Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-23T23:19:25Z No. of bitstreams: 1 Arquivototal.pdf: 1967881 bytes, checksum: 7ca8d86d26370955fcc5937ad9344abc (MD5) / Made available in DSpace on 2018-04-23T23:19:25Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 1967881 bytes, checksum: 7ca8d86d26370955fcc5937ad9344abc (MD5) Previous issue date: 2017-05-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Sesbania virgata is a shrub belonging to a family Leguminosae whose fruit presents seeds with high protein content. Considering the potential of use of this species as a biotechnological tool, the objective this paper was to characterize the seeds of S. virgata (Cav.) Pers., as well as evaluate extracts and fractions for a presence of some biological activities of medical and pharmacological interest. For this, S. virgata seeds were collected in the city of João Pessoa, Paraíba, and had their morpho-physical-chemical characteristics determined. Afterwards, the seeds were delipidated, processed and solubilized in extractive solutions, obtaining 30 extracts and 10 protein fractions, allowing a characterization of the antinutritional factors; protein and amino acid profile; and biological activities. The nutritional characteristics of the seed flour emphasize its high protein content. The optimization of the production of extracts and fractions allowed obtaining the total crude extract (EBT), purification of the protein fraction with lectin activity (FPAL) and the determination of several antinutritional factors. The protein analyzes revealed 60.79% of total protein for the fine flour (FF), 49.34% for EBT and 55.84% for FPAL. With molecular weights between 31-225 kDa, FF, EBT and FPAL presented antioxidant activity. However, they did not present antibacterial activity against the strains investigated, but promoted the inhibition of Candida albicans, C. tropicalis, Aspergillus flavus and Penicillium citrinum. In addition to the high protein content and biological activities, it is important to incorporate new analyzes into the products so that, in the future, they will be viable in the food or pharmaceutical industry, by creating of topical drugs to control fungal contamination or reduction of the damage caused to human health by free radicals and other oxidants. / Sesbania virgata é um arbusto pertencente a família Leguminosae, cujo fruto apresenta sementes com elevados teores proteicos. Considerando o potencial de uso dessa espécie como ferramenta biotecnológica, objetivou-se caracterizar nutricional, antinutricional e bioquimicamente as sementes de Sesbania virgata (Cav.) Pers., bem como avaliar extratos e frações proteicas quanto a presença de algumas atividades biológicas de interesse médico e farmacológico. Para tal, sementes de Sesbania virgata foram coletadas na cidade de João Pessoa, Paraíba, e tiveram suas características morfo-físico-químicas determinadas. Posteriormente, as sementes foram delipidadas, processadas e solubilizadas em soluções extratoras, obtendo-se 30 extratos e 10 frações proteicas, permitindo a caracterização dos fatores antinutricionais, do perfil proteico e aminoacídico, e atividades biológicas. As características nutricionais da farinha enfatizaram seu elevado teor protéico. A otimização da produção dos extratos e frações permitiu a obtenção do extrato bruto total (EBT), a purificação da fração proteica com atividade lectínica (FPAL) e a determinação de fatores antinutricionais diversos. As análises proteicas revelaram 60,79% de proteína total para a farinha fina (FF), 49,34% para EBT e 55,84% para FPAL. Com massas moleculares entre 31-225 kDa, FF, EBT e FPAL apresentaram atividade antioxidante. Contudo, não apresentaram atividade antibacteriana contra as cepas investigadas, mas promoveram a inibição de Candida albicans, C. tropicalis, Aspergillus flavus e Penicillium citrinum. Ressalta-se, além do elevado teor proteico e atividades biológicas apresentados, a importância em incorporar novas análises aos produtos para que, futuramente, apresentem viabilidade de implementação na alimentação ou na indústria farmacêutica, mediante criação de fármacos tópicos para controle da contaminação fúngica ou redução dos danos causados à saúde humana pelos radicais livres e outros oxidantes.

Sementes de Sesbania virgata (Cav.) Pers. acumulam suas reservas de carbono no endosperma na forma de um polissacarídeo de parede celular, o galactomanano. Os galactomananos são polissacarídeos constituídos de uma cadeia principal de resíduos de D-manose ligadas β-1,4, ramificada por resíduos de D-galactose α-1,6 ligados. A mobilização deste ocorre após a germinação e envolve três enzimas hidrolíticas (α-galactosidase, endo-β-mananase e exo-β-manosidase). A α-galactosidase é a primeira enzima atuar sobre o galactomanano hidrolisando as ligações α-1,6 das galactoses ramificadas a cadeia principal de manano (ligados β-1,4), permitindo a ação da endo-β-mananase, que hidrolisará o polissacarídeo a oligossacarídeos, onde a β-manosidase atuará (ligações β-1,4), transformando oligossacarídeos a monossacarídeos a serem utilizados no desenvolvimento do embrião. Buscando a compreensão das características da α-galactosidase e modo de ação sobre o galactomanano, procedeu-se com a purificação, em três etapas,e caracterização bioquímica (pH ótimo, temperatura ótima e aspectos cinéticos) da α-galactosidase de sementes de Sesbania virgata (Cav.) Pers. Além disso, visando evidenciar a modulação da enzima endo-β-mananase pela distribuição de ramificações de galactose no galactomanano (estrutura fina do galactomanano), procedeu-se com hidrólises enzimáticas do galactomanano de Sesbania virgata (Cav.) Pers. utilizando a enzima endo-β-mananase de Aspergillus niger (Megazyme®) somente ou em conjunto com a α-galactosidase semipurificada de Sesbania virgata (Cav.) Pers. (Capítulo 1) ou com a α-galactosidase comercial de Cyamopsis tetragonoloba (Megazyme®), seguido de análise dos oligossacarídeos por HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection). Também procedeu-se com hidrólises enzimáticas de galactomananos de 6 espécies com razão manose:galactose variando de 1:1 a 150:1 com endo-β-mananase de Aspergillus niger (Megazyme®) e análise dos oligossacarídeos produzidos por HPAEC-PAD. A α-galactosidase semipurificada possui, aproximadamente, 42 kDa de peso molecular em condições desnaturantes e, aproximadamente 72 kDa de peso molecular na forma nativa, sugerindo que a enzima assuma estrutura quartenária. A temperatura ótima apresentada se encontra na faixa de 50°C a 55°C, pH ótimo na faixa de 4,4 a 5,4, Km= 1,8276 mM e a velocidade máxima de 0,5024 μmolGal.min-1.mgprot-1. A espectrometria de massas gerou os fragmentos: ALADYV-HSK-RMPGSLGHEE-QDAK-TT-GDIEDNWNSM-TSIADS NDKW-ASYAGPGGWN-DPDMLEVGNG-GMTTEEYR-AP-LLVGCDIR-VAVIL-WNR, estando a proteína referente a esta sequência relacionada à mobilização de reserva. Durante a purificação e sequenciamento interno da α-galactosidase e demais proteínas foram detectadas isoformas da α-galactosidase de pesos moleculares variados (42 kDa a 20 kDa). Sugere-se que estas isoformas encontradas inicialmente na purificação estejam relacionadas com outras funções da α-galactosidase, enquanto as isoformas encontradas após todas as etapas de purificação e identificação por espectrometria de massas estejam relacionadas com ativação e adaptação da α-galactosidase durante todo o processo de mobilização de reservas. Os dados gerados das comparações dos oligossacarídeos produzidos em cada hidrólise sugerem que as ramificações do galactomanano podem modular o reconhecimento de sítios de clivagem pela endo-β-mananase: (1) existe a produção de oligossacarídeos limites de digestão F1, F2 e F3 após hidrólise do galactomanano com endo-β-mananase, como demonstrado para xiloglucanos; (2) os oligossacarídeos F1 possuem proporções distintas quando da hidrólise do galactomanano com endo-β-mananase em diferentes concentrações (ExP I e EXP IV), evidenciando preferência por sítios com menor grau de galactosilação; (3) a presença da α-galactosidase diminui a produção dos oligossacarídeos F2 e F3, mostrando que estes não possuem resistência intrínseca a hidrólise e que a reação atinge o equilíbrio mesmo quando ainda existem sítios de clivagem ainda disponíveis (EXP III); (4) polissacarídeos com estruturas diferentes, razão manose:galactose variando entre 150:1 a 1:1, são digeridos em diferentes taxas de hidrólise pela mesma enzima, evidenciando que a ramificação com galactose dificulta a ação da endo-β-mananase. Dessa forma, sugere-se que a estrutura do polissacarídeo galactomanano também contenha, pelo menos, parte da informação requerida para seu próprio metabolismo, código para a sua degradação, estando esta informação contida na distribuição das ramificações com resíduos de D-galactose. Sendo assim, sugere-se que as diferentes isoformas da α-galactosidase relacionadas à degradação da reserva de galactomanano de sementes de Sesbania virgata (Cav.) Pers. seriam produto da ação proteolítica da própria enzima a fim de melhorar a afinidade da α-galactosidase ao substrato durante o processo de mobilização de reserva. O aumento da afinidade da α-galactosidase ao substrato durante todo o processo de mobilização garantiria a liberação das ramificações com galactose de forma contínua, permitindo e aumentando a eficiência da ação da enzima endo-β-mananase aos sítios de clivagem, garantindo a degradação do polissacarídeo a oligossacarídeos de forma regulada, passível de bloqueio, pelo acúmulo de oligossacarídeos e galactose livre que inibem a ação das enzimas endo-β-mananase e α-galactosidase, respectivamente, e dificultando a ação de microorganismos, propiciando ao embrião a maior quantidade de açúcares para o seu desenvolvimento, aumentando as chances de sucesso no estabelecimento da plântula / The seeds of Sesbania virgata (Cav.) Pers. have an endosperm which accumulates galactomannan as a storage polysaccharide in the cell walls. Galactomannans are composed of a linear backbone of β-(1,4)-linked D-mannose residues with D-galactose α-(1,6)-linkages substitutions. The galactomannans are hydrolysed after protrusion of the radicle. This process is perfomed by three enzymes (α-galactosidase, endo-β-mannanase and exo-β-manosidase). The α-galactosidase is the first enzyme to cleave the polysaccharides, removing the D-galactose residues, allowing the performance of the endo-β-mannanase, which hydrolyses the mannan backbone to mannan oligosaccharides. The last part of the process includes exo-β-manoside, that cleaves the mannan oligosaccharides to mannose residues, which could be used by the embryo during growth. Aiming at understanding the function of ?-galactosidase in the process of galatomanannan degradation, we studied its mode of action on mannans and galactomannans. The α-galactosidase of Sesbania virgata (Cav.) Pers. was purified and characterized (pH and temperature optimum and the enzyme kinetics). We found that the semipurified α-galactosidase molecular weight was 42kDa at denaturating conditions, but in native conditions was 72kDa, suggesting that the enzyme has a quaternary structure. The enzyme optimum pH was between 4,4-5,4, optimum temperature between 50°C-55°C, Km= 1,8276 mM and Vmáx= 0,5024 μmolGal.min-1.mgprot-1. Mass spectrometry measures resulted the following fragments: ALADYV-HSK-RMPGSLGHEE-QDAK-TT-GDIEDNWNSMTSIADS-NDKW-ASYAGPGGWN-DPDMLEVGNG-GMTTEEYR-AP-LLVGCDIR-VAVIL-WNR, being the protein from this sequence related with storage mobilization. Possible α-galactosidase isoforms were detected during the purification, suggesting other functions for the enzyme. The α-galactosidase isoforms detected after all purification steps and with measured mass spectrometry (from 42kDa to 20kDa) should be related to the storage mobilization. We suggest that the α-galactosidase isoforms in Sesbania virgata (Cav.) Pers. seeds represents products of the enzyme self-digestion, this process being correlated with the enzyme/polysaccharide affinity and at last, correlated to the galactomannan mobilization. An extract semipurified from Sesbania virgata (Cav.) Pers. and enriched with α-galactosidase activity, was used along with endo-β-mannanase from Aspergillus niger (Megazyme®) or both endo-β-mannanase and α-galactosidase (semipurified from Sesbania virgata seeds - Chapter 1- or commercial enzyme from Cyamopsis tetragonoloba - Megazyme®) were used to study the fine structure of galactomannans. Hydrolysis of galactomannans from six species with different mannose:galactose (1:1 to 150:1) ratio were performed with endo-β-mananase from Aspergillus niger. The oligosaccharides from all hydrolysis were analyzed by HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection). The hydrolysis fragments data (HPAEC-PAD) suggest that the side-chains of the polysaccharides can modulate the hydrolytic sites recognition on the galactomannan by the endo-β-mannanase. This conclusion is supported by: (1) the presence of limited digest oligosaccharides F1 and dimmers (F2) and trimers (F3) of the F1 oligosaccharides; (2) the presence of different F1 oligosaccharides proportions after hydrolysis with endo-β-mannanase at different concentrations, showing preference on less-branched hydrolytic sites; (3) the α-galactosidase digestion avoided the accumulation of oligosaccharides F2 and F3, showing that these oligosaccharides do not present intrinsic resistance to hydrolysis and that the reaction reaches an equilibrium even when sites of hydrolysis are still available; (4) polymers with different fine structure (ratio mannose:galactose 1:1 to 150:1) were hydrolysed at different rates by the endo-β-mannanase, showing that galactose branching interferes on the enzyme action. Considering that, the branching pattern of the polysaccharide seems to have direct influence on the interaction of the enzyme with substrate; we suggest that the structure of the galactomannan holds part of information required for its own degradation. The higher enzyme x substrate affinity, ensure the galactose branches digestion, improving the endo-β-mannanase action, ensuring the degradation of the polysaccharides to oligosaccharides. This highly regulated degradation process prevents microorganisms predation and increases the plantlet establishement

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.

No description available.

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.

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.

Acetohydroxyacid synthase is a multisubunit enzyme that catalyses the first committed step in the biosynthesis of the branched chain amino acids viz., valine, leucine and isoleucine. In order to understand the structural basis for the observed allosteric feedback inhibition in AHAS, the regulatory subunit of AHAS isozymes I from E. coli was cloned, expressed, purified and the conditions were optimized for solution NMR spectroscopy. IlvN was found to exist as a dimer both in the presence and absence of the feedback inhibitor. Using high-resolution multidimensional, multinuclear NMR experiments, the structure of the dimeric valine-bound 22 kDa IlvN was determined. The ensemble of twenty low energy structures shows a backbone root mean square deviation of 0.73 ± 0.13 Å and a root mean square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, greater than 98% of the backbone φ, ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map. Each protomer exhibits a βαββαβα topology that is a characteristic feature of the ACT domain fold that is observed in regulatory domains of metabolic enzymes. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR timescale. Important structural properties of the unliganded state were probed by H-D exchange studies by NMR, alkylation studies by mass spectrometry and other biophysical methods. It was observed that the dynamic unliganded IlvN underwent a coil-to-helix transition upon binding the effector molecule and this inherent conformational flexibility was important for activation and valine-binding. A mechanism for allosteric regulation in the AHAS holoenzyme was proposed. Study of the structural and conformational properties of IlvN enabled a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis. Solution structural studies of 32 kDa Protease-VPg (PVPg) from Sesbania mosaic virus (SeMV) Polyprotein processing is a commonly found mechanism in animal and plant viruses, by which more than one functional protein is produced from the same polypeptide chain. In Sesbania Mosaic Virus (SeMV), two polyproteins are expressed that are catalytically cleaved by a serine protease. The VPg protein that is expressed as a part of the polyprotein is an intrinsically disordered protein (by recombinant expression) that binds to various partners to perform several vital functions. The viral protease (Pro), though possessing the necessary catalytic residues and the substrate binding pocket is unable to catalyse the cleavage reactions without the VPg domain fused at the C-terminus. In order to determine the structural basis for the aforementioned activation of protease by VPg I undertook the structural studies of the 32 kDa PVPg domains of SeMV by solution NMR spectroscopy. NMR studies on this protein were a challenge due to the large size and spectral overlap. Using a combination of methods such as deuteration, TROSY-enhanced NMR experiments and selective ‘reverse-labelling’, the sequence specific assignments were completed for ~80% of the backbone and 13C nuclei. NMR studies on mutants such as the C-terminal deletion mutant, I/L/V to A mutants in VPg domain were conducted in order to identify the residues important for aliphatic-aromatic interactions observed in PVPg. Attempts were made to obtain NOE restraints between Pro and VPg domains through ILV labelled samples; however these proved unsuccessful. It was observed that ‘natively unfolded’ VPg possessed both secondary and tertiary structure in PVPg. However, 30 residues at the C-terminus were found to be flexible. Even though atomic-resolution structure could not be determined, the region of interaction between the domains was determined by comparing NMR spectra of Pro and PVPg. The conditions for reconstitution of the Protease-VPg complex by recombinantly expressed Pro and VPg proteins were standardised. These studies lay the foundation for future structural investigations into the Protease-VPg complex.