, which were then allosteric effector in ideR function would analyzed to study the overall structural also be discussed. Finally, we propose a variation between the two states and model describing the sequence of events hence the influence of iron. Free energy that govern ideR binding to DNA in the landscapes of the different ligand bound presence of iron.

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Session 4: 4:00 PM – 5:00 PM Multiple thionation of cyclic peptides Hitesh Verma, Jayanta Chatterjee

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Cilengitide and Aplidine are N- the carbonyl oxygen is replaced with a methylated cyclic peptides, which are in sulfur atom, with a slight change in clinical trials phase III against several electron distribution in the ground state, cancers. Extensive research on N- which have been used in studies of methylation of biologically active cyclic biologically active peptides. The peptides revealed that in spite of subtle thioamide NH is a stronger hydrogen loss in bioactivity, N-methylation bond donor than the amide NH, while the remarkably improves the pharmacological sulfur is a weaker hydrogen bond properties of cyclic peptides. Thus, by the acceptor than the amide oxygen. With this substitution of a „H‟ atom with „CH3‟ in mind we will employ a rational group in amide bonds dramatically alters approach of multiple thionation of cyclic the physicochemical and pharmacological peptide backbone and study the impact of properties of cyclic peptides. multiple thionation on the Thioxo peptide bond (thioamide) physicochemical and pharmacological (–ψ[CS–NH]–) represents an isosteric properties of cyclic peptides. replacement of the peptide bond in which

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Session 4: 4:00 PM – 5:00 PM

Identifying key residues of the Ferroxidation Processes in Mycobaterium smegmatis DNA binding Proteins under Starvation Sunanda Margrett Williams

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

DNA binding proteins under starvation generated by these substitutions were (Dps) are dodecameric nano- found be increasing unstable and compartments for iron detoxification and dissociate from the dodecamer in the storage in bacterial cells. In addition, they presence of iron. The iron-loaded proteins characteristically bind DNA and protect it of low, medium and high iron-bound from free radical damage by bypassing forms on analysis, were found to exhibit the Fenton reaction. The mechanism by asparate residues with alternate which these proteins belonging to the conformations of which some could be ferritin protein family oxidize and store directly linked to the active sites of iron is an area of active research. Here, ferroxidation and iron entry. Here, we we use X-ray crystallography to demonstrate that the increased flexibility determine the structures of co-crystals of of the aspartates in presence of iron iron and Dps and dissect the changes in facilitate the movement of iron from entry these ligand bound form with respect to site to the ferroxidaton site. We also the apo-protein. These studies have been propose that the dps-like interface of done in the second mycobacterial Dps these proteins function as an entry point protein from Mycobacterium smegmatis, for chloride ions to the protein shell to MsDps2. Also, we have mapped a route neutralize iron. The ferritin-like (iron for iron atoms from its entry in the entry point) and dps-like trimeric protein shell to the ferroxidation centre, interfaces in these proteins exhibit many by mutations and interaction analysis of a similarities to cation and anion channel conserved arginine residue. The mutants proteins, respectively.

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Student Poster Abstracts

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Population shift mechanism opens up new druggable hotspot in Pregnane X Receptor Aneesh Chandran, Saraswathi

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

The Pregnane X Receptor (PXR), a bound systems to reveal the ligand member of the nuclear receptor induced subtle changes in PXR. MD superfamily, regulates the expression of simulations revealed the existence of two proteins involved in xenobiotic different conformational states, „breathin‟ metabolism. PXR gets activated after and „breathe-out‟. Ligand binding events binding promiscuously to a wide variety shift this conformational equilibrium of compounds with different geometrical towards 'breathe-out' state. Computational and chemical features. The most common solvent mapping has identified a new clinical implications of such promiscuous druggable site whose opening-closing ligand binding and PXR activation is the mechanism directly correlates with shift occurrence of adverse drug-drug in conformational population. Structure- interactions and also the onset of early based virtual screening has identified new stage metabolism of drugs. Therefore, lead compounds, which lock this understanding the molecular basis of druggable pocket in its open-state. MD promiscuity in ligand binding to PXR is simulations further revealed that the crucial for the development of effective presence of lead compound in allosteric PXR antagonists. In this study, we have site disrupts the native PXR-ligand performed molecular dynamic (MD) interaction and thereby destabilize the simulations and analysis of protein PXR-ligand complex formation. structure networks of different ligand

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Comprehending structure, function and interactions across host and pathogen proteomes using sequence- profiles: Implications in target identification for Mycobacterium tuberculosis H37Rv Gayatri Ramakrishnan1,3, Nagasuma R. Chandra2 and Narayanaswamy Srinivasan3

1Indian Institute of Science Mathematics Initiative, 2Department of Biochemistry, 3Molecular Biophysics Unit Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

An imperative step towards perceiving interactions across host and pathogen the biology of an organism is to were also derived with the help of known comprehend its functional repertoire. In transient protein-protein complexes. the context of a pathogen, protein Crucial factors such as functional structure and function recognition importance of a protein, its role in becomes a prerequisite to understand the pathogenesis and its essentiality in growth intricateness in host-pathogen interactions. and survival of the pathogen play key role The availability of the genome sequence in its prioritization as a potential drug of Mycobacterium tuberculosis H37Rv target. Established on the grounds of drug has played a major role in its functional repositioning, we report a target characterization, but the emergence of identification methodology based on multidrug and extensively drug-resistant exploration of the evolutionary strains necessitates further advances in relationship between targets of known understanding pathogenesis and its FDA-approved drugs and M. tuberculosis underlying complexity. Curtailing the proteins, wherein the FDA-approved incompleteness in previously annotated drugs are initially subjected to a filter to proteomes, we report an enriched eliminate the drugs known to act on structural and functional characterization human proteins. Such a withdrawal from with the use of sensitive profile-based the analysis minimizes the chances of remote homology and fold recognition obtaining an anti-target as well as algorithms which comprehend ~95% of enriches the credibility of potential drug the M. tuberculosis proteome. The results targets identified in M. tuberculosis. In obtained served as guiding tools for retrospect, a total of 134 FDA-approved prediction and analysis of transient drugs were identified which could be interactions across host and pathogen at repositioned for 56 potential targets. domain level. 39 domain family Protein-ligand docking studies were also interactions across host and pathogen performed to ensure reliability and could be identified which encompass 83 comprehensiveness of the targets M. tuberculosis proteins and ~1000 identified. human proteins. Additionally, transient

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Development of Selective Protein Phosphatase-1 Inhibitor Ishita Chakraborty, Jayanta Chatterjee

Peptide Chemical Biology Lab, Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

Reversible phosphorylation of proteins is overlapping substrate specificity. PP1 and one of the most important regulatory PP2A account for more than 90% of all mechanisms controlling protein activity. serine/threonine dephosphorylations in Protein kinases phosphorylate protein eukaryotic cells. However, there are no whereas protein phosphatases chemical tools to specifically block the dephosphorylate. Protein phosphatases activity of PP1 in cells. Thus, we aim to PP1 and PP2A belong to PPP develop a cell permeable selective phosphatases, a family of serine/threonine inhibitor of PP1 on the basis of a phosphatases. They are closely related in regulatory protein called Inhibitor-2 that sequence and structure and display specifically inhibits the activity of PP1 in a cell cycle dependent manner.

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Gaussian Network Model study of Protein Kinases: Relationship between sequences, structural fluctuations and functions

Raju Kalaivani and Narayanaswamy Srinivasan Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 E-mail: [email protected]

Protein Kinases (PKs) are a large and protein-protein interactions. 2. diverse family of enzymes, that Fluctuations of PKs as a property of their phosphorylate ~30% of all cellular relatedness were studied. Closely related proteins and are implicated in cancer PKs within the same subfamily showed upon mutations. Despite a conserved higher conservation in dynamics than structural scaffold in PKs, how do kinase distantly related PKs. Dynamics of Src specific regulatory mechanisms function? and EGFR, homologous PKs having Although structural conformations contrasting mechanisms of regulation, correspond to functional states, what were studied. Using only the GNM mechanistic aspects of the conformations perceived fluctuations, we could identify enable the activity/inactivity of the kinase? regions of regulation and protein-protein How do structural motifs achieve specific interaction unique to each of them. 3. modules of function? To this end, we GNM fluctuations were assessed for investigated the structures of PKs using information about the functional sites Gaussian Network Model (GNM) based within a PK. We characterized the Normal Mode Analysis (NMA). 1. We fluctuations of different functional motifs analyzed the GNM perceived fluctuations within the PK. Integrating this of PK structures of varying catalytic information with evolutionary competence. The fluctuations in the active conservation scores, we were able to and inactive states were not identical. predict/recall the functional sites with Systematic differences were found in the specificity and sensitivity > 0.93. In fluctuations: the inactive state fluctuates summary, (a) GNM perceived more than the active state. This was true fluctuations co-vary with sequence, of different structures of the same kinase, structure, function information and individual kinase pairs and a population fluctuation differences provide an insight of kinases. Consequently, we predicted about kinase-specific functional attributes; that the conformational energy of a PK (b) Fluctuations are sensitive to would be unfavourable in the inactive conformational changes (active<- state than in the active state, and proved >inactive). αC helix, αG helix and the same using free energy calculations. activation loop contribute to the Interestingly, the higher fluctuations in fluctuation difference during the switch; the inactive state were contributed (c) Since fluctuation patterns are specifically by αC helix, αG helix and characteristic and function dependent, activation loop regions, which are they can reliably predict/recall implicated in the conformational switch functionally specialized regions in a PK from active to inactive forms and in structure.

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Molecular basis of feedback regulation of pppGpp synthesis

Kirtimaan Syal1, Himanshu Joshi2, Vikas Jain2

1Molecular Biophysics Unit,Indian Institute of Science Bangalore 560 012 2Department of Biological Sciences, IISER, Bhopal

Under stress conditions, bacteria give linked these molecules with Rel CTD stringent response via signaling cascades protein (that contain only the TGS and which help in their survival. (p)ppGpp ACT domains). Our data clearly showed synthesis by RelMSM has been recognized that pppGpp carrying the azido group as the key process in stringent response. interacts and binds to Rel CTD. The RelMSM can synthesize as well as selective binding of pppGpp with Rel hydrolyze the (p)ppGpp. The C-terminal CTD has been further evaluated by half of RelMSM has been shown to contain carrying out the ITC titration. Binding two domains viz TGS and ACT, which site of pppGpp in RelMSM is identified by are involved in the regulation of its mass spectrometry. The azido labeled activity. We have earlier demonstrated molecule has been cross-linked to Rel that an intramolecular crosstalk is CTD and the complex is subjected to responsible to achieve this regulation and trypsin digestion followed by mass involvement of (p)ppGpp is indicated. So spectrometry. Mutational analysis has we suggested that the auto-regulation of been done in search of critical residues synthesis by (p)ppGpp could be achieved and further synthesis assays are carried by binding of the latter to the C-terminal out to measure the activity. We found that domain (CTD) of enzyme. The azido (p)ppGpp auto-regulates its own synthesis derivatives of (p)ppGpp have been and involved in change in conformation synthesized for studying their direct of enzyme resulting in switching of interaction with the protein. We cross synthesis to hydrolysis activity.

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Relation between phenotype, in-vivo solubility, and stability for single-site mutants of CcdB Arti Tripathi, Kritika Gupta, Pankaj Jain, Ajai J Pulianmackal, Prasanth Kumar, Raghavan Varadarajan Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

A quantitative understanding of correlation between in-vitro stability and mutational effects on protein structure, in-vivo activity. function and stability is an important goal The soluble fraction can comprise both of of many protein engineering studies. the folded protein which is active and Saturation mutagenesis is a useful tool to soluble aggregates/partially mis-folded understand the relation between genotype protein which is inactive. To study the and phenotype. Using the 101 amino acid, relation between in-vivo activity and homo-dimeric protein CcdB as a model, solubility, in-vitro activity of selected the effect of mutations on protein activity, mutants was studied by monitoring stability and folding was studied. CcdB binding of the soluble fraction of the cell acts as a toxin in E.coli by binding to lysate to gyrase using SPR. In-vitro DNA Gyrase and killing the cells. Of the activity correlated well with the in-vivo ~ 1600 mutants generated using Site- phenotype, even for those mutants for Saturation Mutagenesis tools (Bajaj et al, which the correlation between the in-vivo 2008, Jain & Varadarajan, 2014), 80 activity and solubility was low. single-site mutants of CcdB with varying Effect of ATP-independent chaperones on activities were previously purified and folding was studied for a few selected characterized. The correlation between in- mutants .When the cellular proteostasis vivo activity, solubility and in-vitro machinery was perturbed by either over- stability was examined and the effect of expression or depletion of ATP- mutations on phenotype, protein folding independent chaperones, change in the and stability was rationalized. In a cell, activity of many mutants was observed. the ability of a mutant to fold to the native Most mutants showed an increase in in- state is affected by many parameters that vivo activity and solubility upon mild include the crowded environment of the over-expression of either Trigger factor or cell, folding assistance by various SecB chaperone indicating an increase in chaperones that buffer mutational effects the folded fraction of the protein which is on protein stability, and quality control active. mechanisms which are involved in This study thus helps in understanding degradation and removal of mis-folded mutational effects on protein stability, proteins from a cell. These factors might activity and folding and provides useful be responsible for the less than perfect correlates for protein design.

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Deciphering Novel Functions of (p)ppGpp and c-di-GMP in Mycobacterium smegmatis Physiology Kuldeepkumar Ramnaresh Gupta

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

In this work, we have attempted to find statistically significant. PM data was out various physiological functions that corroborated by determining the are mediated by second messengers minimum inhibitory concentrations (p)ppGpp and c-di-GMP in (MICs). The MICs for knockouts were Mycobacterium smegmatis. To this end, generally higher than those for wild-type. we have made use of wild-type M. We propose that this apparent increase in smegmatis and its isogenic derivatives the MICs may be due to changes in cell Δrel and Δmsdgc1, which lack (p)ppGpp wall structure of knockouts due to lack of and c-di-GMP, respectively. These (p)ppGpp and c-di-GMP in these strains. second messengers have already been We found that knockouts had altered shown to mediate long-term survival colony morphology and reduced sliding under nutrient deprivation in M. motility and were defective in biofilm smegmatis. We have used a recent formation. Analysis of various cell wall technology, called Phenotype Microarray fractions isolated from both planktonic (PM), to search for novel functions and biofilm cultures showed that amounts mediated by aforementioned second of Glycopeptidolipids (GPLs) and Polar messengers. PM analysis of wild-type and lipids were reduced in the knockouts its isogenic derivatives Δrel and Δmsdgc1 compared to that of wild-type. These showed that knockout strains are growing results indicate that second messengers better in antibiotics. Paired t-test showed (p)ppGpp and c-di-GMP may be involved that the growth difference between the in regulating cell wall properties in M. knockouts and the wild-type was smegmatis.

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Conservation of conformational changes upon binding same ligand in lipocalin protein family B. Lakshmi1, 2, G. Archunan2, N. Srinivasan1

1. Molecular Biophysics Unit,Indian Institute of Science, Bangalore 560 012 2. Department of Animal Science, Bharathidasan University, Tiruchirappalli, India

E-mail: [email protected]

Generally, in protein families, the available crystal structures corresponding functionally important residues are to 35 lipocalins bound to 9 different conserved resulting in the conservation of ligands. The nature and extent of overall function of the homologous conservation of structural changes in proteins. When a protein binds to a small lipocalins upon binding to the same molecule, often the protein undergoes ligand have been analyzed. Interestingly structural changes. The nature of the extent of structural change is not same structural changes can be subtle, among homologous lipocalins upon rearrangement of atoms or even large binding to the same ligand. Detailed scale rigid body movements. In this paper analysis reveals that both the local we address the question of extent of conformational change and the conservation of conformational changes conformational change in the ligand in homologous proteins upon binding to interacting regions are not well conserved the same ligand. The current study has in homologous lipocalins. Our results been conducted by considering the family suggest that extreme caution has to be of lipocalins. The family of lipocalins exercised in modeling conformational consists of sequentially diverse small changes, upon ligand binding, on the extracellular proteins that have the ability basis of 3-D structures of homologues to bind and transport various small and their complexes. molecules. We have analyzed a dataset of

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Display of HIV1 gp120 fragment immunogens on Qβ virus like particles

Mansi Purwar 1, Pranveer Singh1, Sanchari Bhattacharyya 1, Celia C. La Branche2, David C. Montefiori2, Jon Pokorski3 , M.G. Finn3 and Raghavan Varadarajan1

1.Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 2. Department of Surgery, Duke University, Durham, North Carolina 27705 3. Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA

E-mail: [email protected]

b12, one of the few broadly neutralizing were displayed either by conjugation of antibodies against HIV-1, binds to the the purified protein on the surface of the CD4 binding site (CD4bs) on the gp120 VLPs using click chemistry or directly subunit of HIV-1 Env. We have displaying the protein as a fusion to the previously reported design of an E.coli coat protein of the phage. The particles expressed small fragment of HIV-1 gp120, were purified and used for rabbit b122a, which display about 70% of the immunization studies with and without b12 epitope. b122a bound b12 with high adjuvant and boosted with gp120. Titers affinity as well as sera from rabbits against gp120 were highest for the group primed with b122a showed higher primed with b122a1-293-448 displayed breadth of neutralization in a TZM-bl on the particle, and were similar for sera assay against Tier2 and Tier3 viruses. from groups with b122a fragment However, as these immunogens were conjugated to the particle both in the only partially structured as assessed by presence and absence of adjuvant. CD and protease resistance, we attempted However, the group with adjuvant stabilization of b122a by engineering showed higher titers against the priming additional disulfides. A disulfide mutant immunogen. Though sera did not b122a1-293-448 showed 15 fold higher neutralize Tier 2 viruses, sera from affinity for b12 compared to b122a. This particles displaying b122a1-293-448 and was further used for rabbit immunization b122a with adjuvant showed higher studies. Virus like particles or VLPs neutralization titers for tier 1 viruses as resemble intact virions in terms of size compared to other groups. Competition and presentation of antigens, however, binding assays with b12 also showed that they are non-pathogenic, non-replicative sera from these groups contained and hence are safe for administration. In significantly higher amounts of antibodies this work, we attempted to display directed towards the CD4 binding site various gp120 fragment proteins on the than sera from group primed with empty surface of Qβ virus like particles to make particles and boosted with gp120. chimeric VLPs. The fragment proteins

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New Structural forms of a mycobacterial adenylyl cyclase Rv1625c

Nikhil G Bharambe1, Deivanayaga V Barathy1, Rohini Matto2, Sandhya S Visweswariah2, Kaza Suguna1

1. Molecular Biophysics Unit, 2. Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012

E-mail: [email protected]

Rv1625c is one of the 16 adenylyl was also generated by changing a cyclases encoded in the genome of phenylalanine predicted to occur at the Mycobacterium tuberculosis. Rv1625c functional dimer interface to an arginine. exists predominantly as a monomer with This single mutant exists as a dimer in a small amount of dimer in solution. It solution but crystallized as a monomer. was shown earlier that the monomer was Analysis of the structure showed that a active and the dimeric fraction was saltbridge formed between a glutamate inactive. Both fractions of Rv1625c-Wt residue in the N-terminal segment and the crystallized as head-to-head inactive mutated arginine residue hinders dimer domain swapped dimers as opposed to a formation by pulling the N-terminal head-to-tail dimer seen in other functional region to the dimer interface. Both the adenylyl cyclases. About half the structures reported here show a change in molecule is involved in the extensive the dimerization arm region which is domain swapping. The strain created by a involved in the functional dimer serine residue located on a hinge loop and formation. We conclude that the the crystallization condition might have dimerization arm along with other led to this unusual domain swapping. The structural elements like the N-terminal inactivity of the dimeric form of Rv1625c region and certain loops are vital for could be explained by the absence of the determining the oligomeric nature of the required catalytic site in the swapped enzyme which in turn is required for its dimer. A single mutant of the enzyme activity.

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High-conductance states and A-type K+ channels are potential regulators of the conductance-current balance triggered by HCN-channel expression Poonam Mishra and Rishikesh Narayanan

Cellular Neurophysiology Laboratory, Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Hyperpolarization-activated cyclic by the presence of background synaptic nucleotide-gated (HCN) channels are activity. To do this, we inserted thousands non-specific cation channels. An increase of excitatory and inhibitory synapses in HCN-channel conductance reduces spanning the somatodendritic arbor, with excitability of a neuron by decreasing its their distributions constrained by input resistance, whereas the consequent experimental observations. We measured increase in an inward current depolarizes the efficacy of HCN channels, the membrane, propelling a unique independently and in conjunction with conductance-current balance triggered by other channels, in altering RMP and input HCN-channel expression. We employed resistance when the neuron received morphologically realistic, conductance- randomized synaptic bombardments based models of hippocampal neurons to through variable numbers of excitatory, explore certain aspects of this inhibitory or balanced excitatory- conductance-current balance. First, inhibitory synaptic inputs. We found that motivated by the properties of A-type K+ the average RMP was predominantly current, we asked if this current could regulated by the impinging synaptic drive nullify the effect of HCN channels on and the impact of HCN channels on input neuronal resting membrane potential resistance was severely weakened under (RMP). We found that the high-conductance states. Our results somatodendritic gradient of depolarized suggest that the debate on the role of RMP induced by the introduction of HCN HCN channels in altering excitability channels was significantly reduced by the should encompass physiological and insertion of a physiologically relevant pathophysiological neuronal states under gradient in A-type K+ channels. Next, in vivo conditions and the spatiotemporal motivated by the well-established interactions of HCN channels with other modulation of neuronal excitability by ion channels expressed along the high-conductance states observed under dendritic arbor. in vivo conditions, we asked if the conductance-current balance triggered by HCN-channel expression could be altered

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CLAP - An efficient alignment-free method to classify and establish functional relationships among multi- domain Prachi Mehrotra1,2, Vimlakany G Ami3 and Narayanaswamy Srinivasan2

1Indian Institute of Science Mathematics Initiative 2Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 3Bharathidasan University, Trichirapalli.

E-mail: [email protected]

The voids in the sequence and function method and the quality of clusters is space can be potentially filled using assessed. As there are no gold standards computational techniques that directly for full-length protein sequence link the sequences to function. Although classification, we resorted to Gene several sequence comparison methods Ontology and domain architecture based exist, many fundamental and similarity measures to assess our LMS computational limitations arise, especially based Classification of Proteins (CLAP). in multi-domain proteins. The reason for CLAP is freely available as a web-server the limitation is that the existing methods at http://nslab.mbu.iisc.ernet.in/clap/ employ an alignment-based approach focusing on single domains in isolation. Two test data-sets were chosen, On the contrary, proteins are constantly one with tyrosine phosphatases and the regulated by the accessory and tethered other one with SH3 domain containing domains, which contribute to the overall proteins from Pfam (sequence database function. To this end, we have developed version 27.0). The phosphatases family an alignment-free method to compute helped to ascertain whether our method Local Matching Scores (LMS) using performs equally well as the other frequency distribution of fixed length alignment-based approaches, in the case patterns between two sequences. of single domain proteins. SH3 proteins on the other side occur mostly as multi- Instead of globally aligning the domain in nature. Using CLAP, domain sequences, LMS matches sequence architecturally pure clusters with higher patterns of length 5 amino acids and then functional relevance were obtained. scores them based on evolutionary CLAP is ~ 7 times faster than alignment- substitution frequencies. Thus, it will based methods. Thus our method is accurately relate the sequences even if instrumental in providing a biologically domain shuffling, duplication or circular meaningful clustering of any given set of permutation events have occurred. The proteins, utilizing only the sequence normalized scores are hierarchically information. clustered using minimum variance

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Influence of the AgrC-AgrA complex in the response time of Staphylococcus aureus quorum sensing

Rajasree K, Fasim A, Sandeep SK, Arakere G, Gopal B

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

The Staphylococcus aureus agr quorum sensing system plays a major role in the biophysical parameters of AgrC-AgrA transition from the persistent to the interactions in the context of the virulent phenotype. S. aureus agr type (I- conformational features of the AgrC- IV) strains are characterized by mutations AgrA complex. This analysis revealed in the sensor domain of the histidine distinct sequence and conformational kinase AgrC and differences in the features that determine the affinity, sequence of the secreted auto-inducing specificity and kinetics of the peptides (AIP). Here we demonstrate that phosphotransfer reaction. This step, that interactions between the cytosolic domain governs the response time for of AgrC (AgrCCyto) and the response transcriptional re-engineering triggered regulator domain of AgrA (AgrARR) by an AIP stimulus, is independent of the dictate the spontaneity of the cellular agr type and similar for agonist or response to AIP stimuli. The crystal antagonist stimuli. These experimental structure of AgrCCyto provided a basis for data could serve as a basis to validate a mechanistic model to understand AgrC- simulations of the quorum sensing AgrA interactions. This model enabled an response and for strategies that employ analysis of the biochemical and the agr quorum sensing system to combat biofilm formation in S. aureus infection.

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Structural And Mechanistic Insights Into Splicing Factor Sf3b Complex Rakesh Ramachandran1, a, Agnel Praveen Joseph2, b, Ramachandra M Bhaskara3, a, N. Srinivasan4, a

a. Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 . b. National Centre for Biological Sciences, GKVK, Bellary Road, Bangalore

E-mail: [email protected]

Spliceosome SF3b complex is an was done by taking advantage of the new important component of the U2 snRNP as homologous structures solved for the well as U11/U12 di-snRNP. The human components as well as advances in both SF3b complex consists of seven protein structure prediction techniques as components – p14, SF3b49, SF3b145, well as cryo-EM based modeling SF3b155, SF3b10, SF3b130 and SF3b14b. techniques such as flexible fitting. A cryo-EM density map of SF3b complex We also used the patterns of was obtained at a resolution of 9.7 Å by evolutionary conservation observed at the Golas et. al. in 20031. The protein p14 surfaces of protein structures known to mainly consists of an RNA – recognition interact with other proteins as an motif (RRM) domain and is known to additional restraint in our integrative interact with the branch point adenosine. structure modelling approach to increase X-ray and NMR structures have been the accuracy of the model. The functional solved for this protein in complex with an significance at the protein-protein and N-terminal fragment of SF3b155. This is protein-RNA interaction level for each of the only protein-protein complex the components involved in the SF3b structure known in the SF3b complex at complex was also studied. Hence we are the atomic level. also able to provide a mechanistic The current work aims to get understanding of SF3b Complex from the insights into the structure of SF3b structural context and its implications for complex by using integrative structure further understanding of both splicing as modelling techniques guided by the SF3b well as the diseases such as cancer caused cryo-EM density map. From our by defects in some of the proteins of the modelling efforts we were able to localize complex. the components of the SF3b complex in Reference: the cryo-EM density map and delineate 1. Golas, M.M., Sander, B., Will, its molecular architecture. The structural C.L., Luhrmann, R., and Stark, H. coverage of the complex was increased by (2003). Molecular architecture of fitting either partial or complete structures the multiprotein splicing factor for some of the other components. This SF3b. Science 300, 980-984.

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Active dendrites regulate the spatiotemporal spread of signaling microdomains in a hippocampal model neuron Reshma Basak and Rishikesh Narayanan

Cellular Neurophysiology Laboratory, Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Calcium is a key component responsible calcium, with phosphorylated CaMKII for the conversion of incoming neuronal (pCaMKII) displaying the largest spread signals from the electrical to the as a consequence of the positive feedback biochemical realm. Calcium ions mechanism related to the accomplish this by differentially autophosphorylation of CaMKII. Next, to activating several downstream signaling study the role of active dendrites in the pathways, some of which have been regulating signaling microdomains, we implicated in mediating neuronal took up two subthreshold dendritic plasticity. Given this critical importance VGICs, the A-type potassium channels of cytosolic calcium to several (KA) and T-type calcium channels (CaT). physiological processes, it is essential to We independently varied the membrane constrict the spatiotemporal spread of the conductance of either channel and calcium signal, which is tightly regulated quantified its impact on the microdomain by several extrusion mechanisms in the spread of calcium and pCamkII. Whereas cytoplasm and on the plasma membrane. an increase in KA conductance resulted in Here, we consider the calcium- a reduction in the spatial spread and calmodulin-dependent protein kinase amplitude of the calcium signal and a II (CaMKII) pathway and quantitatively more pronounced reduction in pCaMKII, assess the spread of synaptically-driven increasing the CaT conductance induced microdomains of calcium and its increases in the spreads and the downstream molecules. To simplify the amplitudes. Finally, we also noted that morphological and computational the impact of both channels on the complexity of the analyses, we employed spreads and the amplitudes (especially of a four-cylinder model, and used pCaMKII) were significantly higher when Markovian kinetics to assess the role of synaptic stimulation evoked spikes. Our dendritic voltage-gated ion channels study demonstrates that active dendrites (VGIC) on the spatiotemporal spread of could play a critical role in steering the signaling in the CaMKII pathway. We spatiotemporal spread of signaling found that the molecules downstream of molecules, thereby acting as routing calcium had progressively higher instruments that regulate signaling spatiotemporal spread compared to specificity.

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Structural comparison and normal mode analysis of protease-inhibitor complexes suggest high retention of interface structure and flexibility of inhibitor proteins Sneha Vishwanath and N.Srinivasan

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

During evolution, interface residues of region of both proteases and inhibitors protein-protein complexes have been were observed to be structurally observed to be conserved due to conserved. But the interface region of functional as well as structural constraints. inhibitors were observed to be highly But some cases have been reported in the flexible and the interface region of literature, where interface residues show proteases were observed to be highly high sequence variability at the interface rigid, as measured using normal mode and one such example is protease analysis. inhibitors. Here we analyze a dataset of Thus from our observations, we can protease-inhibitor complexes of known conclude that asymmetry is observed in three dimensional structures. The the evolution of interface residues of interface residues of the proteases in the proteases and its corresponding dataset show high sequence conservation interacting inhibitors. Conservation of with 47% of them being highly conserved. residues involved in main chain – main On contrary, interface residues of chain interactions and the conservation of inhibitors show high sequence divergence the structure of the interfacial region, with only 24% of them being highly irrespective of sequence variability, conserved which is on par with the extent indicate the importance of backbone of conservation of non-interacting solvent conformation for the interaction between exposed residues. Moreover, two residues proteases and inhibitors. interacting with each other in a complex We believe that the higher flexibility of can be conserved to different extent; for the inhibitor interface residues is the example an interface residue which is reason behind accommodation of the conserved throughout evolution can sequence variability. Thus we argue here interact with a residue which is highly that high sequence variability and high variable. The residues forming side chain flexibility allows inhibitors to interact – side chain interactions were observed to with many different proteases but the show high variability while residues conserved backbone limits its reactivity to forming main chain – main chain only few or confining to mainly a specific interactions were observed to be family of proteases. conserved. On comparing the structure of complexes at C-alpha level, the interfacial

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C terminal CGNR Zinc finger protein: A novel activator of transcription in Mycobacterium smegmatis Subho Ghosh

Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012

E-mail: [email protected]

Zinc finger proteins have at least one This has been found to be a zinc and pH compact domain stabilized by one zinc dependent DNA binding protein and also atom. Since the discovery of TFIIIA as interact with RNA polymerase. This the 1st zinc finger domain protein, many protein has a zinc dependent tetrameric zinc finger domain proteins have been form. This protein is expressed in log discovered in all three domains of life and phase. On over expression of this protein they have been implied in various in Mycobacterium smegmatis colony biological functions. C terminal CGNR morphology, sliding motility and biofilm zinc finger domain containing proteins formation are affected. Invitro single are present in the genome of many non- round transcription with this protein has pathogenic bacteria and have not been shown an increase in amount of transcript. studied so far. Msmeg_0118 is such a This work elucidates the function of a protein in Mycobacterium smegmatis. novel zinc finger protein.

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Active spines and dendrites regulate spine neck impedance and perturb their indirect estimate along with calcium-handling mechanisms Sufyan Ashhad and Rishikesh Narayanan

Cellular Neurophysiology Laboratory, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 E-mail: [email protected]

Most excitatory inputs onto neurons of the direct measurement and indirect the central nervous system impinge upon estimate of SNI. We found that the specialized post-synaptic compartments presence of A-type K+ channels led to a called spines. Anatomically, these are tiny significant reduction in the SNI, in a protrusions from the dendritic branches manner that was dependent on the comprising of a narrow neck (<0.1 µm synaptic strength and the consequent local diameter and ~1 µm length) attached to a EPSP amplitude. Furthermore, the bulbous head (~1 µm diameter). These presence of A-type K+ (KA) channels also anatomical features endow these resulted in an overestimate of SNI structures with the ability to amplify obtained through the indirect method, synaptic excitatory post-synaptic owing to the dependence of calcium potentials (EPSPs) within the spine head, transients on KA channels. The presence while causing unidirectional attenuation of HCN channels, on the other hand, had of EPSPs when they travel from the spine minimal effect on the direct measurement head towards the parent dendrite. Spine of SNI, but resulted in an underestimate neck impedance (SNI) is a quantitative of SNI obtained through the indirect measure of this electrical transformation method. Finally, we also found that the across the spine neck, and of the role of indirect estimate of SNI was heavily spines in neuronal information processing. modulated by various calcium handling As a consequence of several experimental mechanisms with the density of constraints resulting from their small size, sarcoplasmic endoplasmic reticulum direct measurement of SNI and its pump being the most significant among regulators have been infeasible. parameters that influenced the estimate of Furthermore, estimates of SNI from SNI. Our results suggest that sub- indirect methods, some of which image threshold VGICs and their plasticity changes in spine calcium levels and infer could act as gain modulators for spine that as a direct readout of spine voltage, signals, thereby critically influencing have inherent assumptions that might not neuronal integration and output while be valid. In this study, we employed contributing to encoding and storage of biophysically constrained, conductance- the incoming information. Our results based, morphologically realistic neuronal also emphasize the need to account for models endowed with detailed calcium the presence and localization of VGICs handling mechanisms to explore the role and calcium-handling mechanisms in of voltage-gated ion channels (VGIC) on interpreting indirect estimates of SNI.

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