This thesis reports work in three areas of X-ray crystallography. An initial chapter describes the structure of a protein, the methods based on the use of X-rays and computer analysis of diffraction patterns to determine crystal structure, and the subsequent derivation of the structure of part or all of a protein molecule. Work to determine the structure of the protein cytokine receptor-like factor 3 (CRLF3) leading to the successful generation of a structural model of a significant part of this molecule is then described in Chapter 2. A variety of techniques had to be deployed to complete this work, and the steps undertaken are described. Analysis was performed principally using phaser, using maximum likelihood methods. Areas for improvement in generating non-crystallographic symmetry (NCS) operators in existing programmes were identified and new and modified algorithms implemented and tested. Searches based on improved single sphere algorithms, and a new two-sphere approach, are reported. These methods showed improvements in many cases and are available for future use. In Chapter 4, work on determining the relative importance of low resolution and high intensity data in molecular replacement solutions is described. This work has shown that high intensity data are more important than the low resolution data, dispelling a common perception and helping in experimental design.

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2008-09-26 12:45:02.924

O uso desenfreado dos recursos naturais durante as últimas décadas têm impactado drasticamente o meio ambiente, direcionando a humanidade a investir no desenvolvimento de tecnologias para produção sustentável e ecológica de novas fontes de energia renovável e de produtos verdes. Nesse âmbito, o uso de resíduos derivados da biomassa vegetal tem sido apresentado como uma promissora alternativa à substituição de combustíveis, componentes químicos e polímeros de origem fóssil. Esse material é barato, abundante e não compete direta ou indiretamente com a segurança alimentar. Hoje, mais de 200 compostos químicos e biopolímeros de valor agregado podem ser obtidos a partir do processamento de material lignocelulósico. Todavia, essa tecnologia ainda não é plenamente desenvolvida, afetando sua competitividade econômica, sendo que o maior custo atribui-se à despolimerização enzimática dos polissacarídeos que formam a parede celular vegetal (PCV). Essa etapa requer preparados enzimáticos compostos por diversas enzimas, que agem sinergicamente sobre a complexa estrutura da PCV. Dentre essas enzimas, as pectinases e xilanases desempenham um importante papel na desconstrução dos polímeros pécticos e da hemicelulose. O presente trabalho objetivou o estudo funcional e estrutural de diferentes classes de pectinases e xilanases com potencial biotecnológico, no intuito de contribuir para o desenvolvimento pleno da despolimerização enzimática da PCV. Dentro dessa perspectiva, foram estudadas: uma pectina metilesterase (Sl-PME) e uma endo-poligalacturonase (Sl-EndoPG) do inseto Sphenophorus levis; uma exo-poligalacturonase (Bl-ExoPG) de Bacillus licheniformis; uma xilanase GH10 (MT-Xyn10) e duas GH11 (MT-Xyn11a e MT-Xyn11b) identificadas no metatranscriptoma de um consórcio microbiótico derivado de compostagem de bagaço de cana-de-açúcar. A estrutura cristalográfica da Sl-PME evidenciou alta semelhança com outra PME de inseto. Também foi concluído que as PMEs de inseto são mais similares às bacterianas, quando comparadas às fúngicas e vegetais, principalmente em relação ao sulco catalítico. Além disso, PMEs de inseto, exclusivamente, apresentam uma permutação circular, possívelmente realcionada a um evento de transferência horizontal. A Bl-ExoPG apresentou-se monomérica em solução, com atividade ótima em pH neutro a 60°C, sendo estável em uma ampla faixa de pH (5-10) e com considerável termoestabilidade em elevadas temperaturas. Essa enzima, também, apresentou especificidade por pectina não-metilada, liberando unicamente monômeros de ácido galacturônico. As três xilanases estudadas apresentaram-se monoméricas em solução, com maior atividade entre 30 e 45°C e pHs de 6 a 9, retendo atividade acima de 50% nos pHs 5 e 10. Além disso, todas elas apresentam especificidade por xilano, sendo que a MT-Xyn10 apresentou, também, alta atividade sobre arabinoxilano. A MT-Xyn10 apresentou um conjunto de propriedades enzimáticas bastante atrativas às aplicações industriais, uma vez que é altamente estável em uma ampla faixa de pH (4-10), termoestável em temperaturas de até 50°C e sua ação catalítica produz diversos xilo-oligossacarídeos de alto valor agregado. A análise da estrutura cristalográfica da MT-Xyn11a revela três particularidades estruturais, compartilhadas com a MT-Xyn11b, mas não descritas para outras GH11. Dentre essas particularidades, um loop parece limitar o acesso do substrato ao sítio catalítico, contribuindo diretamente para a baixa afinidade ao substrato apresentada por essas duas enzimas. / Decades of unbridled use of natural resources have drastically affected the global environment, driving humanity to invest in the development of novel technologies for production of sustainable and ecofriendly renewable energy sources and green products. In this context, plant biomass residues have been presented as a promising alternative to fuels, chemicals and polymers derived from fossil reserves. This feedstock is abundant, cheap and does not compete directly or indirectly with food security. Today, more than 200 value-added chemicals and biopolymers can be generated by processing lignocellulosic material. However, this technology is not fully developed yet; its major costs stem from the enzymatic depolymerization of the polysaccharides that constitute the plant cell wall (PCW). This step requires enzymatic cocktails composed of several enzymes that synergistically deconstruct the complex PCW. Among these enzymes, pectinases and xylanases play an important role in the depolymerization of pectic polymers and hemicellulose. The present work is a functional and structural study of different classes of pectinase and xylanases with biotechnological potential. It intends to contribute to the full development of PCW enzymatic depolymerization. With this perspective, we studied a pectin methylesterase (Sl-PME) and an endo-polygalacturonase (Sl-EndoPG) from the insect Sphenophorus levis; an exo-polygalacturonase (Bl-ExoPG) from Bacillus licheniformis; a GH10 xylanase (MT-Xyn10); and two GH11 xylanases (MT-Xyn11a and MT-Xyn11b) from the metatranscriptome of sugarcane bagasse compost-derived microbial consortia. The Sl-PME crystallographic structure showed high similarity with other insect PME. It was also concluded that insect PMEs are more similar to bacterial PMEs than fungi or plant PMEs, especially in relation to the catalytic groove. Moreover, insect PMEs exclusively presented a circular permutation that is possibly related to an event of horizontal gene transfer. Bl-ExoPG is monomeric in solution, with optimal activity on neutral pH and 60°C, being stable in a wide pH range (5-10) and with considerable thermostability at high temperatures. This enzyme, also presented specificity for non-methylated pectin substrates, releasing only monomers of galacturonic acid as catalytic product. All three xylanases studied here are monomeric in solution, with optimal activity between 30°C and 45°C and between pHs 6 and 9, retaining more than 50% of original activity in the pHs 5 and 10. Besides, they all showed specificity for xylan, and MT-Xyn10 also showed high activity on arabinoxylan. MT-Xyn10 revealed a set of enzymatic properties attractive for industrial applications, such as high stability in a wide pH range (4-10), thermostability up to 50°C and released products that are high value-added xilo-oligosaccharides. The MT-Xyn11a crystallographic structure revealed three structural particularities shared with MT-Xyn11b, but not previously described in other GH11. Among these particularities, a loop seems to limit the substrate access to the catalytic site, contributing to the low enzyme affinity presented by both MT-Xyn11a and MT-Xyn11b.

O uso desenfreado dos recursos naturais durante as últimas décadas têm impactado drasticamente o meio ambiente, direcionando a humanidade a investir no desenvolvimento de tecnologias para produção sustentável e ecológica de novas fontes de energia renovável e de produtos verdes. Nesse âmbito, o uso de resíduos derivados da biomassa vegetal tem sido apresentado como uma promissora alternativa à substituição de combustíveis, componentes químicos e polímeros de origem fóssil. Esse material é barato, abundante e não compete direta ou indiretamente com a segurança alimentar. Hoje, mais de 200 compostos químicos e biopolímeros de valor agregado podem ser obtidos a partir do processamento de material lignocelulósico. Todavia, essa tecnologia ainda não é plenamente desenvolvida, afetando sua competitividade econômica, sendo que o maior custo atribui-se à despolimerização enzimática dos polissacarídeos que formam a parede celular vegetal (PCV). Essa etapa requer preparados enzimáticos compostos por diversas enzimas, que agem sinergicamente sobre a complexa estrutura da PCV. Dentre essas enzimas, as pectinases e xilanases desempenham um importante papel na desconstrução dos polímeros pécticos e da hemicelulose. O presente trabalho objetivou o estudo funcional e estrutural de diferentes classes de pectinases e xilanases com potencial biotecnológico, no intuito de contribuir para o desenvolvimento pleno da despolimerização enzimática da PCV. Dentro dessa perspectiva, foram estudadas: uma pectina metilesterase (Sl-PME) e uma endo-poligalacturonase (Sl-EndoPG) do inseto Sphenophorus levis; uma exo-poligalacturonase (Bl-ExoPG) de Bacillus licheniformis; uma xilanase GH10 (MT-Xyn10) e duas GH11 (MT-Xyn11a e MT-Xyn11b) identificadas no metatranscriptoma de um consórcio microbiótico derivado de compostagem de bagaço de cana-de-açúcar. A estrutura cristalográfica da Sl-PME evidenciou alta semelhança com outra PME de inseto. Também foi concluído que as PMEs de inseto são mais similares às bacterianas, quando comparadas às fúngicas e vegetais, principalmente em relação ao sulco catalítico. Além disso, PMEs de inseto, exclusivamente, apresentam uma permutação circular, possívelmente realcionada a um evento de transferência horizontal. A Bl-ExoPG apresentou-se monomérica em solução, com atividade ótima em pH neutro a 60°C, sendo estável em uma ampla faixa de pH (5-10) e com considerável termoestabilidade em elevadas temperaturas. Essa enzima, também, apresentou especificidade por pectina não-metilada, liberando unicamente monômeros de ácido galacturônico. As três xilanases estudadas apresentaram-se monoméricas em solução, com maior atividade entre 30 e 45°C e pHs de 6 a 9, retendo atividade acima de 50% nos pHs 5 e 10. Além disso, todas elas apresentam especificidade por xilano, sendo que a MT-Xyn10 apresentou, também, alta atividade sobre arabinoxilano. A MT-Xyn10 apresentou um conjunto de propriedades enzimáticas bastante atrativas às aplicações industriais, uma vez que é altamente estável em uma ampla faixa de pH (4-10), termoestável em temperaturas de até 50°C e sua ação catalítica produz diversos xilo-oligossacarídeos de alto valor agregado. A análise da estrutura cristalográfica da MT-Xyn11a revela três particularidades estruturais, compartilhadas com a MT-Xyn11b, mas não descritas para outras GH11. Dentre essas particularidades, um loop parece limitar o acesso do substrato ao sítio catalítico, contribuindo diretamente para a baixa afinidade ao substrato apresentada por essas duas enzimas. / Decades of unbridled use of natural resources have drastically affected the global environment, driving humanity to invest in the development of novel technologies for production of sustainable and ecofriendly renewable energy sources and green products. In this context, plant biomass residues have been presented as a promising alternative to fuels, chemicals and polymers derived from fossil reserves. This feedstock is abundant, cheap and does not compete directly or indirectly with food security. Today, more than 200 value-added chemicals and biopolymers can be generated by processing lignocellulosic material. However, this technology is not fully developed yet; its major costs stem from the enzymatic depolymerization of the polysaccharides that constitute the plant cell wall (PCW). This step requires enzymatic cocktails composed of several enzymes that synergistically deconstruct the complex PCW. Among these enzymes, pectinases and xylanases play an important role in the depolymerization of pectic polymers and hemicellulose. The present work is a functional and structural study of different classes of pectinase and xylanases with biotechnological potential. It intends to contribute to the full development of PCW enzymatic depolymerization. With this perspective, we studied a pectin methylesterase (Sl-PME) and an endo-polygalacturonase (Sl-EndoPG) from the insect Sphenophorus levis; an exo-polygalacturonase (Bl-ExoPG) from Bacillus licheniformis; a GH10 xylanase (MT-Xyn10); and two GH11 xylanases (MT-Xyn11a and MT-Xyn11b) from the metatranscriptome of sugarcane bagasse compost-derived microbial consortia. The Sl-PME crystallographic structure showed high similarity with other insect PME. It was also concluded that insect PMEs are more similar to bacterial PMEs than fungi or plant PMEs, especially in relation to the catalytic groove. Moreover, insect PMEs exclusively presented a circular permutation that is possibly related to an event of horizontal gene transfer. Bl-ExoPG is monomeric in solution, with optimal activity on neutral pH and 60°C, being stable in a wide pH range (5-10) and with considerable thermostability at high temperatures. This enzyme, also presented specificity for non-methylated pectin substrates, releasing only monomers of galacturonic acid as catalytic product. All three xylanases studied here are monomeric in solution, with optimal activity between 30°C and 45°C and between pHs 6 and 9, retaining more than 50% of original activity in the pHs 5 and 10. Besides, they all showed specificity for xylan, and MT-Xyn10 also showed high activity on arabinoxylan. MT-Xyn10 revealed a set of enzymatic properties attractive for industrial applications, such as high stability in a wide pH range (4-10), thermostability up to 50°C and released products that are high value-added xilo-oligosaccharides. The MT-Xyn11a crystallographic structure revealed three structural particularities shared with MT-Xyn11b, but not previously described in other GH11. Among these particularities, a loop seems to limit the substrate access to the catalytic site, contributing to the low enzyme affinity presented by both MT-Xyn11a and MT-Xyn11b.

Les acides chlorogéniques (CGAs) représentent une famille d'esters formés d'un dérivé de l'acide cinnamique conjugué à l'acide quinique ou shikimique. Ces métabolites secondaires produits par la voie des phénylpropanoides sont largement répandus chez les végétaux terrestres et sont une source majeure d'antioxydants alimentaires. Les esters hydroxycinnamoyl-CoA sont les précurseurs des CGAs et d'autres composés phénoliques tels que les lignines. Ces intermédiaires activés sont synthétisés à partir d'un acide hydroxycinnamique et du coenzyme A par la 4-coumarate CoA ligase (4CL) appartenant à la superfamille des enzymes formant des adénylates. Nicotiana tabacum 4CL2 a été utilisée pour la production d'esters et sa structure a été résolue par remplacement moléculaire. Deux gènes codant pour des hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransférases chez Coffea canephora ont été clonés. CcHCT et CcHQT, qui appartiennent à la superfamille des acyltransférases acyl-CoA-dépendantes, ont été surexprimées dans E. coli et purifiées à homogénéité. L'analyse par diffraction aux rayons X de cristaux de CcHCT a permis de déterminer sa structure par remplacement moléculaire. Un modèle a été dérivé par homologie de séquence pour CcHQT afin de proposer les déterminants de la préférence pour l'acide quinique ou shikimique. Des modélisations moléculaires ont été réalisées afin d'identifier les résidus potentiellement impliqués dans les intéractions enzyme-substrat. L'analyse par chromatographie liquide haute performance des réactions enzymatiques ont montré que ces enzymes sont capables de synthétiser l'acide 5-O-caféoylquinique mais aussi le diester 3,5-O-dicaffeoylquinique, qui est un composé majeur du grain de café avant mûrissement. La production de variants par mutagenèse dirigée a permis l'identification de résidus importants pour la catalyse des réactions de mono- et de diacylation. L'approche combinée de la biologie structurale et de l'enzymologie s'avère particulièrement utile pour mieux comprendre le rôle de HCT et HQT. / Chlorogenic acids (CGAs) represent a family of esters formed between a cinnamic acid derivative and quinic or shikimic acid. CGAs are secondary metabolites produced via the phenylpropanoid pathway by higher plants and are a major source of dietary antioxidants. Hydroxycinnamoyl-CoA esters are the precursors for CGAs and other phenolic compounds such as lignins. These activated intermediates are synthesized from a hydroxycinnamic acid and coenzyme A by 4-coumarate CoA ligase (4CL), which belongs to the adenylate-forming enzyme superfamily. Nicotiana tabacum 4CL2 was used to produce hydroxycinnamoyl-CoA esters and its structure was solved by molecular replacement. Two genes encoding hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferases from Coffea canephora were cloned. CcHCT and CcHQT, which belong to the acyl-CoA-dependent acyltransferase superfamily, were overexpressed in E. coli and purified to homogeneity. X-ray diffraction analysis of CcHCT crystals resulted in a structural solution by molecular replacement. A homology model was derived for CcHQT in order to propose some determinants of the preference for quinic or shikimic acid. Docking experiments were carried out in order to identify potential residues involved in enzyme-substrate interactions. High performance liquid chromatography analysis of enzymatic reactions showed that these enzymes are capable of synthesizing 5-O-caffeoylquinic acid but also the diester 3,5-O-dicaffeoylquinic acid, which is a major component of the coffee grain before ripening. The production of variants by site-directed mutagenesis enabled the identification of residues important for catalysis of the mono- and diacyltransfer reactions. The combined approach of structural biology and enzymology provides molecular insights into the role of HCT and HQT in CGA biosynthesis.

No description available.

Pantothenate kinase (PanK) is an ubiquitous and essential enzyme that catalyzes the first step in the universal Coenzyme (CoA) biosynthesis pathway. In this step, pantothenate (Vitamin B5) is converted to 4′-phosphopantothenate, which subsequently forms CoA in four enzymatic steps. In bacteria, three types of PanK’s have been identified which exhibit wide variations in their distribution, mechanisms of regulation and affinity for substrates. Type I PanK is a key regulatory enzyme in the CoA biosynthesis pathway and its activity is feedback regulated by CoA and its thioesters. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on type I PanK from Mycobacterium tuberculosis (MtPanK) was initiated and the structure of this enzyme in complex with a CoA derivative has been reported earlier. To further elucidate the structural basis of the enzyme action of MtPanK, several crystal structures of the enzyme in complex with different ligands have been determined in the present study. In conjunction to this, solution studies on the enzyme were also carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion method was used for crystallization in all cases. The X-ray intensity data were collected using a MAR Research imaging plate system mounted on a Rigaku RU200 and Bruker-AXS Microstar Ultra II rotating anode X-ray generator. The data were processed using the HKL and MOSFLM and SCALA from the CCP4 suite. The structures were solved by the molecular replacement method using the program AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT and the refined structures were validated using PROCHECK and MOLPROBITY. Secondary structure was assigned using DSSP, structural superpositions were made using ALIGN and buried surface area was calculated using NACCESS. Solution studies on CoA binding and catalytic activity were carried out using Isothermal titration calorimetry (ITC). To start with, the crystal structures of the complexes of MtPanK were determined with (a) citrate, (b) the non-hydrolysable ATP analog AMPPCP and pantothenate (initiation complex), (c) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (end complex), (d) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (intermediate complex), (e) CoA, (f) ADP prepared by soaking and co-crystallization, which turned out to have identical structures and (g) ADP and pantothenate. Unlike in the case of the homologous E.coli enzyme (EcPanK), AMPPCP and ADP occupied different, though overlapping, locations in the respective complexes; the same was true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK was found to be substantially preformed while that of EcPanK exhibited considerable plasticity. The difference in the behavior of the E.coli and M.tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action and the changes in the locations of ligands exhibited by M.tuberculosis pantothenate kinase are remarkable and novel. The movement of ligands exhibited by MtPanK during enzyme action appeared to indicate that the binding site of the enzyme was less specific for a particular type of ligand than EcPanK. Kinetic measurements of enzyme activity showed that MtPanK had dual substrate specificity for ATP and GTP, unlike the enzyme from E.coli which showed a much higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes was provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate (2) GDP and phosphopantothenate (3) GDP (4) GDP and pantothenate (5) AMPPCP and (6) GMPPCP and the structures of the complexes of the two enzymes involving CoA and different adenyl nucleotides. The explanation was substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. Dual specificity of the type exhibited by this enzyme is rare and so are the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands and specificity. The crystal structures of MtPanK in binary complexes with nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP) provided insights about the natural location and conformation of nucleotides. In the absence of pantothenate, the NDP and the NTP bound with an extended conformation at the same site. In the presence of pantothenate, as seen in the initiation complexes, the NTP had a closed conformation and an altered location. However, the effect of the nucleotide on the conformation and the location of pantothenate were yet to be elucidated as the natural location of the ligand in MtPanK was not known. This lacuna was sought to be filled through X-ray analysis of the binary complexes of MtPanK with pantothenate and two of its derivatives, namely, pantothenol and N-nonyl pantothenamide (N9-Pan). These structures demonstrated that pantothenate, with a somewhat open conformation occupied a location similar to that occupied by phosphopantothenate in the “end” complexes, which was distinctly different from the location of pantothenate in “closed” conformation in the ternary “initiation” complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appeared to play a critical role in the movement of ligand that took place during enzyme action. The structure analysis of the binary complexes with the vitamin and its derivatives completed the description of the locations and conformations of nucleoside di and triphosphates and pantothenate in different binary and ternary complexes. These complexes provide snapshots of the course of action of MtPanK.

No description available.

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.