Membrane proteins represent about one third of all known vertebrate proteins and over half of the current drug targets. Knowledge of their three-dimensional (3D) structure is worth millions of pounds to the pharmaceutical industry. Yet experimental structure elucidation of membrane proteins is a slow and expensive process. In the absence of experimental data, computational modelling tools can be used to close the gap between the numbers of known protein sequences and structures. However, currently available structure prediction tools were developed with globular soluble proteins in mind and perform poorly on membrane proteins. This thesis describes the development of a modelling approach able to predict accurately the structure of transmembrane domains of proteins. In this thesis we build a template-based modelling framework especially for membrane proteins, which uses membrane protein-specific information to inform the modelling process.Firstly, we develop a tool to accurately determine a given membrane protein structure's orientation within the membrane. We offer an analysis of the preferred substitution patterns within the membrane, as opposed to non-membrane environments, and how these differences influence the structures observed. This information is then used to build a set of tools that produce better sequence alignments of membrane proteins, compared to previously available methods, as well as more accurate predictions of their 3D structures. Each chapter describes one new piece of software or information and uses the tools and knowledge described in previous chapters to build up to a complete accurate model of a transmembrane domain.

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 Å resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

X-ray lasers bring us into a new world in photon science by delivering extraordinarily intense beams of x-rays in very short bursts that can be more than ten billion times brighter than pulses from other x-ray sources. These lasers find applications in sciences ranging from astrophysics to structural biology, and could allow us to obtain images of single macromolecules when these are injected into the x-ray beam. A macromolecule injected into vacuum in a microdroplet will be affected by evaporation and by the dynamics of the carrier liquid before being hit by the x-ray pulse. Simulations of neutral and charged water droplets were performed to predict structural changes and changes of temperature due to evaporation. The results are discussed in the aspect of single molecule imaging. Further studies show ionization caused by the intense x-ray radiation. These simulations reveal the development of secondary electron cascades in water. Other studies show the development of these cascades in KI and CsI where experimental data exist. The results are in agreement with observation, and show the temporal, spatial and energetic evolution of secondary electron cascades in the sample. X-ray diffraction is sensitive to structural changes on the length scale of chemical bonds. Using a short infrared pump pulse to trigger structural changes, and a short x-ray pulse for probing it, these changes can be studied with a temporal resolution similar to the pulse lengths. Time resolved diffraction experiments were performed on a phase transition during resolidification of a non-thermally molten InSb crystal. The experiment reveals the dynamics of crystal regrowth. Computer simulations were performed on the infrared laser-induced melting of bulk ice, giving a comprehension of the dynamics and the wavelength dependence of melting. These studies form a basis for planning experiments with x-ray lasers.

The genus Salmonella consists of pathogenic gram negative organisms which infect intestines of birds, animals and humans. They are the causative agents of salmonellosis which is characterised by diarrhoea, nausea, fever and abdominal cramps. If not treated in time, salmonellosis can also be fatal. Salmonella genus is divided into two species Salmonella bongori and Salmonella enterica. Salmonella enterica is further divided into six subspecies out of which the subspecies enterica has many of the pathogenic serovars of this species. Salmonella typhimurium is a server in the subspecies enterica of Salmonella enterica species. Transmission of salmonellosis takes place through contaminated food and water. When the organism enters a host, it encounters a range of hostile environments such as acidic pH, lack of oxygen as well as immune response of the host. In order to establish infection, the bacterium needs to survive under stressful conditions and propagate itself. Various proteins are induced in cells under unfavourable conditions that protect them in such situations. One such group of proteins belongs to the Universal Stress Protein (USP) family. Universal Stress Proteins are a set of proteins induced in organisms when it is exposed to a variety of environmental insults including heat shock, nutrient starvation, presence of toxic compounds, etc. Although survival in adverse conditions is mediated by induction of this group of proteins, the precise mechanism of cellular protection has not been elucidated yet. The functional role of a protein is directly related to its three-dimensional structure and hence important insights can be gained regarding the role of these proteins by determining their structures. The structures of two Universal Stress Proteins from S. typhimurium; a single domain protein, YnaF and another tandem USP domain protein, YdaA were determined by X-ray crystallography and biochemical analysis was carried out on them. Guided by structure, plausible roles for both the proteins in stress tolerance of S. typhimurium have been proposed. Additionally, work was also carried out on phosphomannose isomerise from S. typhimurium. Phosphomannose isomerase is a housekeeping enzyme which catalyses the interconversion of mannose-6-phosphate and fructose-6-phosphate. Mannose is important for mannosylation of various lipids and proteins which form an important component of bacterial and fungal cell walls. Presence of a functional phosphomannose isomerise enzyme is important as it helps the organism survive adverse conditions by forming a strong cell wall which shields it from harmful environments. Moreover, phosphomannose isomerase was also found to be essential for virulence of Leishmania mexicana and Cryptococcus neoformans. The structure of phosphomannose isomerase from S. typhimurium was determined in our laboratory in the year 2009. However, in the earlier studies, the catalytically important residues had not been identified and mechanism of isomerisation was not established. Structural analysis, site directed mutagenesis and biochemical assays were used to identify key residues in the active site of StPMI. Identification of these residues might help in deciphering the catalytic mechanism which will eventually be useful to develop inhibitors that arrest the growth of Salmonella as well as other microorganisms. The work reported in this thesis describes the efforts made to enhance our understanding of functional aspects of the two Universal Stress Proteins, YnaF and YdaA and phosphomannose isomerase from S. typhimurium. Chapter 1 begins with a brief introduction to the kinds of unfavourable environments encountered by microorganisms and their strategies of adaptation. This is followed by a review of the literature on Universal Stress Proteins, which are induced in many organisms in response to arrest of or perturbations in the growth rate. Structural, biochemical and evolutionary aspects of members of the family have also been discussed. Subsequently, a brief description of the earlier work carried out on another enzyme important in stress tolerance, phosphomannose isomerase, has been documented. A detailed account of mechanisms of isomerisation carried out by aldose ketose isomerases and identification of important strategies for determination of mechanism of phosphomannose isomerase catalysed reaction have then been provided. The chapter ends with a summary of aims and objectives of the present work. Chapter 2 describes the various experimental techniques and computational methods used during the course of this thesis work. Isolation of plasmids, overexpression and purification of protein, site directed mutagenesis, biochemical assays, crystallisation of proteins, X ray diffraction data collection form a part of the experimental aspect and have been described in detail. Brief descriptions of the programs used and principles behind computational methods used for structure determination (including data processing, phasing, model building and refinement), validation and analysis have also been provided. Chapter 3 includes the structural and functional studies carried out on YdaA, a tandem USP domain protein from S. typhimurium. Expression, purification, crystallisation and structure determination of YdaA in its native and ADP bound forms are described in the chapter. Biochemical assays with radiolabelled ATP showed that YdaA was an ATPase. The crystal structure of YdaA complexed with ATP revealed the presence of ADP (hydrolysis product of ATP) only in the C-terminal domain of the protein. Based on structural analysis and presence of ATP binding motif in the C-terminal domain, it could be hypothesized that ATP hydrolysis activity of the protein is confined to the C-terminal domain of the protein. The N-terminal domain of the protein was found to play another interesting role. A zinc binding site could be identified in the N terminal domain based on structural analysis and elemental X-ray absorption studies done at the synchrotron. Site directed mutagenesis and biochemical experiments suggested that zinc binding in the N-terminal domain was not related to ATPase activity of the C-terminal domain. Additionally, an intermediate of lipid A biosynthesis pathway UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetyl glucosamine was found bound to the N-terminal domain of YdaA. Lipid A is the membrane anchor of polysaccharides in the outer membrane of gram negative organisms and the intermediate occurs at the committed step of the pathway. However, no similarities could be identified between YdaA and members of the relevant biosynthetic pathway. Therefore, YdaA is unlikely to play a catalytic role in the same pathway but can function as a carrier molecule. A plausible link between the N- and C-terminal domains of YdaA could be identified by structural analysis. Many catalytically suitable residues from the N-terminal domain were found to be close to the β-phosphate of ADP bound to the C-terminal domain. Hence YdaA was identified to be a zinc binding ATPase which might play some yet unidentified role in lipid A biosynthesis pathway. Chapter 4 describes the attempts made towards understanding the functional role of YnaF, a single domain USP from S. typhimurium. A description of the expression, purification, crystallisation and X ray diffraction techniques used for structure determination of YnaF and its single site mutant have been provided in detail. Gel filtration, dynamic light scattering studies and the crystal structure determination of YnaF showed a tetrameric organisation of four USP protomers stabilised in the centre by chloride ions. Additionally, YnaF crystallised with a bound ATP even though ATP was not included in the crystallisation cocktail. Biochemical assays on YnaF with radiolabelled ATP showed that it was inactive with respect to ATP hydrolysis. When selected mutations that disrupt chloride binding were made, YnaF was converted to an active ATPase. The crystal structure of the mutant complexed with an ATP analogue revealed key differences at the active site in comparison with that of the wild type and allowed identification of residues that might be important for ATP hydrolysis in this group of proteins. Hence YnaF might play the role of a sensor protein in some signal transduction pathway involving chloride ions in bacteria. A structure based analysis and comparison of USPs from the Protein Data Bank with the structures of YnaF and YdaA is summarised at the end of this chapter. Chapter 5 describes the efforts carried out towards determination of mechanism of isomerisation catalysed by phosphomannose isomerise (PMI). Earlier reports suggest that the enzyme catalyses the reversible isomerisation of mannose-6-phosphate and fructose-6-phosphate via formation of a cis-enediol intermediate. The structure of phosphomannose isomerase from S. typhimurium has been reported by our laboratory. The enzyme is a monomer with three domains; a catalytic domain, a carboxy terminal domain and an α-helical domain. Residues from the catalytic domain were found to coordinate a zinc ion. Overexpression, purification, co crystallisation experiments and soaking studies carried out on crystals of PMI and its single site mutants are outlined in this chapter. The structure of a complex of PMI with mannose-6-phosphate at pH 7.0 revealed the presence of a blob of density close to the zinc binding site which was confirmed to be the active site by analysis of conservation of residues in the site. Based on site directed mutagenesis, activity studies and analysis of structure of PMI, zinc was identified to play an important role in maintaining the structural integrity of the active site. Electrostatic surface analysis of the structure of PMI revealed that the zinc ion might also play the role of anchoring phosphate moiety of the substrate in a highly negatively charged active site pocket. Activity assays following site directed mutagenesis studies eliminated the role of Glu264 in catalysis and implicated two lysines, Lys86 and Lys132 as the possible base in the reaction. The plausible role of a highly conserved residue Arg274 was also proposed based on comparison of structures of wild type and mutant PMIs. The future prospects of the work are briefly discussed towards the end of the thesis. Further experiments and analysis required to obtain better understanding of the functions of these proteins have been discussed. The Appendix section describes extensive crystallisation attempts that were carried out on the enzyme sorbitol-6-phosphate-dehydrogenase from S. typhimurium which catalyses the isomerisation reaction between sorbitol-6-phosphate and glucose-6-phosphate using NADPH as the cofactor. Needle shaped crystals were obtained which diffracted to a poor resolution of 7-8 Å at our in house X ray facility. Attempts to improve the quality of the crystals like co crystallisation with substrate and its analogues, soaking in various compounds and seeding are briefly described. The following manuscripts based on work described in this thesis have been published or will be communicated for publication. 1. Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance. Bangera M., Panigrahi R., Sagurthi S.R., Savithri H.S., Murthy M.R.N. Journal of Structural Biology, 2015 Mar; 189 (3): 238-50. 2. Structural and functional insights into phosphomannose isomerise: role of zinc and catalytic residues. Bangera M., Savithri H.S., Murthy M.R.N. Manuscript under preparation

Understanding the molecular structure of DNA was considered as greatest achievement in modern biology. It helped in understanding fundamental cellular processes such as replication of DNA, nature of the genetic code and transcription. It also led to technological advancements such as DNA sequencing, genetic engineering and gene cloning. The DNA molecule is highly polymorphic in nature and its structure is dependent on environment, base composition and sequence context. B-DNA, A-DNA, Z-DNA and curved or kinked DNA are some of the well characterized double helical polymorphs. B-DNA is the most prevalent structure in vivo and it can undergo small local variations and global variations. In this thesis we refer to distinct structural property of any particular DNA sequence as deviation from fibre model B-DNA structural parameters or random sequence DNA. Structural properties of DNA are an outcome of the linear arrangement of the 4 chemically different nucleotide bases and the characteristic features of the two grooves (minor and major) arising due to the asymmetric position of glycosidic bonds of base pairs. DNA structure and properties are expected to vary along its length. Several structural features have been defined for DNA duplex, while DNA stability, bendability and intrinsic curvature are well studied and found to be biologically relevant. These three sequence dependent properties differ in their nature and information content and can be studied both at local and global levels, depending on the length of DNA fragment being examined. Majority of the work in this thesis focuses on the analysis of these three DNA structural features in promoter regions of different eukaryotic systems and their relationship with gene expression. The thesis work is divided in to five sections briefly described below. The sections discuss prevalence of the three structural features, DNA stability, bendability and intrinsic curvature in the promoter regions of six eukaryotic systems namely S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human. The relationship between DNA structural features of promoter regions of S. cerevisiae with gene expression variability is discussed, followed by application of the structure-based promoter prediction algorithm ‘PromPredict’ in annotating promoter regions of six different eukaryotes. Finally, an analysis of structural features of the flanking sequences of transcription factor binding sites (TFBSs) of six transcription factors and their relationship with the DNA binding affinity is discussed. Each of the projects described below will appear as a separate chapters in the thesis. An overview of the eukaryotic transcription machinery, promoter elements and different DNA structural properties are discussed in the introduction of the thesis (chapter 1). The structural properties of DNA in the promoter regions of eukaryotic genes (chapter 2)Earlier studies in the lab reported that, apart from sequence motifs, promoter re- gions have distinct structural properties, such as lower stability, lesser bendability and more curvature compared to other genomic regions. But those studies were on small datasets and few model systems. Advancement in high-throughput tech- niques has made availability of transcription start site information for many model systems. This work was initiated with the aim of investigating the structural fea- tures in different eukaryotic systems belonging to different domains of life. The quantitative analysis of three different structural features of promoter regions of six different model systems S. cerevisiae, C. elegans, D. melanogaster, zebrafish, mouse and human has been carried out. Further, the composition of different k-mers (k=3, 4 and 6) A-tracts and G-quadruplexes has been studied. The analysis allowed us to understand the similarities and differences in struc- tural features of promoter sequences in different model systems. The core promoter sequences of S. cerevisiae, C. elegans, D. melanogaster, zebra fish, mouse and hu- man have been observed to be less stable and have lower preference for nucleosome formation. S. cerevisiae, C. elegans and D. melanogaster promoter sequences have been shown to be less bendable whereas zebrafish, mouse and human promoter se- quences are flexible in terms of bendability towards major groove as predicted fDNase 1 sensitivity model. S. cerevisiae, C. elegans, D. melanogaster core promoter regions have AT rich oligomers, whereas mouse and human core promoter regions have GC rich oligomers and G-quadruplex motifs. DNA structural features of TATA-containing andTATA-less promoters (chapter 3)Eukaryotic genes can be broadly classified as TATA-containing and TATA-less based on the presence of TATA-box in their promoter sequences. Experiments on both classes of genes have reported that, they have differences in regulation of gene ex- pression and cellular functions. In this chapter, the differences in compositional and structural features of TATA-containing and TATA-less promoters in the above mentioned model systems are discussed. The results suggested that DNA structural features of TATA-containing and TATA-less promoters are distinctly different in all eukaryotes. The TATA-containing promoters are less stable, more flexible and more curved compared to TATA-less promoters in lower eukaryotes. In mouse and hu- man genes, DNA duplex stability and G-quadruplex motifs are very distinguishing features in the two classes of promoters. DNA structural properties of eukaryotic promoter regions and gene expression variability (chapter 4) Gene expression is regulated by various external (environment and evolution) and internal (genetic) factors. Presence of sequence motifs, such as TFBSs and TATA- box, as well as DNA methylation has been implicated in the regulation of expression of some genes in vertebrates, but a large number of genes lack these sequences. Ear- lier analyses (described in previous sections) in S. cerevisiae, have shown that their promoter sequences have special structural properties, such as low stability, less bendability and more curvature compared to other genomic regions. These strutural features may play a role in transcription initiation and regulation of gene expression. This project was carried out to understand 1. What is the relationship between DNA structural features and gene expres- sion? 2. What is the relationship between gene expression and bidirectionality of a pro- moter region? For this purpose, the information of seven different gene expression variability measures, stochastic noise, responsiveness, stress response, trans variability, mu- tational variance, interstrain variation and expression divergence have been com- pared with structural features in the promoter regions. It is observed that a few of the variability measures of gene expression are linked to DNA structural prop- erties, along with nucleosome occupancy, TATA-box presence and bidirectionality of promoter regions. Interestingly, gene responsiveness is shown to be most, inti- mately correlated with DNA structural features and promoter architecture. The study highlights the importance of sequence dependent structural features in gene regulation. Promoter prediction in eukaryotes using DNA duplex stability (chapter 5) Structural property-based algorithms can discriminate promoter sequences from non-promoter sequences and are far better than sequence motif-based predictors. Compared to other structural features, low stability is found to be the most preva- lent feature in promoter regions. “PromPredict” (in-house algorithm) uses the din- ucleotide free energy values obtained from differential melting stability of DNA du- plexes as a predictor of promoters and has been successfully used earlier to annotate promoter sequences in prokaryotes and rice. Comprehensive analysis of the perfor- mance of PromPredict in S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human as well as TATA-containing and TATA-less promoter regions of S. cere visiae with TSS data and 48 eukaryotic systems with translation start site (TLS) data revealed that differential stability is a good criterion for promoter prediction. DNA structure in flanking sequences of consensus motifs modulate transcription factor binding (chapter 6) Sequence specific DNA-protein interactions are essential for specific expression pat- terns during the development. There are several factors contribute to DNA-binding specificities of transcription factors (TFs). They include structure and flexibility of TFs, cofactors, chromatin environment and DNA sequence. Along with actual tran- scription factor binding sites (TFBSs), their sequence context (flanking sequences) is also shown to play a major role in gene regulation. Most of the studies have ad- dressed the sequence context at global level but very little is understood about the role of sequences flanking TFBSs in binding of transcription factors. This project was initiated with the aim of understanding the effect of flanking sequences of TFBSs in transcription factor binding affinity. In vitro DNA binding information of six different transcription factors (with three types of DNA bind- ing domains, Zinc finger (GATA4), home domain (AbdA, AbdB and Ubx) and bZIP (fos-jun and Nfil3)) was provided by Aseem Ansari’s lab. The compositional and structural features (minor groove width, propeller twist, wedge and free energy) are compared with the DNA binding profiles of 12mers (or 8mers) of six different transcription factors. It has been observed that some of the DNA structural proper- ties of flanking sequences are strongly correlated with binding affinity. For GATA4 sequences, binding affinity is negatively correlated to GC content or minor groove width at their 5′ -flanking region, showing the significance of narrow minor groove at 5′ -region. On the other hand, the binding affinity of bZIP proteins is negatively correlated to wedge angles, whereas in case of homeodomain proteins, it is posi- tively correlated to propeller twist and GC content. Thus, this study highlights the differential preference for flanking sequences outside the core binding motifs of six different TFs, which interact with DNA through α-helix. ‘The relationship between transcription pre-initiation complexes and gene ex- pression variability in S. cerevisiae’ is briefly described in the appendix section of the thesis. General conclusion Overall, the results presented in this thesis indicate that DNA sequence based structural features are unique to promoter regions and play an important role in gene regulation. Local structural features of flanking sequences of transcription factor binding sites are also instrumental in determining the DNA binding affinity of transcription factors.

The sequence of a polypeptide chain determines its structure which in turns determines its function. A protein is stabilized by multiple forces; hydrophobic interaction, electrostatic interactions and hydrogen bond formation between residues. While the above forces are non-covalent in nature the protein structure is also stabilized by disulfide bonds. Structural features such as naturally occurring cavities in proteins also affect its stability. Studying factors which affect a protein’s structural stability helps us understand complex sequence-structure-function relationships, the knowledge of which can be applied in areas such as protein engineering. The work presented in this thesis deals with various and diverse aspects of protein structure. Chapter 1 gives an overall introduction on the topics studied in this thesis. Chapter 2 focuses on a unique, non-regular, structural feature of proteins, viz. protein cavities. Cavities directly affect the packing density of the protein. It has been shown that large to small cavity creating mutations destabilize the protein with the extent of destabilization being proportional to the size of cavity created. On the other hand, small to large cavity filling mutations have been shown to increase protein stability. Tools which analyze protein cavities are thus important in studies pertaining to protein structure and stability. The chapter presents two methods which detect and calculate cavity volumes in proteins. The first method, DEPTH 2.0, focuses on accurate detection and volume calculation of cavities. The second method, ROBUSTCAVITIES, focuses on detection of biologically relevant cavities in proteins. We then study another aspect of protein structure – the disulfide bond. Disulfide bonds confer stability to the protein by decreasing the entropy of the unfolded state. Previous studies which attempted to engineer disulfides in proteins have shown mixed results. Previously, disulfide bonds in individual secondary structures were characterized. Analysis of disulfides in α-helices and antiparallel β-strands yielded important common features of such bonds. In Chapter 3 we present a review of these studies. We then use MODIP; a tool that identifies amino acid pairs which when mutated to cysteines will most likely form a disulfide bond, to analyze disulfide bonds in parallel β-strands. A direct way to analyze sequence-structure relationships is via mutating individual residues, evaluating the effect on stability and activity of the protein and inferring its effect on protein structure. Saturation mutagenesis libraries, where all possible mutations are made at every position in the protein contain a huge amount of information pertaining to the effect of mutations on structure. Making such libraries and screening them has been an extremely resource intensive process. We combine a fast inverse PCR based method to rapidly generate saturation mutagenesis libraries with the power of deep sequencing to derive phenotypes of individual mutants without any large scale screening. In Chapter 4 we present an Illumina data analysis pipeline which analyzes sequencing data from a saturation mutagenesis library, and derives individual mutant phenotypes with high confidence. In Chapter 5 we apply the insights derived from structure-function studies and apply it to the problem of protein engineering, specifically immunogen design. The Human Immunodeficiency Virus adopts various strategies to evade the host immune system. Being able to display the conserved epitopes which elicit a broadly neutralizing response is the first step towards an effective vaccine. Grafting such an epitope onto a foreign scaffold will mitigate some of the key HIV defenses. We develop a computational protocol which grafts the broadly neutralizing antibody b12 epitope on scaffolds selected from the PDB. This chapter also describes the only experimental work presented in this thesis viz. cloning, expressing and screening the epitope-scaffolds using Yeast Surface Display. Our epitope-scaffolds show modest but specific binding. In a bid to improve binding, we make random mutant libraries of the epitope-scaffolds and screen them for better binders using FACS. This work is on-going and we aim to purify our epitope-scaffolds, characterize them biophysically and eventually test their efficacy as immunogens.

Local field potentials (LFPs), extracellular potentials that reflect localized electrical activity, have long been used as a window to understand the behavioural dependence and mechanistic aspects of brain physiology. A principal premise that has driven the interpretation of LFPs is that they largely reflect the synaptic drive that impinges on neurons located in the vicinity of the recording microelectrode. An implicit, yet critical, assumption that led to the emergence of this premise is that dendrites, the structures onto which most synaptic inputs project, are purely passive compartments. However, there is a growing body of evidence demonstrating that dendrites express a plethora of active conductance, like voltage-gated ion channels, several of which are active in the subthreshold regime. These subthreshold-activated ion channels and their intra-neuronal localization profiles play widely acknowledged regulatory roles in the physiology, plasticity and pathophysiology of synapses and neurons. Despite this, the implications for the existence of these subthreshold conductances on constituent oscillatory patterns in LFPs and on the phase of neuronal spiking with reference to oscillating LFPs have surprisingly remained unexplored. The aim of this thesis is to examine if there exists a role of subthreshold conductances in regulating LFPs and the phase of spikes with reference to these LFPs. To address this, we chose to study LFPs and spikes from the CA1 region of the rat hippocampus, with hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels forming the specific subthreshold conductance of focus. The reasons behind these choices were manifold. First, CA1 pyramidal neurons are arranged in a laminar open-field configuration, making the interpretation of the source-sink formation in this region relatively tractable. Second, the dendrites of these neurons are endowed with a multitude of subthreshold conductances whose expression profiles, physiology and plasticity have been characterized in great detail. Third, this brain region has been implicated in coding for episodic and spatial memories. The phase of the spikes of the CA1 pyramidal neurons, with reference to the LFP, is believed to serve as a code that can be used to decode the location of the animal. Given that the most dominant LFP pattern seen in the CA1 region during such active exploration (and possibly encoding of spatial memories) consists of oscillations in the 4–10 Hz theta frequency band, we decided to focus our study on theta-frequency LFPs. Finally, consistent with the choice of the specific band of LFP frequencies, we focused on HCN channels because of their predominantly dendritic expression and their ability to bestow resonance and impedance phase lead, both in the theta-frequency range, on CA1 pyramidal neurons. In exploring the role of HCN channels on LFPs, we used a multi-compartmental morphologically realistic CA1 pyramidal neuron model and introduced an HCN channel conductance gradient that was constrained with several experimental measurements. This neuron was driven by dendritic excitatory synapses and perisomatic inhibitory synapses, both theta-modulated with a phase difference of +60º between their arrivals timings. We increased the excitatory synaptic conductance with distance from the soma to account for the fact that irrespective of the location of the synapse in the dendrites, the unitary excitatory post-synaptic potential remains the same at the soma. Employing these model configurations, we generated 25 different synaptic distributions on the same neuronal morphology to account for the input variability and for each of these models, we recorded transmembrane currents from all the compartments, for 8–10 cycles of the theta-modulated inputs. To model LFPs using the forward modelling scheme of line source approximation, we designed a cylindrical neuropil of 40 µm height and 100 µm radius and inserted a virtual linear electrode with 7 contact points distributed on the probe at the canter of the neuropil such that we could compute the LFP at all the strata of the CA1 region. Accounting for the volume of the neuropil and the density of neurons in this region, we took 440 instances of the morphology, rotated them at uniformly distributed angles, and distributed the somata of these model neurons within the neuropil. Each of these 440 neurons received transmembrane currents from one of the 25 models picked uniformly. With a passive model, where we did not introduce HCN channels, we expectedly observed the formation of a source-sink structure that expressed as a progressive phase shift spanning different strata, owing to the perisomatic inhibitory currents coupled with the dendritic excitatory currents. On introducing a somatodendritic gradient of HCN conductance with identical input conditions, we observed a phase lead in the LFPs across all the layers, with the magnitude of the lead increasing with distance from the soma in a manner that was correlated with the increase in HCN conductance. Next, we computed spike phases, for each of the 25 neuron models, with reference to the stratum pyramidale (SP) LFP for model configurations with and without HCN channels. We found that the spikes showed a phase lag in the presence of a gradient of HCN channels when compared to the spike phases measured from the passive neuron models. Finally, we computed the coherence of spikes across all the 25 passive or 25 active (with HCN channels) neuron models and found that the presence of HCN channels greatly enhanced spike phase coherence across neurons. Together, these results demonstrate that the presence of HCN channels introduces a lead in the theta-frequency LFP phase, a lag in the associated spike phase, and a significant enhancement of spike phase coherence. Exploring the robustness of these findings to the model configuration, we first found these conclusions to be robust to increases in neuropil size (400-µm diameter neuropil with 1797 neurons, and 1-mm diameter neuropil with 11297 neurons). Next, we introduced heterogeneities in the population of neurons (in terms of morphology as well as passive and active properties) that formed the neuropil, and found our conclusions to be invariant to such degeneracy in the underlying neuronal population. It has been observed that under certain pathological conditions like epilepsy, an entire population of CA1 neurons can undergo intrinsic plasticity, such as global (i.e., across the entire neuronal topograph) downregulation of HCN channels. To assess the impact of such up/downregulation on LFPs, we respectively increased/decreased HCN channel conductance globally in our model neurons, and found the magnitude of the lead in the LFP phase to progressively increase with HCN-channel conductance. Similarly, the magnitude of the spike-phase lag and the spike phase coherence also progressively increased as functions of HCN-channel conductance. Although such population-level global intrinsic plasticity is observed under pathological conditions, a more physiological scenario would be when a single neuron, in the process of encoding new inputs (such as encoding spatial or episodic memories), undergoes intrinsic plasticity. To assess this, we increased or decreased HCN-channel conductance specifically in a single neuron placed closest to the electrode, while leaving the HCN expression in other neurons of the neuropil at the baseline level. Expectedly, we did not find significant changes in LFP amplitude or phase, but we did find a significant lag in the spike phase preference of the neuron that underwent an upregulation of HCN conductance. Another physiological scenario is when the rat experiences a reward or exhibits anxiety-like behaviour, which can lead to changes in hormonal or neuromodulator concentrations. These changes, functioning through the activation of G-protein coupled receptors and the consequent elevation of cytosolic cyclic adenosine monophosphate (cAMP) concentrations, could shift the half-maximal activation voltage ( V1/2 ) of HCN channels to a more depolarized potential. Would such a shift in V1/2 impact LFPs and spike phases in a manner similar to that observed with increasing the conductance of HCN channels? Assessing this within our modeling framework, we found that shifting the V1/2 by +5 mV resulted in an increased lead in the LFP phase, an increased lag in the spike phase and an enhanced spike phase coherence compared to the case with a hyperpolarized V1/2 . What are the biophysical mechanisms that underlie these robust changes observed in LFPs and spike phases observed as a consequence of these several ways of increasing the current through HCN channels? We reasoned that our observations could be explained by one of the two distinct changes conferred on CA1 pyramidal neuron physiology by the presence of HCN channels. First, in the presence of HCN channels, the voltage response of CA1 pyramidal neurons shows a phase lead with reference to a sinusoidal current input (inductive phase lead) in the theta frequency range. Second, HCN channels regulate the excitability of these cells by decreasing the input resistance and impedance amplitude. To delineate the differential role of the inductive changes vs. changes in excitability, we replaced HCN channels by a faster variant (HCNFast) such that neuronal excitability remained the same while abolishing the inductive phase lead in the theta band. On doing so, we found that the lead in the LFP phase and the lag in the spike phase brought about by HCN channels was partially reversed when HCN conductance values were low. However the reversal was not substantial when HCN conductance values were high, suggesting that the inductive phase component dominates at lower HCN channel conductances, whereas the excitability component plays a critical role at higher HCN conductances. Akin to intrinsic plasticity mentioned above, under certain pathological conditions, an entire population of neurons can undergo scaling of their excitatory or inhibitory synapses. In assessing the implications for such synaptic plasticity, we first found that our conclusions on the roles of HCN channels in introducing a lead in the LFP phase, a lag in the spike phase and an enhancement of spike phase coherence were invariant to the specific values of synaptic conductances, or the phase difference between excitatory and inhibitory theta-modulated inputs. While these observations further established the robustness of the changes brought about by HCN channels to LFPs and associated spikes, we next asked whether synaptic plasticity, mediated by changes in subthreshold synaptic conductances, could itself bring about changes in the LFP and spike phase. Expectedly, we found that scaling up of excitatory synapses introduced a mild lag in the LFP phase and a lead in the spike phase, whereas scaling up of inhibitory synapses introduced a lead in the LFP phase and a lag in the spike phase. Finally, we observed a critical role of the arrival phase of inhibition with reference to excitation in altering both, the stratum pyramidale LFP and associated spike phases, with the magnitude of change in both the LFP and the spike phase roughly following the magnitude of the shift in the excitatory-inhibitory phase difference. However, in contrast to changes observed with HCN-channel plasticity, there was no significant change in spike phase coherence with any of the three forms of synaptic changes explored. Together, our results identify definite roles for HCN channels and synaptic receptors in phase-coding schemas and in the formation and dynamic reconfiguration of neuronal cell assemblies and present a clear case for the incorporation of subthreshold-activated ion channels, their gradients, and their plasticity into the computation of LFPs. Given the rich expression of several subthreshold ion channels — including HCN, A-type potassium and T-type calcium — in neuronal dendrites, future work could focus on the impact of subthreshold channels on LFPs recorded in different brain regions under different behavioral states. This thesis is organized into seven chapters. Chapter 1 provides the motivations for the study, introduces the aim of the study and poses the specific questions asked in our endeavor to understand the role of subthreshold conductances in regulating LFPs and spike phases. Chapter 2 discusses the physiological foundations and relevant literature that places the questions posed in the first chapter in the context of the aim of the thesis, with an emphasis on the literature on HCN channels. In chapter 3, we introduce the computational and theoretical foundations required to model neurons and to compute LFPs. In chapter 4, we look at the consequences of the presence of a non-uniform density of somatodendritic HCN channels on LFPs and spike phase and test the robustness of the effects observed. In chapter 5, we present our assessment of the impact of intrinsic plasticity/modulation of HCN channels on LFPs and spike phases, also exploring the biophysical mechanisms underlying such an impact. In chapter 6, we test if the observed effects still hold under synaptic plasticity, and assess the regulation of LFPs and spike phases by synaptic changes. In chapter 7, we summarize and conclude the results presented in the preceding chapters and provide some potential directions for future studies.

Cellular response to environmental changes involves a wide repertoire of complex signalling systems often resulting in up and down regulation of various genes. These mechanisms are generally conserved in a variety of organisms. These pathways are also constantly rewired in various organisms, which aid them in maintaining homeostasis and result in species-specific adaptation mechanisms. Protein kinases are central to these mechanisms and orchestrate a multitude of these pathways. This thesis aims to understand the selective forces behind evolution of signalling pathways. More specifically, this thesis focuses on structural and domain architecture differences of protein kinases. Protein kinases are one of the most populated families of proteins in many organisms and it constitutes about 2-3% of proteomes of most of the eukaryotic organisms. These kinases have evolved over ~400 million years and regulate nearly all major signalling pathways. Classification of kinases enables convenient association of kinases to the function and signalling pathway in which they participate. The current scheme of classification is based on the amino acid sequence of the catalytic region, which consists of about 200-300 residues. This scheme proposes division into 7 groups which show gross level similarities in function such as the TK group, which constitutes all tyrosine kinases, or AGC group which constitutes kinases regulated by second messengers. These groups are further divided into ~280 subfamilies providing us insights into function and regulation at a much finer level. This enables ascertaining information about signalling pathways, protein-protein interactions or substrates the kinase phosphorylates. Chapter 1 provides an elaborate introduction to the various types of protein kinases and their roles in signalling processes. This chapter discusses how protein kinases work in a concerted manner with several other players of a signalling pathway to generate a regulated response to external stimuli. Furthermore, it highlights both the evolutionary aspects and dynamical nature of such pathways. The subsequent part of this chapter deals with protein kinases, their evolution, regulation and structural features crucial to catalysis. Protein kinases are regulated in many ways ¬regulation is achieved from within the catalytic domain and also by means of additional domains tethered to the catalytic domain. The regulatory switch is triggered by various cellular and molecular events such as phosphorylation of specific residues, changes in spatial-temporal localization and altered redox states to name a few. The effects of regulatory domains on the overall function have also been discussed. The chapter concludes by highlighting structural analysis carried out to understand the regulatory aspect of kinases and uses this information in rational drug discovery. Chapters 2 and 3 report identification and analysis of a repertoire of protein kinases encoded in the genomes of two of the organisms which are frequently used in comparative genomics. Chapter 2 focuses on the distribution of kinases in Takifugu rubripes, a teleost fish which is a widely used model system for studying human genes. Use of remote homology detection methods identified 519 kinases in fugu. Although the group-wise distribution of kinases shows high similarity to that of human kinases, subfamily distribution shows considerable differences in 22 subfamilies. They are either under or over-represented in fugu. Most noticeable difference is seen for the DYRK subfamily, which is eight times higher in fugu than human. Detailed analysis of the DYRKs revealed interesting insights into and explained partially their high representation in fugu. Only about ten of these kinases classified into these subfamilies showed high sequence similarity and conserved localization signals to the human kinases and kinases commonly found in other eukaryotes such as C.elegans, S.cereviseae and D.melanogaster. Disparity at the level of genome may be attributed to the observation of unique domain architectures characteristic of this genome. A comparison of domain architectures of kinases documented in Pfam with that of the kinases in Takifugu also revealed two kinases with unique domain architectures in fugu; they are associated with Galectin domain and YkyA domains. Despite inconsistencies in the distribution, human and Takifugu kinases subfamilies remarkable similarity is observed in the MAP kinase pathway, which is ubiquitously found across eukaryotic organisms. Nearly 83% of the proteins in this pathway show more than 30% sequence identity between the two organisms thus, validating the use of Takifugu as a model system to study human signalling pathways. While addressing the possibilities of similar expansions of kinases in other teleosts, it was noticed that the Danio rerio genome (zebrafish) had a massively expanded kinome with ~1200 kinases. Chapter 3 explores the possible reasons for the expansion of kinome with kinases specific to Zebrafish. For e.g., the number of kinases from one subfamily (CAMK) is roughly similar to the total number of protein kinases encoded in the human genome. Further, the PIM kinase subfamily is the sole subfamily, which is massively over-represented (~30 times) in this genome. A detailed analysis of PIM kinases of zebrafish revealed that the sequences are divergent from the canonical PIM kinases. Despite this difference, the specific residues, which dictate the functional properties specific to PIM kinases, are highly conserved. These PIM kinases are usually constitutively active, features of which are conserved in PIM kinases of zebrafish as well. Unlike canonical PIM kinases in other eukaryotes, the post-transcriptional regulation of these PIM kinases might be different due to the absence of regulatory regions in the 3'UTR regions of the PIM gene. However, conservation of a S261 phosphorylation site highlights regulation by phosphorylation, which compensates for the constitutively active nature. A massive expansion of the substrate pool of PIM kinases in this genome seems to correlate well with the expansion. Since PIM kinases regulate large number of growth related pathways, we believe that, this might be associated with high regenerative capacity of organs observed in this fish, which makes it an ideal model to study most cancers. While the earlier two chapters primarily focused on the kinase catalytic domain and organism specific changes; the next two chapters address the contribution of domains tethered to the catalytic domain in the overall function of the kinase. Deviations from canonical kinase domain architectures indicate expansion in the functional repertoire of kinases. Chapter 4 is a study on human kinases from the latest revised version of the human genome sequence data. The initial part of the chapter focuses on the differences in the kinase repertoire upon revision of the human genomic data. Seven sequences gleaned from the earlier genomic data are absent and 16 new sequences are added to the kinome dataset according to the latest human genome sequence data. In addition, differences in transcripts for 23 kinases have led to differences in overall length and sub-family classification of these kinases. The identification of the kinome data from this latest version was a mandatory step prior to the study of outlier kinases due to variations in gene transcripts. The domain architectures of the human kinases have been compared with known subfamily-specific domain architectures, in order to identify outliers. Based on the type of domain architecture these outliers have been classified as “rogue” or “hybrid” kinases. Hybrid architecture represent kinases showing high sequence similarity within the kinase domain to a known sub¬family of kinases with the acquisition of non-kinase domains typically found in one of the other subfamilies of kinases. On the other hand rogue architectures belong to kinases with domain architectures not observed in any of the kinase sub-families. A total of 23 outliers have been identified in the human genome-13 hybrids and 10 rogues. The presence of such "hybrid" and "rogue" kinases makes classification of kinases into subfamilies a daunting task and hence necessitates a new method for classification using the full-length sequences. The use of one such alignment-free method, ClaP (Appendix), using full length sequences has been validated for classification of kinases. A similarity metric obtained from full protein sequence comparison further improved the existing methods of classification for 29 kinases, which utilize only the catalytic domain of kinases. Classification based on catalytic domain is incomplete without the knowledge of associated domains, which also have an important role in function. This necessitates a new approach in classification of kinases for function annotation-an integrated one that uses information from the full-length sequence of each kinase. Chapter 5 extends the learning from chapter 4 and aids in identification of 74 "Hybrid" and 18 "Rogue" kinases in other model eukaryotes, Mus musculus, C.elegans, S. Cerevisiae, D. melanogaster and Takifugu rubripes which show significant variations in the overall functions. These sequences due to their hybrid nature might facilitate cross-talk between signalling pathways. Thus annotating the function of each of these 92 outliers has highlighted the use of domain recombination in wiring new pathways and re-wiring existing pathways. Also, these sequences because of their hybrid nature cannot be classified under any of the existing sub-families. Therefore, it has been proposed in this chapter that they be classified as separate sub-family containing sequences with hybrid properties. To validate this, the ClaP method has been extended where the pair-wise distances between two sequences (using full length sequence) has been used to generate phylogenetic trees which have then been subjected to hierarchical clustering to generate sub-family based clusters. Further, a Shannon entropy based score has been used to identify clusters that contain sequences from diverse sub-families grouped together. Upon analysis of these clusters, it was observed that the hybrid and rogue kinases specifically cluster within four clusters with high entropy (constitute large number of sub-families) validating their status as emergent sub-families. In addition, more hybrids and rogues have been identified in these clusters, which have long regions without any domain assignments. Such sequences may contain domain families deviant from those that are currently known and information on their function can be obtained from further genomic studies in future. Lastly, the prevalence of such hybrid and rogue kinases in the genome of a protozoan parasite, P. falciparum has been studied in detail. The role of hybrids and rogues in host-pathogen interaction has been explored. Chapter 6 presents an in-depth analysis of the possible role of charge-neutralization around phosphosites in protein kinases and its substrates. This analysis was a follow up of a study and in collaboration with Dr.Warwicker's group in Manchester, which identified positively charged residues around phosphosites in kinase substrates. The current study not only aims to address the importance of charge neutralization around phosphosites, but also uses this feature for prediction of phosphosites in known structures of kinase substrates. A dataset of phosphosites mapped on a 3-D structure has been used to calculate peak electrostatic potentials around phosphosites based on the solution of a non-linear Poisson-Boltzmann equation. A comparison of peak potentials around phosphosites with that of non-phosphosites reveals a higher positive peak potential at ~10.0 Å radius around the phosphosite. This variation is significantly higher around tyrosine residues in comparison to Ser/Thr residues phosphosites. Further, this distinction in peak potential around the phosphosite is attributed to only certain families like protein kinases and pyruvate kinases. The concept of charge neutralization will therefore show greater success in prediction of phosphosites in such families in comparison to other families with phosphosites. The functional importance of such charge neutralizations has been studied in great detail in the protein kinase domain family due to prior knowledge that certain phosphorylation events contribute to conformational change, which may be correlated to the changes in peak potentials upon phosphorylation. Phosphorylation at certain sites within the kinase catalytic domain often mediates onset of certain signalling events including regulating activity levels of kinases, mediating protein-protein interactions and altering their localization. Therefore, by means of studying conservation patterns of such phosphosites or neutralizing residues, the variations in signalling pathways in homologues with differences in conservation patterns, have been highlighted. Among domain families which do not show clear differences in peak potentials between phosphosites and non-phosphosites, it was noted, in a few cases, that negatively charged ligands bind to the protein in the vicinity of phosphosites, in the un-phosphorylated forms of the protein. Structural studies on a few cases in ligand bound forms indicate a competitive mechanism between phosphorylation and ligand binding which helps in switching between different functional forms. Therefore, the role of phosphorylation as a regulatory mechanism for modulating ligand binding in such domain families has been highlighted. Chapter 7 of the thesis reports a study on disease causing mutations in kinases. So far 180 kinases have been reported to contain disease causing mutations. This chapter particularly focuses on understanding the deleterious effects of non-synonymous missense mutations in kinases. Mutations at certain sites are enriched as seen by the concentration of disease phenotypes upon mutations at these sites in comparison to others. Interactions involving Arginines in sub-domains VIB, VIII, IX and XI are perturbed which affect catalysis. Structural explanation of 10 such mutations, which occur in important sub-domains and not directly implicated in catalysis has been provided. Apart from analyzing the various evolutionary and structural aspects of protein kinases in this thesis an attempt has been made to provide a deeper structural understanding of Msh (MutS Homologues) proteins involved in eukaryotic chromosomal segregation. Chapter 8 deals with Msh4-Msh5 complex, which are eukaryotic homologues of the MutS family of proteins in bacteria. MutS proteins form homodimeric complexes in bacteria that aid in mismatch repair process. There are six MutS homologues in eukaryotes, which form hetero-dimers. Two of the homologues are Msh4 and Msh5, which form hetero-dimeric complexes which is a pre-requisite for its function. They are involved in chromosomal segregation during meiosis-I and aid in resolving Holliday junction DNA. Till date no structure of this complex is available and the exact mode of binding is unclear. In addition, Msh4 and Msh5 display asymmetry in DNA and ATP binding sites. These insights are derived from the severity in phenotypes upon mutation of various residues in these proteins. This work is in collaboration with Dr. Nishant from IISER, Trivandrum. The questions addressed in chapter 8 of the thesis are: What are the structural features that contribute to the asymmetry in function between Msh4 and Msh5 in DNA and ATP binding? Can a structural explanation be provided for each of the 27 mutations causing severe phenotypes (cross-over defects/viability) to predict their role in function of the Msh4-Msh5 complex? Can a prediction be provided for the mode of binding of the Holliday junction DNA? Can residues occurring at interface regions of Msh4 and Msh5 be identified on the basis of the structure which affects the complexation of Msh4 and Msh5? These questions are addressed by homology modelling of the Msh4-Msh5 complex using the Msh2-Msh6 complex as template. Structural explanations have been provided for 23 out of 27 mutations with severe phenotypes. Certain residues in Msh5 are shown to form tighter network of interactions than their counterparts in Msh4 and therefore likely to have a more prominent role in DNA and ATP binding which corroborate with the observed asymmetry in mutant functions. A volume based calculation has been used to suggest a possible mode of binding of the Holliday junction within the cavity of the complex. Finally, the model has been used to predict interface residues that play a crucial role in complexation and function. Experiments are being carried out in Dr. Nishant's laboratory to mutate these residues to validate the model. Chapter 9 summarizes the entire thesis work and also clearly states the chief conclusions from various chapters. Apart from studies embodied in the thesis, the author has been involved in one other study, which is provided as appendix.

Structuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66. Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds. Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property.

The three-dimensional structures of biological macromolecules and molecular assemblies are becoming increasingly important with the changing methodologies of drug discovery. The structures aid in understanding of protein function at the molecular level: be it a macromolecular assembly, a cytosolic enzyme or an intermembrane receptor molecule. X-ray crystallography is the most powerful technique to obtain the three-dimensional structures of such molecules at or near atomic resolution. With such a wide-spread importance, crystallography is an integral part of structural biology and also of the current drug discovery programs. The present thesis mainly deals with application of the crystallographic techniques for understanding the structure and function of adenylosuccinate lyase (ASL) from bacterial pathogens Salmonella typhimurium and Mycobacterium tuberculosis as well as its non-pathogenic counterpart Mycobacterium smegmatis. Studies were also carried out to understand the structure-function relationship of the protease in the plant virus Sesbania Mosaic Virus (SeMV). The thesis has been divided into six chapters. The first chapter contains an introduction to nucleotide synthesis and ASL superfamily of enzymes known as the aspartase/fumarase superfamily based on the published literature. Chapter 2 provides the details of the techniques used for the investigations presented in this thesis. Chapters 3-5 deal with the structural and functional studies carried out on ASL from the three bacterial organisms. Chapter 6 deals with the simulation studies carried out on SeMV protease. Mechanism and importance of nucleotide synthesis is introduced in Chapter 1, with special emphasis on purine de novo and salvage pathways. ASL is introduced as an important enzyme for purine synthesis. Its superfamily, the aspartase/fumarase superfamily of enzymes is described in detail with respect to its structure, function and pathophysiology. Objectives of the present study are outlined towards the end of the chapter. The experimental and computational techniques utilized during the course of my research are described in Chapter 2. These techniques include gene cloning, protein expression and purification, kinetic and biophysical characterization of proteins, crystallization, X-ray diffraction, data collection and processing, structure solution, refinement, model building, validation and structural analysis, phylogenetic studies, molecular docking and molecular dynamic simulation studies. Adenylosuccinate lyase is an important enzyme participating in purine biosynthesis. With the emergence of drug resistant variants of various pathogens, ASL has been recognized as a drug target against microbial infections. Chapter 3 deals with the structural and functional characterization of ASL from Salmonella typhimurium. Two constructs of the StASL gene were cloned and expressed leading to the purification of truncated (residues 1-366) and full-length (residues 1-456) polypeptides. Crystallization of the two polypeptides resulted in three independent structures. The full-length structure was very similar to the E. coli ASL structure consistent with 95% amino acid sequence identity between the two polypeptides. However, the truncated structures showed large distortions, especially of the active site residues, accounting for the catalytic inactivity of the truncated polypeptide in spite of retaining all residues considered important for function. The full-length ASL was catalytically active. A unique feature observed in StASL, not reported in other ASLs, was its allosteric regulation by the substrate. Kinetic studies also revealed hysteretic behavior of the enzyme. The electron density map of the full-length structure showed two novel densities on the molecular 2-fold axis into each of which a molecule of cadavarine could be fitted. Docking studies revealed a ligand-binding site at the inter-subunit interface between the two observed densities which might represent a potential allosteric site. Combining the structural and kinetic results, a possible morpheein model of allosteric regulation of StASL was hypothesized. Chapter 4 deals with the crystallographic and kinetic investigations on ASL from Mycobacterium smegmatis and Mycobacterium tuberculosis. MsASL and MtbASL were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at 2.16 Å resolution. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. Most of the active site residues were found to be conserved with the exception of Ser 148 and Gly 319 of MsASL. Ser 148 is structurally equivalent to a threonine in most other ASLs. Gly 319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active when compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria, their high GC containing genomes as well as with their dependence on other salvage pathways for the supply of purine nucleotides. Chapter 5 deals with the identification of the catalytic residues important for ASL catalysis in view of the earlier conflicting reports on the identity of these residues. pH-dependent kinetic studies were performed on full-length StASL. The theory behind these studies is also described in this chapter. Two residues with pKa values of 6.6 and 7.7 were identified as essential for the enzymatic activity. These results were interpreted along with structural comparison of MsASL and other superfamily enzymes with ordered C3 loops. They suggest that His 149 and either Lys 285 or Ser 279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. The final Chapter 6 of the thesis deals with the structural and dynamic studies carried out on Sesbania mosaic virus (SeMV) protease. The chapter begins with a general introduction to viruses, followed by a brief summary of SeMV. The goal of this study is to understand the interactions between the protease and VPg at a structural level using the information available from biochemical studies. Crystallographic studies initiated for the mutant H275APro and Y315APro were unsuccessful due to the insolubility of the proteins. Co-crystallization or soaking experiments of wild type protease with cognate peptides were unsuccessful due to the inability of the enzyme to bind to its substrates in the absence of VPg. Higher resolution structure of wild type protease did not yield any new insights when compared to the earlier reported structure determined at a lower resolution. In the absence of structural insights, molecular dynamic simulations were carried out on wild type protease structure and in silico generated mutants using GROMACS package. The studies showed the importance of flipping of residue Phe 301 and opening-closing of the loop region corresponding to residues 301-308 for the catalytic mechanism. The thesis concludes with Future perspectives of the various studies carried out on ASL and SeMV protease. The atomic coordinates determined from the work presented in this thesis have been deposited in the PDB and the assigned PDB codes are reported in the respective chapters. Publications cited in the thesis are listed in the Bibliography section.