No description available.

No description available.

X-ray crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules at atomic resolution. Crystallography can reliably provide the answer to many structure related questions, from global folds to atomic details of bonding. Crystallographic techniques find wide applications in understanding macromolecular assembly, enzyme mechanism, mode of activation of enzymes, substrate-specificity, ligand-binding properties, domain movement etc. The knowledge of accurate molecular structures is also a prerequisite for rational drug design and for structure based functional studies to aid the development of effective therapeutic agents. The current thesis can be broadly divided into two major parts. The first four chapters deal with assembly studies that have been carried out on Sesbania mosaic virus and the next two chapters describe the structure and function of a stationary phase survival protein, SurE from Salmonella typhimurium. In both studies X-ray crystallographic techniques have been used extensively for the structural studies. Viruses are obligate parasites with a proteinaceous capsid enclosing the genetic material. For genetic economy, several copies of capsid proteins self assemble to form complex virus capsids. Due to their intricate symmetric structures, viruses are considered as minute marvels of molecular architecture and study of virus structures serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes at near-atomic resolution, and is thus an extraordinarily powerful tool for understanding the function of these biological systems. Protein-protein interactions, protein-nucleic acid interactions, metal-ion mediated interactions, interactions between capsid proteins and auxillary or scaffolding proteins and particle maturation or post processing of capsid protein subunits are various elements that play a role in capsid assembly. Many structural and sequential motifs have been proposed as important conformational switches of capsid assembly. A functional analysis of these motifs by way of mutations in the capsid protein and structural studies of these mutants can provide further insight into capsid assembly pathways. Interaction between capsid protein subunits can determine the size and robustness of the capsid. Analysis of protein-protein interactions can help in understanding the principles of self-assembly. Arresting capsid assembly by disrupting intersubunit interactions and trapping the assembly intermediates will be helpful to delineate the changes that happen in capsid protein during the course of assembly and understand assembly pathways. Sesbania mosaic virus (SeMV) is a plant virus with a positive sense single-stranded RNA genome and belongs to the Sobemovirus genus. The protein and nucleic acids of SeMV can be separated and reassembled in vitro. Also, expression of the coat protein (CP) gene of SeMV in E. coli leads to the formation of virus like particles (VLPs). Therefore, SeMV is an excellent model system to study the assembly pathways that lead to the formation of complex virus shells. Earlier structural and functional studies on the native virus and the recombinant capsid protein and its various mutants have revealed the following: SeMV is a T=3 virus with chemically identical A-, B- and C-subunits occupying quasi equivalent positions in the icosahedral asymmetric unit of the virus particle. The A-type subunits form pentamers at the five-fold, and the B- and C- type subunits form hexamers at the icosahedral three-fold axes. The amino terminus of the polypeptide is ordered from residue 72 in the A- and B- subunits whereas it is ordered from residue 44 in the C-subunit. The disordered segment in all the subunits has an arginine rich motif (N-ARM). The segment ordered only in C-subunits has a -annulus structure that promotes intersubunit interactions at the quasi six-fold and a -segment (A). The virus is stabilized by protein-protein, protein–RNA and Ca2+ mediated protein-protein interactions. Virus like particles (VLPs) formed by the expression of full length CP encapsidate 23 S E. coli rRNA and CP mRNA. Expression of a deletion mutant lacking the N-terminal 65 residues (rCP∆N65) which results in the removal of the N-ARM, the -annulus and the A leads to the formation of stable T=1 particles. The -annulus, which was earlier believed to be an important molecular switch controlling the assembly of T=3 VLPs was found to be dispensable. The N-ARM, though important for RNA encapsidation, was not essential for capsid assembly . Depletion of Ca2+ ions led to slight swelling of virus particles and significantly reduced stability. Extensive studies on the VLPs suggested that the assembly is most likely initiated by the dimers of the capsid protein. Following a brief account of the historical highlights in the field of structural virology, a review of current literature on the available crystal structures of viruses and various assembly studies on viruses that have been carried out with emphasis on role of nucleic acid mediated interactions, protein-protein interactions and role of specific residues and ion-mediated interactions in assembly are presented in Chapter I of the thesis. A separate section in this chapter deals with the disassembly experiments that have led to the formation of smaller oligomers of spherical viruses. This chapter also gives an account of the earlier work that has been carried out on SeMV, which is the model system of study for the present thesis. Chapter II describes in detail the structural studies on the β-annulus deletion mutant of SeMV. A unique feature of several T = 3 icosahedral viruses is the presence of a structure called the β-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T = 3 capsid assembly. In order to examine the importance of the β-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48–59 involved in the formation of the β-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Δ48–59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of size close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6Å resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the β-annulus. Therefore, the β-annulus is not essential for T = 3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the β-annulus could be closely similar. Chapter III begins with a detailed description of the interfacial residue mutations that have been carried out in SeMV with the aim of disrupting assembly and trapping an assembly intermediate. These mutations were performed in rCP as well as rCP∆N65 gene. Among these, a single point mutation of a Trp 170 to a charged residue (either Glu or Lys) arrested virus assembly and resulted in stable dimers of the capsid protein. The chapter also gives an account of the biophysical characterization of these mutants. rCP∆N65 dimer mutants showed a characteristic 230 nm peak in CD spectral studies which may be due to the interactions of a stretch of aromatic residues in the capsid protein. The isolated dimers were more susceptible to trypsin cleavage compared to the assembled capsids due to the exposed basic amino terminus. Thermal melting studies showed that the isolated dimer mutants were much less stable when compared to the assembled capsids, probably due to the loss of intersubunit interactions and Ca2+ mediated interactions. The structure of one of the isolated dimer mutant- rCP∆N65W170K was solved to a resolution of 2.65Å. Chapter IV describes the crystal structure analysis of the rCP∆N65W170K mutant dimer and compares its structure with the dimers of native virus, T=3 and T=1 VLPs. A number of structural changes occur especially in the loop and interfacial regions during the course of assembly. The dimer in solution was “more relaxed” than the dimer that initiates assembly. Ca2+ ion is not bound and consequently the C-terminal residues are disordered. The FG loop, which interacts with RNA, was found to be flexible and adopts a different conformation in the unassembled dimer. The present thesis also deals with the structural and functional studies of a phosphatase, SurE, the stationary phase survival protein from Salmonella typhimurium. Chapter V provides a general introduction on Salmonella, which is a mesophilic food borne pathogen, its general features, classification and stress responses. This chapter also gives an account of stationary phase in bacteria and stress responses. A brief description about phosphatases and their classification is also presented in this chapter. Following this, a review of the current literature on the structural, biochemical and functional role of stress related proteins and phylogenetic and enzymatic studies of various homologues of SurE are described in detail. Chapter VI deals with the detailed crystal structure analysis of SurE, the first stationary phase survival protein from a mesophilic organism. SurE, of Salmonella typhimurium forms part of a stress survival operon regulated by the stationary phase RNA polymerase alternative sigma factor. SurE is known to improve bacterial viability during stress conditions. It functions as a phosphatase specific to nucleoside monophosphates. Here we report the X-ray crystal structure of SurE from Salmonella typhimurium (St SurE). The protein crystallized in two forms- orthorhombic F222 and monoclinic C2. The two structures were determined to resolutions of 1.7Å and 2.7Å, respectively. The protein exists as a domain swapped dimer. The residue Asp 230 is involved in several interactions that are probably crucial for domain swapping. A divalent metal ion is found at the active site of the enzyme, which is consistent with the divalent metal-ion dependent activity of the enzyme. Interactions of the conserved DD motif present at the N-terminus with the phosphate and the Mg2+ present in the active site suggest that these residues play an important role in enzyme activity. The divalent metal ion specificity and the kinetic constants of SurE were determined using the generic phosphatase substrate- para- Nitro Phenyl Phosphate. The enzyme was inactive in the absence of divalent cations and was most active in the presence of Mg2+. Thermal denaturation studies showed that St SurE is much less stable compared to its homologues and an attempt was made to understand the molecular basis of the lower thermal stability based on solvation free energy. The thesis concludes with a brief summary of the entire work that have been presented and future prospects. The various crystallographic, biochemical and biophysical techniques employed in the investigations are described under the section experimental techniques in Appendix I and the NCS matrices used in the structure solution of the β-annulus deletion mutant are listed in Appendix II.

Assembly of virus capsid protein (CP) into icosahedrally symmetric particles is an intriguing and elegant process. In most cases of virus assembly, a large number of identical protein subunits self-assemble to generate a shell that protects the viral genome. Studies on virus assembly have resulted in a new scientific technique that uses these proteinaceous shells as nano-particles for a variety of biological applications. The current thesis deals with understanding the factors that govern the assembly of the Sesbania mosaic virus (SeMV) and a pleomorphic virus, Tobacco streak virus (TSV). CP of SeMV, a T=3 plant virus, consists of a disordered N-terminal R-domain and an ordered S-domain. The importance of the R-domain in the assembly was probed by replacement with polypeptides such as the B-domain of Staphylococcus aureus protein A and polypeptides P10 and P8 of SeMV. These chimera assembled into T=3 or larger virus like particles (VLPs). Addition of divalent cations resulted in the formation of heterogeneous nucleoprotein complexes that disappeared upon treatment with EDTA/RNAse. One of the chimeras (N∆65-B) purified in a dimeric form by affinity chromatography assembled into T=1 VLPs during crystallization. The three dimensional structure of these VLPs showed that they were devoid of divalent ions and the B-domain was disordered. These studies demonstrate the importance of N-terminal residues, metal ions in virus assembly and robustness of the assembly process. Also, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. Tobacco streak virus (TSV) is a polymorphic virus and a major plant pathogen. TSV capsids encapsidate the tri-partite ss-RNA genome of the virus in three spheroidal particles of diameters 27, 30 and 33 nm, respectively. CPs of ilarviruses are also involved in genome activation. The labile nature of ilarviruses has posed difficulties in their structure determination. This thesis describes the first crystal structure of truncated TSV-CP. The core of TSV CP conforms to the canonical β-barrel jelly roll tertiary structure found in other viral coat proteins. Dimers of CP with swapped C-terminal arms (C-arm) were observed in the two crystal structures determined. The C-arm was found to be flexible and responsible for the polymorphic and pleomorphic nature of TSV capsids. Mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was also found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses.

No description available.

1. Viruses are obligate parasites that hijack the host cell machinery to synthesize their own gene products and for their propagation. In order to establish a successful viral infection, viruses have evolved different strategies to evade host check points. Further more, their success also relies in employing varied strategies to express maximum number of functional proteins from their small constrained genome. Polyprotein processing is a widely used strategy of expression by many plant viruses. With limited information available on this aspect for sobemoviruses, the present study was undertaken. 2. The present thesis deals with the mechanism of Sesbania mosaic virus (SeMV) polyprotein processing and functional characterization of the polyprotein domains. SeMV infects Sesbania grandiflora that belongs to the Fabaceae family. It is a positive sense ssRNA virus with a genome length of 4149 nucleotides. The genome encodes four potential overlapping open reading frames (ORFs). ORF1 codes for an 18 kDa protein that is proposed to be involved in the movement of the virus. ORF 3 codes for the coat protein (CP) that encapsidates the viral genomic RNA to form the viral particles. The central ORF codes for polyprotein that has a serine protease domain at its Nterminus that cleaves the polyprotein at specific E-T/S sites to release the functional domains. So far only in SeMV, the E. coli expressed polyprotein, Protease-VPg-RdRp was shown to undergo processing at E325-T326, E402-T403 and E498-S499 releasing protease, VPg, P10 and RdRp domains respectively. 3. Based on the arrangement of the central ORF, the genome organization of SeMV was earlier shown to be like that of SCPMV type. However, recent sequencing data from the laboratory showed that the organization of SeMV gRNA was like that of CfMV type. This would imply that in SeMV the central two ORFs will be translated to give two polyproteins, 2a (Protease-VPg-C-terminal domain) and 2ab (Protease-VPg-RdRp) the C-terminus of 2a and N-terminus of RdRp being different from what was reported previously. Therefore, in the light of the new genome organization for SeMV, the mechanism of processing of polyprotein 2a and 2ab needs to be revisited. 4. SeMV protease domain was shown to require natively unfolded VPg at its Cterminus for its activity. Aromatic stacking interactions between protease and VPg (via W43 residue) were shown to confer the activity to the protease. However, the residues in the protease domain involved in these interactions have not been identified. 5. The objectives of the present studies are • To elucidate the mechanism of processing of polyproteins 2a and 2ab in E. coli and in planta. • To identify residues in the protease domain involved in mediating aromatic stacking interactions with VPg. • To functionally characterize the C-terminal domain of polyprotein 2a. 6. Polyprotein 2a when expressed in E. coli, from the new cDNA clone, got cleaved at the earlier identified sites E325-T326, E402-T403 and E498-S499 to release protease, VPg, P10 and P8 respectively. The specificities of the cleavage sites were established by mutational analysis. 7. Additionally, a novel cleavage was identified within the protease domain at position E132-S133. The polyprotein 2a that was mutated for this site (ΔN70 2a-E132A) showed no release of P8 protein though the polyprotein was intact for E498-S499 site. Unlike other cleavage site mutants, ΔN70 2a-E132A mutant also revealed large accumulation of intact polyprotein, again implying that the mutation not only abolished the proteolytic cleavage at that site but hampered the processing at other sites. The results confirmed that the cleavage at N-terminus of the protease/polyprotein is crucial for an efficient processing in particular for the cleavage between P10-P8. 8. Interestingly, though the sites in polyprotein 2ab are exactly the same as identified in polyprotein 2a, the former got cleaved between Protease-VPg but not between VPg-RdRp. This cleavage site appeared to be rather masked in polyprotein 2ab. Also, the cleavage at E132-S133 site appeared to be rather slow. These results indicate to a differential cleavage pattern, governed probably by the conformation of 2ab. In other words, the local context of the cleavage site and just not the sequence per se could be playing a key role in 2ab polyprotein processing. 9. Products, corresponding to all cleavages identified in E. coli (E132-S133, E325-T326, E402-T403 and E498-S499) were also detected in infected Sesbania leaves. Products corresponding to the sizes of ΔN132 Protease and ΔN132 Protease-VPg were detected suggesting that the removal of the membrane anchoring domain from the protease does occur in planta. Also, detection of band corresponding P8, confirmed that the cleavage between P10-P8 indeed occured in planta too. 10. The trans cleavage experiments suggested that not all of the four cleavages in polyprotein 2a occur in trans (intermolecular). Cleavages at E132-S133 and E498-S499 do not occur in trans impling that cleavages at these sites could only occur in cis (intramolecular) by auto-proteolysis of the polyprotein. 11. The Thr at P1’ did not make a site trans cleavable. Interestingly, SeMV protease was found to cleave even an E-S site in trans but only when present at positions 324-325 and 402-403, suggesting that trans cleavage in SeMV is governed by the context rather than the Thr at P1’position of the cleavage site. The E498-S499 site was found to be highly stringent not only for the mode of its cleavage (cis cleavage) but also for its sequence (E-S only). A Thr substitution for Ser at this site, made it non cleavable in cis. 12. The results reveal that the polyprotein processing in SeMV is regulated by a number of strategies, viz. a) availability of the cleavage site depending on the conformation of the flanking domains (E132-S133 and E402-T403 cleavages in 2ab). b) Mode of recognition (cis or trans). c) Context/position of the cleavage site. 13. Based on the sequences of all four cleavage sites identified, a consensus has been drawn for SeMV serine protease cleavage site, i.e., N/Q-E-T/S-X (where X is an aliphatic residue) at P2-P1-P1’-P2’ position respectively. 14. With a view to understand the structural reasons for such high specificity, the residues in the S1 and S2 binding pocket, that recognize the substrate P1 and P2 residues respectively, were identified based on the structural comparison of SeMV protease with other Glu/Gln specific proteases. Mutational analysis of these residues clearly demonstrated that H298, T279 and N308 of the S1-binding pocket that would bind the substrate glutamate are crucial for the protease activity. R309 that forms the S2 binding pocket is also crucial for protease activity. 15. Also, the P2 (Asn/Gln) residue recognized by R309 plays an important role in determining the substrate specificity. A positively charged residue Lys was not tolerated at this position. SeMV protease was also shown to efficiently cleave the peptide bond C-terminus to an uncharged Gln in vivo suggesting that it is a Glu/Gln specific protease. 16. An interesting feature of the SeMV protease domain is the presence of a disulphide bond that holds the S1-binding pocket. However, unlike for the cellular counterparts like trypsin, the disulphide was found to be not essential for either the SeMV protease activity or structural stability. 17. Protease and VPg domains were proposed to be involved in aromatic interactions that conferred activity to the protease. The structure of protease revealed a stack of aromatic residues (W271, F269. Y315 and Y319) exposed to the solvent. Mutational analysis was performed to identify their role in mediating the interactions and hence the activity of protease. H275, though not a part of exposed aromatic stack in the protease, was chosen for mutational analysis as it lies close to the W271 in sequence and is conserved in the protease domain across all the known sobemoviruses. The in vivo and trans cleavage assays suggested that residues W271 and H275 but not Y315 or Y319 are crucial for protease activity. 18. The Far-UV CD spectrum of protease-VPg is characterized by a positive peak at 230 nm, signifying the aromatic interactions. Far-UV CD spectral analysis of the aromatic mutants showed that W271 and H275, but not F269 and Y319 are the major contributors of the 230 nm positive peak, confirming the direct involvement of these residues in the stacking interactions with W43 of VPg. Thermal stability studies, fluorescence spectroscopy and 1D-NMR spectroscopy studies also confirmed the histidine aromatic interactions between W271, H275 of protease with W43 of VPg. 19. The loss in aromatic interactions in the mutants caused Protease-VPg to aggregate, suggesting that the aromatic interactions between protease and VPg not only conferred activity to the protease but also the active oligomeric status. 20. In silico analysis of the C-terminal domain showed that it has no significant similarities with any known functional proteins. The region corresponding to P8 was amplified and cloned in pRSET C vector, over-expressed and purified. 21. The purified His-tagged P8 showed mass abnormality on the SDS-PAGE. However, the mass spectrometric analysis of the purified protein showed that it had a molecular mass of 9.766 kDa as is expected for a His-tagged P8. P8 is highly basic, which could possibly explain its anomalous behaviour on the SDS-PAGE. The purified recombinant P8 protein was found to be natively unfolded. In vitro binding studies revealed that P8 had nucleic acid binding property. The protein was also found to be phosphorylated both in vitro and in vivo conditions. 22. Interestingly, P18, (a precursor of P8) but not P8, was found to possess an inherent ATP hydrolyzing property. Optimum conditions for the ATPase assay were found to be Tris HCl pH 8.0, 37 ºC, 5 mM MgCl2. The activity was linear upto 20 mins. P18 could utilize all NTPs and dNTPs. Studies revealed that ATPase activity resided in the P10 domain of P18, though P8 region could enhance the activity. Conclusively, the results demonstrate that the C-terminal domains of polyprotein 2a have ATPase and nucleic acid binding activity and could therefore have possible roles in movement and replication.

This thesis describes the tertiary structures and dynamic studies of two protein systems. The first is human γC -crystallin protein, which is present in the nucleus of the human eye lens and the other is the plant viral protein VPg (an intrinsically disordered protein) in its free as well as its protease bound forms. The structural studies described here have been carried out using high-resolution solution NMR spectroscopic methods. Project I: Determination of solution structure and dynamics of Human γC-crystallin (HGC) using NMR spectroscopy The crystallins are the most abundant proteins in the eye lens of vertebrates. These proteins are packed in short-range spatial order to provide the transparency and appropriate refractive index gradient that are required for vision. The crystallins belong to two gene families, which are categorized as the alpha and beta/gamma crystallins respectively. The classification on the basis of molecular size and structure results in the proteins being referred to as alpha, beta and gamma crystallins. Again, each of the crystallins has two or more subtypes. The stoichiometry of the subtypes of α, β and γ crystallins varies with the age of the organism, but the order of abundance remains as β > α > γ irrespective of age. The most abundant crystallins in the nucleus (central region) of eye lens are the γ -crystallins. In the human lens, only three members of the γ− crystallin family are mainly expressed i.e. γS- (HGS), γC - (HGC) and γD - (HGD). HGS is expressed postnatally and thus is present mainly in the cortical region of the lens unlike HGC and HGD crystallins, which are present in the nucleus. It is known that aging and some cataract-associated genetic mutations alter the structure of these proteins. Other point mutations result in minimum structural perturbation but with drastically lowered solubility. Mutation in the human γC -crystallin leads to congenital cataract such as Coppock-like cataract, while structural information is available for HGD & HGS but no structure is available for HGC. However, recently a model structure has been reported for HGC based on a mouse orthologous. Based on this model structure, it was argued that HGC is an insoluble protein and was explained by lower magnitude of dipole moment and fluctuation in N-terminal domain of the model structure. However it is shown that HGC is very soluble protein. Solution structure of human γC-crystallin has been determined from an analysis of multidimensional triple resonance NMR spectroscopy using distance restraints from unambiguously assigned 1H-1H NOE peaks and dihedral angle restraints from HNHA and HNHB spectra. 15N relaxation average T1 and T2 correspond to 0.729 ± 0.02 and 0.060 ± 0.04 second from 15N backbone relaxation study, which gives average rotational correlation time 10.87 ns that shows human γC-crystallin is monomer in solution of molecular weight 21 kDa (173 residues). The ensemble of 20 lowest energy structures shows a root mean square deviation of 0.60 ± 0.12 Å for the backbone atoms, and 1.03 ± 0.09 Å for all heavy atoms. The comparison between the calculated NMR structure with backbone chain atoms C`, Cα and NH, of the x-ray crystal structure of the mouse γC - crystallin shows that the structure determined here of human γC-crystallin is very similar with an RMSD of 1.3 Å, which is not surprising given the 84.5% amino acid sequence identity between the two proteins. More importantly, the NMR structure reported here shows the subtle differences in the orientation of specific residues as well as the domain interface between the human and mouse orthologs. The orientation of the calculated dipole moment for this NMR structure differs from earlier reported for model structure. However it is similar to the other known soluble proteins. The determined solution structure of human γC-crystallin also enables us to estimate the effect of cataract-associative mutations on the structure and properties of the protein. Several such mutations are already known, and the work presented here could likely shed light on the molecular basis of these cataracts. Project II: Solution structural studies of intrinsically disordered protein VPg in free and bound forms from Sesbania mosaic virus Sesbania mosaic virus (SeMV) is a plant virus, which infects the Sesbania grandiflora tree. SeMV belongs to Sobemovirus genus, which is not defined under any family. The length of this viral genome is ~4kb. This viral genome has four open-reading frames (ORF). ORF1 and ORF2 encode movement and coat proteins, respectively. ORF2 is again split into two ORFs i.e. ORF2a and ORF2b by a -1 shift in the reading frame and encode two polypeptide chains. These polypeptide chains generate several functional proteins upon polyprotein processing. Polyprotein processing is a mechanism employed by animal and plant viruses to produce several functional proteins from a single polypeptide chain. The two polyproteins expressed are catalytically cleaved by a serine protease, thus releasing the four proteins: VPg (viral protein genome linked), RdRP (RNA dependent RNA polymerase), P10, and P8. VPg (“Viral Protein genome linked”) as its name suggests, is covalently linked to the 5` end of the viral RNA. VPgs are generally known to be intrinsically disordered proteins and have many interacting partners. Intrinsically Disordered Proteins (IDPs) are not explained by the 3D structure–function dogma. However, they are important for biological functions such as molecular recognition, signal transduction and regulation. It is known that SeMV protease becomes inactive in the absence of the VPg domain at its C-terminal. VPgs of animal viruses are well studied as compared to VPgs of plant virues. The size of VPg varies across the Sobemovirus genus. It is important to know the structure of VPg since it is necessary for protease activity. The studies conducted here focus on the structural analysis of the VPg in its free and bound forms with protease (VPg complex) as well as some aspect of full-length ProVPg. For structural studies, two constructs of VPg as fusion protein with Cytb5 tag, one lacking 23 residues at its C-terminal using the pET21a(+) plasmid vector have been designed. Sub-cloning was also done to add a thrombin recognition site to remove the hexa-His tag from new constructs of full-length ProVPg and protease (PRO). These proteins were highly expressed, isotopically labeled and purified for NMR study. The sample used for structural studies of the ProVPg 23 complex was prepared using selectively protonated Ile, Leu and Val; and isotopically labeled i.e. 2H, 13C, and 15N-VPg 23 protein. VPg in its free form is an intrinsically disordered protein and this has been confirmed by its dynamic nature observed using solution NMR spectroscopy. VPg binds to its partner protease and adopts a 3D-structure, which has been shown here. The tertiary structure has been determined using distance restraints from 1HN-1HN NOEs and methyl 1HN NOEs, and dihedral angle predicted from analysis of chemical shift values. The tertiary structure of ProVPg 23 complex has one β -sheet composed of three antiparallel β-strands and an α-helix. The ensemble of 20 lowest energy structures shows a root mean square deviation of 0.42 ± 0.09 Å for the backbone atoms, and 1.09 ± 0.11 Å for all heavy atoms for residues 15 to 50 that are primarily involved in structure formation. On the other hand RMSD is 2.34 ± 0.72 Å for the backbone and 2.55 ± 0.60 Å for all heavy atoms for all residues including both termini. That the tertiary fold of VPg both in full-length ProVPg and when complexed with protease domain (PRO) are the same has been shown here. The NMR structure reported here provides a structural basis for the origin of resonances in the up-field region of one–dimensional proton spectrum of full length ProVPg. The binding surface based on the structures of ProVPg 23 complex determined here and X-ray structure of PRO; has been determined using HADDOCK. The structural model here of full length ProVPg 23 shows the presence of aromatic interaction between Trp271 of PRO and Trp46 of VPg, which is consistent with the earlier biochemical studies.

A unique mechanism of protein oligomerization is domain swapping. It is a feature found in some proteins wherein a dimer or a higher oligomer is formed by the exchange of identical structural segments between protomers. Domain swapping is thought to have played a key role in the evolution of stable oligomeric proteins and in oligomerization of amyloid proteins. This thesis deals with studies to understand the significance of hinges involved in domain swapping for protein oligomerization and function. The stationary phase survival protein SurE from Salmonella typhimurium (StSurE) and Sesbania mosaic virus (SeMV) coat protein have been used as models for studies on domain swapping. This thesis has been divided into eight chapters. Chapter 1 provides a brief introduction to domain swapping, while Chapters 2 to 6 describes the studies carried out on StSurE protein, Chapter 7 deals with studies on SeMV coat protein. The final Chapter 8 provides brief descriptions of various experimental techniques employed during these investigations. Chapter 1 deals with a brief introduction to domain swapping in proteins. Examples where different domains are exchanged are cited. Then it describes physiological relevance of domain swapping in proteins and probable factors which promote swapping. Finally it also discusses the uncertainties that are inevitable in protein structure prediction and design. Chapter 2 describes the structure of Salmonella typhimurium SurE (StSurE; Pappachan et al., 2008) determined at a higher resolution. The chapter also deals with the sequence and structure based comparison of StSurE with other known SurE homolog structures. A comparative analysis of the relative conservation of N- and C-terminal halves of SurE protomer and variations observed in the quaternary structures of SurE homologs are presented. Then a brief introduction is provided on function of StSurE. The conserved active site of StSurE that might be important for its phosphatase activity is described. A plausible mechanism for the phosphatase activity as proposed by Pappachan et al. (2008) is presented. Crystal structures of StSurE bound with AMP, pNPP and pNP that was determined with the view of better understanding the mechanism of enzyme function is presented. These structures provide structural evidence for the mechanism proposed by Pappachan et al. (2008). Finally a substrate entry channel inferred from these structures is discussed. SurE from Salmonella typhimurium (StSurE) was selected for studies on domain swapping as there is at least one homologous structure (Pyrobaculum aerophilum - PaSurE) in which swapping of the C-terminal helices appears to have been avoided without leading to the loss of oligomeric structure or function. It was of interest to examine if an unswapped dimer of StSurE resembling PaSurE dimer could be constructed by mutagenesis. To achieve this objective, a crucial hydrogen bond in the hinge involved in C-terminal helix swapping was abolished by mutagenesis. These mutants were constructed with the intention of increasing the flexibility of the hinge which might bring the C-terminal helices closer to the respective protomer as in PaSurE. Chapter 3 presents a comparative analysis of the hinges involved in C-terminal helix swapping in PaSurE and StSurE. Based on the comparison of structure and sequence, crucial residues important for C-terminal helix swapping in StSurE were identified as D230 and H234. The chapter describes the construction of mutants obtained by substituting D230 and H234 by alanine and their biophysical characterization. Finally it describes structural studies carried out on these mutants. The mutation H234A and D230A/H234A resulted in highly distorted dimers, although helix swapping was not avoided. Comparative analysis of the X-ray crystal structures of native StSurE and mutants H234A and D230A/H234A reveal large structural changes in the mutants relative to the native structure. However the crystal structures do not provide information on the changes in dynamics of the protein resulting from these mutations. To gain better insights into the dynamics involved in the native and mutants H234A and D230A/H234A, MD simulations were carried on using GROMACS 4.0.7. Chapter 4 deals with a brief description of the theory of molecular dynamics, followed by results of simulation studies carried out on monomeric and dimeric forms of StSurE and dimeric forms of its mutants H234A and D230A/H234A. The conformational changes and dynamics of different swapped segments are discussed. Crystal structures of H234A and D230A/H234A mutants reveal that they form highly distorted dimers with altered dimeric interfaces. Chapter 5 focuses on comparison of dimeric interfaces of the native StSurE and hinge mutants H234A and D230A/H234A. Based on the analysis, three sets of interactions were selected to investigate the importance of the interface formed by swapped segments in StSurE mutants H234A and D230A/H234A. One of the selected sites corresponds to a novel interaction involving tetramerization loop in the hinge mutants H234A and D230A/H234A resulting in a salt bridge between E112 – R179’ and E112’ – H180 (prime denotes residue from the other chain of the dimeric protein). This salt bridge seems to stabilize the distorted dimer. It is shown by structural studies that the loss of this salt bridge due to targeted mutation restores symmetry and dimeric organization of the mutants. Loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE not only leads to large structural changes but also alters the conformation of a loop near the active site. It is of interest to understand functional consequences of these structural changes. StSurE is a phosphatase, and its activity could be conveniently monitored using the synthetic substrate para nitrophenyl phosphate (pNPP) at pH 7 and 25 ºC. Chapter 6 deals with the functional studies carried out with various StSurE mutants. The studies suggest that there is a drastic loss in phosphatase activity in hinge mutants D230A, H234A and D230A/H234A, while in the salt bridge mutants the function seems to have been restored. Few of these mutants also exhibit positive cooperativity, which could probably be due to altered dynamics of domains. Sesbania mosaic virus (SeMV) is a plant virus, belonging to genus sobemovirus. SeMV is a T=3 icosahedral virus (532 symmetry) made up of 180 coat protein (CP) subunits enclosing a positive-sense RNA genome. The asymmetric unit of the icosahedral capsid is composed of chemically identical A, B and C subunits occupying quasi-equivalent environments. Residues 48 – 59 of the N-terminal arms of the C subunits interact at the nearby icosahedral three-fold axes through a network of hydrogen bonds to form a structure called the “β-annulus”. Residues 60 – 73 form the “βA-arm” that connects the N-terminal β-annulus to the rest of the protomer. Various studies on SeMV-CP suggest that different lengths of the N-terminal segments affect the assembly of virus. It might be possible to exploit this flexibility of the N-terminus in SeMV-CP to introduce swapping of this segment between two 2-fold related C subunits as is found in Rice yellow mottle virus (RYMV), another sobemovirus, with which SeMV shares significant sequence similarity. Chapter 7 focuses on attempts made to examine the mutational effects planned to introduce domain swapping. The strategy used for introducing swapping in SeMV-CP was based on the sequence of the βA-arm or the hinge involved in swapping of β-annulus in RYMV. TEM images of the mutant virus like particles obtained suggest that they are heterogeneous. These mutants could not be crystallized, probably due to the heterogeneity. However, the assembly of the expressed proteins to virus like particles was profoundly influenced by the mutations. Chapter 8 discusses various crystallographic, biophysical and biochemical techniques used during these investigations. Finally the thesis concludes with Conclusions and Future perspectives of the various studies reported in the thesis. In summary, I have addressed the importance of amino acid residues and interactions of hinges involved in domain swapping for the quaternary structure and function of proteins.

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Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this study are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.