Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-16Bitstream added on 2014-06-13T18:29:23Z : No. of bitstreams: 1 ozatojunior_t_me_sjrp.pdf: 797973 bytes, checksum: 60aec21696ccdd3f5f3f798127a445e0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho consistiu no seqüenciamento e caracterização molecular das Cadeias Abertas de Leitura (Open Reading Frames - ORFs) 2 e 3 do genoma do isolado São Paulo do Southern bean mosaic virus (SBMV-SP), completando-se o seqüenciamento de todo o genoma desse isolado. A ORF 2 codifica uma poliproteína (serino protease – VPg – RNA polimerase RNA-dependente) e a ORF 3 um produto com função desconhecida. O seqüenciamento da ORF 2 apresentou 2889 nucleotídeos, incluindo-se o códon de terminação UGA, com 962 aminoácidos deduzidos e massa molecular estimada de aproximadamente 105 kDa. Dentro da ORF 2, localiza-se a ORF 3 contendo 398 nucleotídeos, incluindo-se o códon de terminação UAA, com 132 aminoácidos e massa molecular estimada de aproximadamente 15 KDa. A análise feita a partir das seqüências das ORFS 2 e 3 do genoma do SBMV-SP, quando comparadas com outras espécies do mesmo gênero e isolados do SBMV, depositadas no GenBank, mostrou que a ORF 2 apresenta maior identidade (91,4% na seqüência de nucleotídeos e 95,0% na seqüência de aminoácidos deduzidos) com o isolado de Arkansas. Resultado similar foi obtido em relação à ORF 3 com valores de identidade de 97,0% tanto para as seqüências de nucleotídeos e aminoácidos deduzidos. Dados de filogenia corroboram os dados de identidade. Regiões conservadas do gênero Sobemovirus também foram identificadas, tais como Sítio de Ligação à Capa Protéica (CBPS), a tríade catalítica da serino protease (H-D-S) e a seqüência de heptanucleotídeos (TTTAAAC). A expressão em Escherichia coli da porção C-terminal da RNA Polimerase RNA Dependente (RpRd) produziu uma proteína de fusão de aproximadamente 67 kDa no sistema pMAL c2-x e de 30 kDa no sistema pET 28a. Quando a proteína de fusão foi injetada em coelhos houve a produção de anti-soro específico para a proteína recombinante. / The present work consisted of the sequencing and molecular characterization of the Open Reading Frames (ORFs) 2 and 3 from the São Paulo isolate genome of Southern bean mosaic virus (SBMV-SP), completing the sequencing of all the genome of this isolate. The ORF 2 encodes a polyprotein (serine protease - VPg - RNA dependent RNA polimarase) and the ORF 3 one product with unknown function. The sequencing of the ORF 2 reveals 2889 nucleotides, including the stop codon (UGA), with 962 deduced amino acids e and estimated molecular weight of approximately 105 kDa. Nested in the ORF 2 were found the ORF 3 with 398 nucleotides, including the stop codon (UAA), with 132 deduced amino acids and estimated molecular weight of approximately 15 kDa. The analysis made from the sequences of the ORFs 2 and 3 from the SBMV-SP genome, when compared with other species of the same gender and isolates of SBMV, deposited in the GenBank, showed that the ORF 2 presents higher identity (91,4% in the nucleotide sequence and 95,0% in the deduced amino acids sequence) with Arkansas isolate. Similar result was obtained in relation to the ORF 3 with identity values of 97,0% for the nucleotides and deduced amino acids sequences. Phylogeny data corroborate the identity data. Conserved regions of the Sobemovirus gender had also been identified such as Coat Protein Binding Site (CPBS), serine protease catalytic triad (H-D-S) and heptanucleotide sequence (TTTAAAC). The Expression in Escherichia coli of the C-terminal region of the RNA dependent RNA polimerase produced a fusion protein of approximately 67 kDa in pMAL c2-x system and a 30 kDa protein in pET 28a system. When the fusion protein ...(Complete abstract click electronic access below)

Orientador: José Osmar Gaspar / Banca: Mário Tyago Murakami / Banca: Marinônio Lopes Cornélio / Resumo: No Brasil, foram descritas mais de dez viroses em feijoeiro, citando-se aquela causada pelo Southern bean mosaic virus (SBMV) do gênero Sobemovirus. Este vírus tem uma restrita gama de hospedeiras, confinada quase exclusivamente a espécies da família das leguminosas, sendo algumas de interesse econômico como o feijoeiro comum e a soja. O SBMV é a espécie tipo do gênero, possui partículas isométricas (28-30nm) contendo RNA genômico de 4-4,5kb com polaridade positiva e proteína capsidial com massa molecular de 29-30 kDa. O genoma do SBMV é constituído por quatro Open Reading Frames (ORFs) com sobreposição entre elas. A ORF 1 codifica uma possível proteína do movimento, a ORF 2 uma poliproteína, que por clivagem, origina três produtos funcionais: uma serino protease, uma proteína ligadora do genoma viral (VPg) e uma polimerase com atividade relacionada à replicação do RNA viral (RNA polimerase RNA-dependente, RpRd). Dentro da ORF 2 encontra-se a ORF 3, que codifica uma proteína de função desconhecida. A ORF 4 codifica a proteína capsidial, traduzida a partir de um RNA subgenômico. O presente trabalho consiste na expressão da proteína P1 do SBMV-SP em Escherichia coli, sua purificação e a posterior utilização para a produção de antissoro policlonal. As atividades realizadas compreenderam a purificação das partículas virais do SBMV isolado São Paulo (SBMV-SP) a partir de folhas infectadas de Phaseolus vulgaris 'Jalo', obtenção do RNA viral a partir de partículas purificadas, produção de cDNA utilizando primer anti-senso, amplificação do gene da proteína do movimento, purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, corte enzimático com as enzimas Bam HI e Hind III, subclonagem nos vetores de expressão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In Brazil, more than ten bean viroses were described, including the one caused by Southern bean mosaic virus (SBMV) from genus Sobemovirus. This virus has a strict host range, confined almost exclusively to species from leguminosas family, and some of them have economic importance, like the common bean and soya. SBMV is the type species from this genus, has isometric particles (28-30nm) that contains genomic RNA of 4-4,5kb with positive polarity and a capsidal protein with molecular mass of 29-30kDa. SBMV genome consistis of four Open Reading Frames (ORFs) with overlap between them. ORF 1 encodes a possible movement protein, ORF 2 a poliprotein, which origin three functional products by clivage: a serin protease, a viral genomic biding protein (VPg) and a polimerase. Inside ORF 2 is situated the ORF 3, that encodes a protein with unknow function. ORF 4 encodes the capsidal protein, translated by a subgenomic RNA. The activities performed until this moment were the purification of SBMV isolated São Paulo(SBMV-SP) viral particles, obtention of viral RNA from SBMV purified partcles, cDNA production using anti-sense primer, amplification of the supposed gene of movement protein, purification of the amplified fragment and later biding on expression vector pGEM-T. The recombinat vector were transformed in Escherichia coli cells competent and than purified and digested with Bam HI and Hind III enzime. The released fragment was subcloned in pMAL and pET28a, which were used on competent cells for expression tests. Analysis of gel poliacrialamida showed the efficiency of tests of expression. Using pET28a was observed an expression of a protein of approximately 22 kDa (17 kDa relating to MP + 5 kDa relating to the tail of histidine) which was purified in resin Ni-NTA by affinitive chromatography.Using pMAL was observed an expression of a protein of approximately... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-22Bitstream added on 2014-06-13T19:55:51Z : No. of bitstreams: 1 mariutti_rb_me_sjrp.pdf: 594995 bytes, checksum: 89516139d0654c33b1221cfd31a27c6d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / No Brasil, foram descritas mais de dez viroses em feijoeiro, citando-se aquela causada pelo Southern bean mosaic virus (SBMV) do gênero Sobemovirus. Este vírus tem uma restrita gama de hospedeiras, confinada quase exclusivamente a espécies da família das leguminosas, sendo algumas de interesse econômico como o feijoeiro comum e a soja. O SBMV é a espécie tipo do gênero, possui partículas isométricas (28-30nm) contendo RNA genômico de 4-4,5kb com polaridade positiva e proteína capsidial com massa molecular de 29-30 kDa. O genoma do SBMV é constituído por quatro Open Reading Frames (ORFs) com sobreposição entre elas. A ORF 1 codifica uma possível proteína do movimento, a ORF 2 uma poliproteína, que por clivagem, origina três produtos funcionais: uma serino protease, uma proteína ligadora do genoma viral (VPg) e uma polimerase com atividade relacionada à replicação do RNA viral (RNA polimerase RNA-dependente, RpRd). Dentro da ORF 2 encontra-se a ORF 3, que codifica uma proteína de função desconhecida. A ORF 4 codifica a proteína capsidial, traduzida a partir de um RNA subgenômico. O presente trabalho consiste na expressão da proteína P1 do SBMV-SP em Escherichia coli, sua purificação e a posterior utilização para a produção de antissoro policlonal. As atividades realizadas compreenderam a purificação das partículas virais do SBMV isolado São Paulo (SBMV-SP) a partir de folhas infectadas de Phaseolus vulgaris ‘Jalo’, obtenção do RNA viral a partir de partículas purificadas, produção de cDNA utilizando primer anti-senso, amplificação do gene da proteína do movimento, purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, corte enzimático com as enzimas Bam HI e Hind III, subclonagem nos vetores de expressão... / In Brazil, more than ten bean viroses were described, including the one caused by Southern bean mosaic virus (SBMV) from genus Sobemovirus. This virus has a strict host range, confined almost exclusively to species from leguminosas family, and some of them have economic importance, like the common bean and soya. SBMV is the type species from this genus, has isometric particles (28-30nm) that contains genomic RNA of 4-4,5kb with positive polarity and a capsidal protein with molecular mass of 29-30kDa. SBMV genome consistis of four Open Reading Frames (ORFs) with overlap between them. ORF 1 encodes a possible movement protein, ORF 2 a poliprotein, which origin three functional products by clivage: a serin protease, a viral genomic biding protein (VPg) and a polimerase. Inside ORF 2 is situated the ORF 3, that encodes a protein with unknow function. ORF 4 encodes the capsidal protein, translated by a subgenomic RNA. The activities performed until this moment were the purification of SBMV isolated São Paulo(SBMV-SP) viral particles, obtention of viral RNA from SBMV purified partcles, cDNA production using anti-sense primer, amplification of the supposed gene of movement protein, purification of the amplified fragment and later biding on expression vector pGEM-T. The recombinat vector were transformed in Escherichia coli cells competent and than purified and digested with Bam HI and Hind III enzime. The released fragment was subcloned in pMAL and pET28a, which were used on competent cells for expression tests. Analysis of gel poliacrialamida showed the efficiency of tests of expression. Using pET28a was observed an expression of a protein of approximately 22 kDa (17 kDa relating to MP + 5 kDa relating to the tail of histidine) which was purified in resin Ni-NTA by affinitive chromatography.Using pMAL was observed an expression of a protein of approximately... (Complete abstract click electronic access below)

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

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.

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