É conhecido que a força iônica do meio desempenha um papel fundamental na estrutura de vesículas aniônicas de DMPG (dimiristoil fosfatidilglicerol) em dispersões aquosas. A baixa força iônica (~ 6 mM), as dispersões de DMPG exibem várias características anômalas, que foram interpretadas como a abertura de poros na bicamada ao longo da larga região de transição de fase gel-fluida (de ~ 18°C a 30°C). Aqui, revisitamos o sistema de DMPG em tampão a baixa força iônica, mas com dispersões obtidas após a extrusão por filtros de 100 nm, portanto menos polidispersas. Para enfatizar as interações eletrostáticas entre as cabeças polares dos lipídios, que não estarão blindadas pela presença de sais na solução, estudamos dispersões de DMPG em água pura, de modo a monitorar os agregados presentes na dispersão, e suas interações. As dispersões em água foram caracterizadas antes e depois da extrusão. Para tal, utilizamos diversas técnicas experimentais, em diferentes temperaturas: espalhamento de luz estático (SLS) e dinâmico (DLS), calorimetria diferencial de varredura (DSC), Ressonância Paramagnética Eletrônica (RPE) de marcadores de spin incorporados aos agregados, espalhamento de raios-X a altos e baixos ângulos (WAXS e SAXS), e medidas de viscosidade, turbidez, mobilidade eletroforética e condutividade elétrica. Resultados das várias técnicas com dispersões extrusadas de DMPG em tampão mostraram que o comportamento anômalo é observado de forma similar ao de dispersões não extrusadas. Entretanto, o pico de SAXS em muito baixo ângulo é visto de 5 a 45°C, e não apenas na região de transição de fase, portanto não deve ser modelado como a distância entre poros na bicamada lipídica que se abririam nesta região. A distância de repetição relacionada a este pico diminui na região de transição de fase, e com o aumento da concentração lipídica. Medidas de DSC indicaram que, em água, a região de transição de fase da vesícula de DMPG é ainda mais ampla, começando em torno de 10°C, mas ainda terminando em ~ 30oC. No entanto, a alta condutividade elétrica, viscosidade, mobilidade eletroforética, raio efetivo, e a baixa turbidez, vistas apenas na região de transição de fase do DMPG em tampão, são encontradas até altas temperaturas em água, quando a bicamada lipídica já se encontra na fase fluida. Medidas de RPE e WAXS mostraram a transição da membrana de uma fase mais rígida/imóvel/organizada para uma fase mais frouxa/móvel. Dados de espalhamento de luz, RPE e SAXS mostram que, similar ao DMPG em tampão, em água, o DMPG organiza-se como vesículas esféricas, unilamelares, mas possivelmente menores e mais carregadas, exibindo fortes interações vesícula-vesícula. Nas medidas de SAXS, o pico de Bragg na região de muito baixo ângulo foi visto em todas as temperaturas (de 5 a 60°C), sendo que a distância de repetição diminui para temperaturas maiores do que 10oC. Os resultados obtidos para dispersões em água, reforçam o comportamento anômalo observado anteriormente para dispersões em tampão em baixa força iônica. De acordo com eles, propomos a existência de vesículas altamente deformadas e ionizadas a partir de uma certa temperatura, T1 para o DMPG em água e Tmon em tampão baixa força iônica, sendo que em água a forte repulsão eletrostática PG--PG- levaria a fortes deformações e interações vesícula-vesícula, em uma ampla extensão de temperaturas. / It is known that the ionic strength plays a fundamental role in the structure of DMPG (dimyristoyl phosphatidylglycerol) anionic vesicles in water medium. At low ionic strength (~ 6 mM), DMPG dispersions display several anomalous characteristics, which were interpreted as the opening of bilayer pores along the wide bilayer gel-fluid transition region (from ~ 18°C to 30°C). Here, we revisit DMPG in buffer at low ionic strength, but with dispersions obtained after the extrusion by 100 nm filters, thus less polydisperse. To emphasize electrostatic interactions between the polar head-groups, which will not be shielded by ions in solution, we studied DMPG dispersions in pure water to monitor the aggregates in the dispersion and their interactions. Water dispersions were characterized before and after extrusion. For such, we used several experimental techniques, at different temperatures: light scattering, both static (SLS) and dynamic (DLS); differential scanning calorimetry (DSC); electron spin resonance (ESR) of spin labels incorporated into the aggregates, Small and Wide Angle X-Ray Scattering (SAXS and WAXS); and viscosity, turbidity, electrophoretic mobility and electrical conductivity measurements. Several techniques with extruded dispersions of DMPG in buffer showed that the anomalous behavior is also observed. However, the SAXS peak at very low angles is seen from 5 to 45°C, and not only in the phase transition region, therefore it should not be modeled as the distance of correlated pores in the lipid bilayer that would open in this region. The repeating distance related to this peak decreases in the phase transition region, and with increasing lipid concentration. DSC indicates that, in water, the bilayer gel-fluid transition is even wider, starting around 10oC but still ending ~ 30oC. However, high electric conductivity, viscosity, electrophoretic mobility, effective radius and low turbidity found only in the gel-fluid transition region for DMPG in buffer, are found at higher temperatures in water, when lipid bilayers are already in the fluid state. ESR and WAXS measurements evidenced the transition of the membrane from a more rigid/immobile/organized phase to a more soft/mobile phase. Light scattering, ESR and SAXS data showed that, similar to DMPG in buffer, in water, DMPG is organized as spherical unillamelar vesicles, but possibly smaller, highly charged, displaying strong vesicle-vesicle interactions. With SAXS the Bragg peak at very low angles was seen at all temperatures (from 5 to 60°C) with the repetition distance decreasing at temperatures higher than 10 ° C. The results obtained for water dispersions reinforce the anomalous behavior previously observed for buffer at low ionic strength dispersions. According to them, we propose the existence of highly deformed and ionized vesicles from a certain temperature, T1 for DMPG in water and Tmon in buffer at low ionic strength. In water the strong PG- - PG- electrostatic repulsion would lead to strong deformations and vesicle-vesicle interactions, over a wide range of temperatures.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-18Bitstream added on 2014-06-13T19:00:57Z : No. of bitstreams: 1 cortezgutierrez_ho_dr_sjrp.pdf: 1171634 bytes, checksum: dab1a18fa9cc0c38b49fe73954595b2d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O objetivo deste trabalho é investigar a localização de energia e o aprisionamento do breather no modelo de Peyrard Bishop com o potencial original de Morse, potencial simétrico de Morse e potenciais quárticos em cadeias homogêneas e não homogêneas usando o Teorema do limite anticontinuum. No caso não homogêneo, a impureza é introduzida pela profundidade do potencial. Foi observado que o modelo SPB apresenta pequenas amplitudes e menor densidade de energia comparada com o modelo PB. Para potenciais quárticos simétricos e assimétricos foi verificado numericamente que não temos transição de fase usando o conceito de amplitude média das vibrações. Entretanto, um potencial híbrido formado por um quártico e Morse pode fornecer uma grande amplitude de vibração das fitas de DNA . Finalmente, no sistema não homogêneo, se verificou a hipótese de aprisionamento por meio de uma simulação de interação do breather móvel com a região de TATA box para uma cadeia longa de DNA, correspondente à seqüencia de nucleotídeos que codifica a insulina. Este resultado deve ajudar a estender a aplicação dos breathers discretos a sistemas biológicos que levam em conta reações bioquímicas localizadas (como, por exemplo, reações enzimáticas) em macromoléculas biológicas. O modelo PB pode ser modificado para explicar a existência de movimentos localizados de grande amplitude. O decaimento da função potencial para valores grandes mostraram uma vibração localizada no equilíbrio de grande amplitude. Isso é feito alterando o potencial on site. / The objective of this work is to investigate the energy localization and the breather trapping in the Peyrard-Bishop model with the original Morse potential, symmetric Morse potential and quartics potentials in homogeneous and inhomogeneous chains. In the inhomogeneous case, the impurity is introduced by the depth of the potential. It was observed that the SPB model shows small amplitude and lower density of energy compared with the PB model. Quartics potentials, for symmetric and asymmetric cases, do not show phase transition. It is verified by numerical calculation using the concept of mean amplitude of the vibrations. Meanwhile, a potential hybrid formed by a quartic and Morse is ideal for DNA applications. Finally, it was verified the trapping hypotheses through the simulation of interaction of mobile breather with the region of TATA box for a long chain of DNA corresponding to the nucleotide sequence that encodes the insulin. This result should help to extend the application of discrete breathers the biological systems that take into account located biochemical reactions (such as enzymatic reactions) in biological macromolecules. In addition, the PB model can be modified to explain the existence of highly localized large amplitudes motions of the base pairs in DNA. We do this change the on site potential.

Foi realizado um estudo por espectroscopia de ressonancia paramagnetica eletronica de solucoes de complexos formados por melanina sintetica, obtida por auto-oxidacao de l-dopa (dihidroxi-l-fenilalanina), e albumina de soro bovino (bsa). Investigaram-se os efeitos devidos a alteracoes de ph, de concentracao e conformacao da bsa, e presenca de O IND.2, tomando como base as variacoes das formas e intensidades das linhas espectrais dos radicais livres intrinsecos da melanina e dos induzidos por radiacao visivel-uv. Verificou-se uma dependencia da intensidade do sinal com o estado de protonacao do pigmento. Na presenca da albumina a inducao de radicais livres por luz foi maior, o que nao observou-se quando esta foi denaturada. A inducao e decaimento dos radicais fotoinduzidos foi monitorada por espectro de varredura temporal, observando-se uma cinetica que envolveu processos mais lentos com o aumento da concentracao da proteina. A partir de medidas de saturacao progressiva, verificou-se um comportamento de saturacao homogeneo para a melanina pura e nao homogeneo quando bsa e O IND.2 estavam presentes, acompanhados de variacoes nos tempos de relaxacao. Os resultados foram interpretados com base em um modelo que supoe a existencia de processos de transferencia de eletrons da melanina excitada para o O IND.2 e transferencia de carga entre o pigmento e a proteína. / Synthetic melanin (obtained by L-DOPA auto-oxidation) and melanin-bovine serum albumin complexes were studied by electron paramagnetic resonance spectroscopy (EPR). The melanin intrinsic and Iight-induced free radicals by visible-UV were studied. The dependence of the EPR line shapes on the pH value, albumin conformation and concentration, and O² presence was analyzed. It was found that the EPR signal intensity depended on the pigment protonation state. The light free radical induction in the melanin-albumin solutions was higher than in pure melanin solution. This effect was only observed when the protein was in its natural form. The induction and decay of the light-induced radicals were monitored by time-resolved experiments. A slower kinetic was observed with increasing of the albumin concentration. Progressive saturation measurements showed a homogeneous saturation behavior for pure melanin and inhomogeneous in the protein or O² presence, showing also changes in time relaxations. The results were interpreted assuming the existence of electron transfer processes from excited melanin to O² and charge exchange between the pigment and the protein.

Foi realizado um estudo por espectroscopia de ressonancia paramagnetica eletronica de solucoes de complexos formados por melanina sintetica, obtida por auto-oxidacao de l-dopa (dihidroxi-l-fenilalanina), e albumina de soro bovino (bsa). Investigaram-se os efeitos devidos a alteracoes de ph, de concentracao e conformacao da bsa, e presenca de O IND.2, tomando como base as variacoes das formas e intensidades das linhas espectrais dos radicais livres intrinsecos da melanina e dos induzidos por radiacao visivel-uv. Verificou-se uma dependencia da intensidade do sinal com o estado de protonacao do pigmento. Na presenca da albumina a inducao de radicais livres por luz foi maior, o que nao observou-se quando esta foi denaturada. A inducao e decaimento dos radicais fotoinduzidos foi monitorada por espectro de varredura temporal, observando-se uma cinetica que envolveu processos mais lentos com o aumento da concentracao da proteina. A partir de medidas de saturacao progressiva, verificou-se um comportamento de saturacao homogeneo para a melanina pura e nao homogeneo quando bsa e O IND.2 estavam presentes, acompanhados de variacoes nos tempos de relaxacao. Os resultados foram interpretados com base em um modelo que supoe a existencia de processos de transferencia de eletrons da melanina excitada para o O IND.2 e transferencia de carga entre o pigmento e a proteína. / Synthetic melanin (obtained by L-DOPA auto-oxidation) and melanin-bovine serum albumin complexes were studied by electron paramagnetic resonance spectroscopy (EPR). The melanin intrinsic and Iight-induced free radicals by visible-UV were studied. The dependence of the EPR line shapes on the pH value, albumin conformation and concentration, and O² presence was analyzed. It was found that the EPR signal intensity depended on the pigment protonation state. The light free radical induction in the melanin-albumin solutions was higher than in pure melanin solution. This effect was only observed when the protein was in its natural form. The induction and decay of the light-induced radicals were monitored by time-resolved experiments. A slower kinetic was observed with increasing of the albumin concentration. Progressive saturation measurements showed a homogeneous saturation behavior for pure melanin and inhomogeneous in the protein or O² presence, showing also changes in time relaxations. The results were interpreted assuming the existence of electron transfer processes from excited melanin to O² and charge exchange between the pigment and the protein.

<p>Radiation damage limits the resolution in imaging experiments. Damage is caused by energy deposited into the sample during exposure. Ultrashort and extremely bright X-ray pulses from free-electron lasers (FELs) offer the possibility to outrun key damage processes, and temporarily improve radiation tolerance. Theoretical models indicate that high detail-resolutions could be realized on non-crystalline samples with very short pulses, before plasma expansion.</p><p>Studies presented here describe the interaction of a very intense and ultrashort X-ray pulse with material, and investigate boundary conditions for flash diffractive imaging both theoretically and experimentally. In the hard X-ray regime, predictions are based on particle simulations with a continuum formulation that accounts for screening from free electrons.</p><p>First experimental results from the first soft X-ray free-electron laser, the FLASH facility in Hamburg, confirm the principle of flash imaging, and provide the first validation of our theoretical models. Specifically, experiments on nano-fabricated test objects show that an interpretable image can be obtained to high resolution before the sample is vaporized. Radiation intensity in these experiments reached 10^14 W/cm^2, and the temperature of the sample rose to 60000 Kelvin after the 25 femtosecond pulse left the sample. Further experiments with time-delay X-ray holography follow the explosion dynamics over some picoseconds after illumination.</p><p>Finally, this thesis presents results from biological flash-imaging studies on living cells. The model is based on plasma calculations and fluid-like motions of the sample, supported by the time-delay measurements. This study provides an estimate for the achievable resolutions as function of wavelength and pulse length. The technique was demonstrated by our team in an experiment where living cells were exposed to a single shot from the FLASH soft X-ray laser.</p>

The work presented in this thesis has striven toward improving the capability to study proteins using infrared (IR) spectroscopy. This includes development of new and improved experimental and theoretical methods to selectively observe and simulate protein vibrations. A new experimental method of utilising adenylate kinase and apyrase as helper enzymes to alter the nucleotide composition and to perform isotope exchange in IR samples was developed. This method enhances the capability of IR spectroscopy by enabling increased duration of measurement time, making experiments more repeatable and allowing investigation of partial reactions and selected frequencies otherwise difficult to observe. The helper enzyme mediated isotope exchange allowed selective observation of the vibrations of the catalytically important phosphate group in a nucleotide dependent protein such as the sarcoplasmic reticulum Ca2+-ATPase. This important and representative member of P-type ATPases was further investigated in a different study, where a pathway for the protons countertransported in the Ca2+-ATPase reaction cycle was proposed based on theoretical considerations. The transport mechanism was suggested to involve separate pathways for the ions and the protons. Simulation of the IR amide I band of proteins enables and supports structure-spectra correlations. The characteristic stacking of beta-sheets observed in amyloid structures was shown to induce a band shift in IR spectra based on simulations of the amide I band. The challenge of simulating protein spectra in aqueous medium was also addressed in a novel approach where optimisation of simulated spectra of a large set of protein structures to their corresponding experimental spectra was performed. Thereby, parameters describing the most important effects on the amide I band for proteins could be determined. The protein spectra predicted using the optimised parameters were found to be well in agreement with experiment. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>

Radiation damage limits the resolution in imaging experiments. Damage is caused by energy deposited into the sample during exposure. Ultrashort and extremely bright X-ray pulses from free-electron lasers (FELs) offer the possibility to outrun key damage processes, and temporarily improve radiation tolerance. Theoretical models indicate that high detail-resolutions could be realized on non-crystalline samples with very short pulses, before plasma expansion. Studies presented here describe the interaction of a very intense and ultrashort X-ray pulse with material, and investigate boundary conditions for flash diffractive imaging both theoretically and experimentally. In the hard X-ray regime, predictions are based on particle simulations with a continuum formulation that accounts for screening from free electrons. First experimental results from the first soft X-ray free-electron laser, the FLASH facility in Hamburg, confirm the principle of flash imaging, and provide the first validation of our theoretical models. Specifically, experiments on nano-fabricated test objects show that an interpretable image can be obtained to high resolution before the sample is vaporized. Radiation intensity in these experiments reached 10^14 W/cm^2, and the temperature of the sample rose to 60000 Kelvin after the 25 femtosecond pulse left the sample. Further experiments with time-delay X-ray holography follow the explosion dynamics over some picoseconds after illumination. Finally, this thesis presents results from biological flash-imaging studies on living cells. The model is based on plasma calculations and fluid-like motions of the sample, supported by the time-delay measurements. This study provides an estimate for the achievable resolutions as function of wavelength and pulse length. The technique was demonstrated by our team in an experiment where living cells were exposed to a single shot from the FLASH soft X-ray laser.

Shigella flexneri attacks cells of the intestinal tract, causing over 1 million deaths annually from bacterial dysentery. A type III secretion system (T3SS) initiates the host-pathogen interaction and transports virulence factors directly into host cells via a needle complex (NC) comprising an extracellular needle and membrane-spanning basal body. Rings formed by the single-pass membrane proteins MxiG and MxiJ are arranged concentrically within the inner membrane ring (IMR) of the NC. The Neterminal domain of MxiG (MxiG-N) is the predominant IMR cytoplasmic structure, however it was structurally and functionally uncharacterised. Determination of the solution structure of MxiG-N in this study revealed it to be a forkhead associated (FHA) domain, although subsequent analyses of conserved residues suggested it does not have the canonical role in cell-signalling via phospho-threonine recognition. Subsequent positioning of the structure in the electron microscopy (EM) density for the S. flexneri NC supported models with 24-fold symmetry in the IMR. Both MxiG and MxiJ also have significant periplasmic domains, which were purified to homogeneity in this study, facilitating preliminary characterisation of their structures and intermolecular interactions. In addition, the entire IMR within the context of intact basal bodies was isolated and visualised in vitro by EM. The essential function of MxiG-N could be to localise the putative cytoplasmic ring (Cering) at the base of the T3SS. Although absolutely required for secretion, the Csring component, Spa33, was structurally uncharacterised. The crystal structure of the Cvterminal domain of Spa33 (Spa33-C) was determined in this study, showing an intertwined dimer that aligned with homologous structures and exhibited a novel interaction with the N-terminus of the ATPase regulator, MxiN. Subsequently, Spa33-C was identified as an altemative translation product of spa33 that formed a 2: 1 complex with Spa33 in vitro. This complex oligomerised further, demonstrating for the first time that Spa33 has the propensity to form the ordered, high molecular weight assemblies that would be required for C-ring formation in S. flexneri.

Genetic material of bacteria is subject to damage due to multitudinous factors, both extrinsic and intrinsic in origin. Mechanisms for the maintenance of genomic integrity are thus essential for a bacterium to survive. Bacterium also requires appropriate minor changes in the genetic material so as to adapt to the changing environments. Structural and related studies of two proteins from mycobacteria, one involved in recombinational DNA repair (RecA) and the other involved in SOS response which helps in adaptation to stress (LexA) form the subject matter of the thesis. The available literature on structural and related studies on RecA and LexA are reviewed in the introductory chapter. The action of RecA involves transition to an active filament formed in association with DNA and ATP, from an inactive filament in the absence of DNA. The structure of the inactive filament was first established in E. coli RecA (EcRecA). The interaction of RecA with non-hydrolysable ATP analogues and ADP were thoroughly characterised and the DNA binding loops were visualised in this laboratory using the crystal structures involving the proteins from Mycobacterium tuberculosis (MtRecA) and Mycobacterium smegmatis (MsRecA). A switch residue, which triggers the transformation of the information on ATP binding to the DNA binding regions, was identified. The 20 residue C-terminal stretch of RecA, which is disordered in all other relevant crystal structures, was defined in an MsRecA-dATP complex. The ordering of the stretch is accompanied by the generation of a new nucleotide binding site which can communicate with the original nucleotide binding site of an adjacent molecule in the filament. The plasticity of MsRecA and its mutants involving the switch residue was explored by studying crystals grown under different conditions at two different temperatures and, in one instance, at low humidity. The structures of these crystals and those of EcRecA and Deinococcus radiodurans RecA (DrRecA) provide information on correlated movements involving different regions of the molecule. MtRecA has an additional importance as an adjuvant drug target in Mycobacterium tuberculosis. Apart from recombination, another important property of RecA is its coprotease activity whereby it stimulates the inherent cleavage of a certain class of proteins. One of the substrates for the coprotease activity of RecA is LexA. LexA is a transcriptional repressor involved in SOS response in bacteria. LexA performs its function through an autoproteolysis stimulated by RecA, resulting in the derepression of the genes under its control. Structural studies on LexA from E. coli have shown that it has an N-terminal domain involved in binding to DNA and a C-terminal domain involved in catalysis and dimerisation. LexA mediated SOS response in bacteria has been shown in many cases to be responsible for the resistance gained by bacteria on treatment with antibiotics. In that respect, LexA is considered to be a potential drug target in Mycobacterium tuberculosis. Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions and reported in the thesis, provide insights into hitherto underappreciated details of molecular structure and plasticity (Chapter 2). In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP is central for all the biochemical activities of RecA. The strengths of interaction of the ligands with the P-loop reveal significant differences. This in turn affects the magnitude of the motion of the ‘switch’ residue, Gln195 in M. tuberculosis RecA, which triggers the transmission of ATP-mediated allosteric information to the DNA binding region. M. tuberculosis RecA is substantially rigid compared with its counterparts from M. smegmatis and E. coli, which exhibit concerted internal molecular mobility. Details of the interactions of ligands with the protein, characterised in the structures, could be useful for design of inhibitors against M. tuberculosis RecA. Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from M. tuberculosis, M. smegmatis and E. coli and their ATP complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural frame work within which RecA molecules from different eubacteria function (Chapter 3). Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins. As described in Chapter 4, full length LexA, the N-terminal and C-terminal segments defined by the cleavage site, two point mutants involving changes in active site residues (S160A and K197A) and another involving change at the cleavage site (G126D) were cloned, expressed and purified. The wild type protein cleaves at basic pH. The mutants do not autocleave at basic pH even after incubation for 12 hours. The wild type and the mutant protein dimerise and bind DNA with equal facility. The C-terminal segment also dimerises, but has a tendency to form tetramer as well. The full length proteins including the mutants and the C-terminal segment crystallised. The structure of the crystals obtained for mutant G126D could not be solved. Each of the other crystals, four in number, contained only the catalytic core and a few residues preceding it, indicating that the full length proteins underwent cleavage, at the canonical cleavage site or elsewhere, during the long period involved in the formation of the crystals. Crystals obtained from the solutions of the wild type protein and the C-terminal segment contains dimers of the catalytic core. Crystals obtained using the active site mutants appear to contain different type of tetramers. One of them involves the swapping of the peptide segment preceding the catalytic core. Models of tetramerisation of the full length protein similar to those observed for the catalytic core are feasible. A model of a complex of MtLexA with M. tuberculosis SOS box could be readily built. In this complex, the mutual orientation of the two N-domains of the dimer is different from that in the EcLexA-DNA complex.

The work presented in this thesis describes the determination of structures of peptides and proteins at atomic resolution. Nuclear Magnetic Resonance (NMR) spectroscopy was used as the principal method of investigation. The thesis is divided into three parts. Part I of the thesis consists of chapters 1 to 4, and deals with structural studies of two novel conotoxins. Part II of the thesis consists of chapter 5 and deals with structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2. Part III of the thesis consists of Appendices A to D. Appendix A describes implementation of a novel pulse sequence for determination of disulfide connectivity using long-range 13 C–13 C scalar couplings across disulfide bonds. Appendices B, C and D contain supplementary infor- mation (acquisition parameters and chemical shifts) for the structural studies presented in parts I and II of the thesis. Part I: Structural studies of novel conotoxins from Conus monile Chapter 1 gives a brief overview of the conotoxins and their structural studies. The first half of the chapter describes biosynthesis, classification schemes, nomenclature, com- monly observed post-translational modifications and applications of conotoxins. The latter half of this chapter summarizes the challenges involved in the structural studies of conotoxins in light of the recent developments in integrated transcriptomic and venomic studies of conotoxins. The key homonuclear and heteronuclear NMR experiments that are employed for structural studies of conotoxins are summarized. Emphasis was laid on describing the spectral features and the structural information that can be gleaned from these experiments. Finally, the current mass spectrometric and NMR methods available for determination of disulfide connectivity are discussed Chapter 2 describes sample preparation and preliminary biophysical characteriza- tion of a conotoxin Mo3964 that contains a hitherto uncharacterized cysteine framework (C–CC–C–C–C). The sequence of Mo3964 was identified at the nucleic acid level as a cDNA clone. Analysis of the signal sequence revealed that the toxin belongs to the M-superfamily, while the cysteine framework bears more resemblance to O- and K- super- family of conotoxins. Structural studies were initiated to determine the disulfide connec- tivity, tertiary structure and biological activity. The gene corresponding to the mature toxin sequence was cloned in a bacterial expression vector pET21a(+) as a C-terminal tag to the cytochrome b5 fusion protein host system. The fusion protein was obtained by recombinant expression using the bacterial expression host E. coli BL21(DE3) and the mature toxin was obtained by either enzymatic or chemical cleavage of the fusion protein followed by size exclusion chromatography and reverse phase HPLC. Proton 1D NMR spectra of the purified peptide exhibited sharp lines and good spec- tral dispersion indicating that molecule was well folded. Formation of disulfide bonds in the mature toxin was ascertained by high resolution mass spectra of intact and chemically modified Mo3964. The peptide toxin exhibited remarkable stability to chemical denatu- ration and proteolytic digestion. Spectroscopic studies clearly showed that Mo3964 pos- sesses a very stable and well defined structure as long as its disulfide bonds are intact. Analytical size exclusion chromatography and Multi Angle Light Scattering (MALS) studies showed that Mo3964 exists in solution as monomer albeit with a non-globular structure. Electrophysiological studies showed that Mo3964 inhibits outward potassium currents in rat Dorsal Root Ganglion (DRG) neurons and increases the reversal potential of rat voltage gated sodium channel rNav 1.2 stably expressed on Chinese Hamster Ovary (CHO) cells at peptide concentrations as low as 10 nM. Chapter 3 describes the determination of disulfide connectivity and tertiary stricture of Mo3964. Initial attempts to determine disulfide connectivity using direct fragmenta- tion of the intact peptide in the mass spectrometer failed due to the relatively large size of the molecule and its resistance to endoproteases. Partial reduction alkylation based methods failed as the first stage of partial reduction gave rise to a mixture of various single disulfide bond reduced species which could not be separated from each other. Subsequently, information about the disulfide connectivity was obtained using a method that does not necessitate separation of such a mixture of single disulfide bond reduced species. This method involves partial reduction, cyanylation of the reduced cysteines and alkali mediated cleavage of the peptide backbone on the N-terminus of cyanylated cysteines. Structural studies were carried out using homonuclear and heteronuclear NMR meth- ods. The hydrogen bond network and hence topology of the molecule was determined with high accuracy using the long-range HNCO-COSY experiment that correlates hydrogen- bond donor-acceptor pairs. This experiment utilizes the three bond heteronuclear scalar coupling, i.e., the h3JN C O′ coupling across the hydrogen bonds. All these restraints proved crucial to the assignment of the disulfide connectivity in Mo3964, given its novel cysteine framework. The structure of Mo3964 was calculated using a total of 549 NOE distance restraints, 84 dihedral angle restraints and 28 hydrogen bond distance restraints. The tertiary structure was constructed from the disulfide connectivity pattern 1–3, 2–5 and 4–6, that is hitherto undescribed for the M–superfamily conotoxins. The ensemble of structures showed a backbone Root Mean Square Deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (φ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The remarkable stability and anomalous spectral properties exhibited by Mo3964 could be rationalized using the disulfide connectivity and the tertiary structure. The tertiary structural fold has not been described for any of the known Conus peptides. Further, a search for structures similar to that of Mo3964 using the web server DALI returned no hits indicating that the peptide scaffold of Mo3964 has no structural homologues. Hence, the conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules. The structure of Mo3964 was submitted to the Protein Data Bank (PDB ID: 2MW7)[1]. Chapter 4 describes the structural studies of a 17 residue, single disulfide containing conopeptide Mo1853. The samples for structural studies were obtained either by chemical synthesis or by recombinant expression methods. Structural studies using homonuclear solution NMR methods revealed that Mo1853 exists as two equally populated cis and trans X–Pro conformers which are in slow exchange regime, compared to the chemical shift timescale. Sequence specific assignments were obtained for both the conformers by analysis of homonuclear 2D 1 H,1H–DQF–COSY,1H,1 H–TOCSY, 1H,1 H–NOESY and 1H,1 H–ROESY spectra. Temperature dependence of chemical shifts was measured and coalescence was observed for two amide protons at 318 K. At this temperature, the rate of exchange and the free energy of activation were determined to be 59 Hz and ≈ 67.2 kJ mol−1 respectively. The evidence for this conformational equilibrium was also observed as exchange correlation peaks in the 2D- NOESY and ROESY spectra. Tertiary structures of both the cis and trans conformers were determined using distance restraints, backbone dihedral angle restraints, the disulfide bond restraint and the cis or trans conformation of the X–Pro peptide bond. Tertiary structures of both the conformers consist of a 29-membered macro-cyclic ring formed by 9 amino acid residues which are cyclized by side chain to side chain disulfide bond. The conformation of the X–Pro peptide bond which is located within this macro-cyclic ring causes the cis structure to be compact and the trans structure to be in an extended form. Analysis of the tertiary structures indicated that the trans conformer is stabilized by hydrogen bonds while the cis conformer is likely to be stabilized by hydrophobic interactions. This was further corroborated by the fact that at lower temperatures, the hydrophobic interactions became weaker reducing the population of the cis conformer with respect to that of the trans conformer. Preliminary electrophysiological studies carried out on rat DRG neurons indicate that Mo1853 transiently reduces late outward potassium currents. Part II: Structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2 Chapter 5 presents the solution NMR studies of the Smr domain from MutS2 of H. pylori , henceforth called as HpSmr. In H. pylori , MutS2 is involved in suppression of homologous recombination and its Smr domain was shown to be necessary for this activity. As of date, in spite of the availability of structural information for the Smr domain, unambiguous identification of the residues involved in metal binding, DNA binding and catalysis remains elusive. Structural studies were carried out on two different constructs of HpSmr viz., HpSmr– (His)6 and GSHM–HpSmr, with and without the hexahistidine tag respectively. Se- quence specific assignments of HpSmr–(His)6 were obtained at two different sample pH conditions viz., pH 8.0 and pH 5.35 using the standard suite of triple resonance NMR experiments. Since, valines and leucines constitute about 25% of the total number of amino acid residues in HpSmr–(His)6 , stereospecific assignments were obtained for di- astereotopic methyl groups of these residues by preparing a fractionally 13C labeled sample of HpSmr–(His)6 . Solution structure of HpSmr–(His)6 at pH 8.0 was determined using 766 NOE restraints, 170 backbone dihedral angle restraints and 70 hydrogen bond distance restraints. The tertiary structure exhibits the canonical α/β sandwich fold ex- hibited by all the other known structures of Smr domains. Further, NMR studies and analytical gel filtration studies indicated the presence of pH dependent conformational exchange in HpSmr that involves strand to coil transition in the C-terminal β-strand. In order ascertain that the conformational equilibrium is not at an artifact caused by the C-terminal hexa-histidine-tag, HpSmr protein construct GSHM–HpSmr, which does not have the hexa-histidine-tag, was prepared. Conformational exchange was observed in this construct as well. The preliminary NMR evidence suggests that the conformational exchange is caused by pH dependent cis–trans isomerization of a semi-conserved Proline residue Pro66 . We have hypothesized that the pH dependent modulation of the activity of Smr domain of MutS2 can be advantageous to H. pylori . Such a regulation could help the bacteria to achieve optimal rate of homologous recombination in response to changes in pH, which is necessary for maintaining homeostasis and tiding over stress conditions. Part III: Appendix Appendix A describes an NMR pulse program LRCC_CH2 that was designed with the aim of determining disulfide connectivity using long-range 13C–13 C (C β –C β ′ ) couplings across the disulfide bond. This experiment is a modification of an earlier experiment pub- lished by Bax and co-workers designed to measure the side-chain χ3 dihedral angle in me- thionines. The experiment described here is optimized for the detection of 3 bond scalar coupled methylene carbons. The details of modifications introduced in LRCC_CH2, its product operator analysis, a representative spectrum acquired on [U-13C,15 N]–Mo3964, short-comings and future directions are described. The C programming code that was used to implement the pulse program is also included in the appendix. Appendices B, C and D contain the supplementary information (acquisition pa- rameters for the NMR experiments and chemical shifts) for the structural studies carried out on Mo3964, Mo1853 and HpSmr.