No description available.

The conformational energy differences have been determined for ethylene glycol, 2- chloroethanol, and 2,2- dichloroethanol in the neat liquid, DMSO, and H20 with Raman spectroscopy. Spectra in the 0-H valence region were utilized to determine the energy difference between interand intramolecularly hydrogen bonded species. It was found that the solvent effect on the relative stabilities of the gauche and trans rotamers of the alcohols differ significantly. The results also indicate that, unlike ethylene glycol, there is significant intramolecular hydrogen bond formation in the halogenated alcohols in the neat liquid phase. Stronger intramolecular hydrogen bond formation was observed in dichloroethanol than in 2-chloroethanol.

xiv, 82 p., ill. (some col.) / Cellular processes are carried out by proteins, which often utilize conformational changes for function. In theory, conformational changes can be harnessed to promote, prevent or monitor cellular processes. Such changes in protein active centers require perturbations through interactions with other proteins, small molecules or through energy input into the system, for example light. The work presented incorporates rational design and crystallographic elucidation of two-state conformational changes in two proteins, green fluorescent protein (GFP) and malate synthase (MS). GFP indicators were previously developed to quantitate the thiol/disulfide redox status within cells. Cysteine residues were introduced in close proximity on the surface of GFP and allow the formation of a disulfide bond. The indicators provide a fluorescent readout of the ambient thiol/disulfide equilibrium, however thermodynamic studies showed the resulting thiol/disulfide to be unusually stable (-287 mV) in comparison to the cellular redox buffer glutathione (-240 mV). In order to produce a family of redox indicators suitable for use in less reducing environments, amino acids were inserted near the introduced cysteine pair in order to destabilize the disulfide. The resulting family of redox indicators, termed roGFP-iX, exhibit midpoint potentials in the more desirable range of -229 to -246 mV. Crystallographic analysis indicates that roGFP-iX indicators undergo much larger two-state conformational changes than the original indicators. Surprisingly, a cis-peptide was discovered between the cysteine and the inserted residue which in combination with the conformational changes helps to explain the reduced stability of the disulfide. Malate synthase is an important virulence factor for certain microbes and carries out the Claisen condensation between glyoxylate and acctyl-CoA to produce malate. Crystal structures of Mycobacterium tuberculosis and Escherichia coli malate synthase isoform G had previously been determined with substrates or products bound. To determine the conformational changes necessary for substrate binding and product release, crystal structures of Escherichia coli malate synthase isoform A were determined in both the apo and acetyl-CoA/inhibitor bound forms. The crystallographic models revealed two-state conformational changes in the part of the active-site loop necessary for substrate binding, which has important implications for drug design. This dissertation includes my unpublished co-authored materials. / Adviser: S. James Remington

xviii, 724 p. : ill. (some col.) / Structural trends applicable to crystal engineering were studied in three classes of thiourea-based compounds. The aim of the study was to identify, predict, and ultimately design reliable single-molecule structural features, which could then be used to engineer crystals with desirable properties. In one class of compounds, this goal was achieved: N-alkyl and N-aryl derivatives of N,N'-bis(3-thioureidopropyl)piperazine adopted an identical conformation in the solid state, which resulted in near-identical crystal packing. A second class of closely related compounds, N-substituted tris(2-thioureidoethyl)amines, showed no such reliability in the solid state, likely because the parent structure lacked hydrogen-bonding functionalities sufficient to control intramolecular structure. In the third class of compounds that we studied, 1-benzoyl-3-(2-pyridyl)thioureas, substitution patterns were often predictive of molecular conformation; however, these intramolecular trends did not lead to recognizable crystal packing motifs. Nevertheless, certain physical properties observed in this last class of compounds--color, solubility, and often crystallinity--were conformer-specific, interestingly without any apparent relevance to crystal lattice structure. Solution-state and solid-state conformational trends in these 1-benzoyl-3-(2-pyridyl)thioureas have been documented, and speculations as to the source of color in one of the two observed conformations have been noted. / Committee in charge: Shih-Yuan Liu, Chairperson; Kenneth M. Doxsee, Advisor; Michael M. Haley, Member; Catherine J. Page, Member; Paul J. Wallace, Outside Member

abstract: Proteins are essential for most biological processes that constitute life. The function of a protein is encoded within its 3D folded structure, which is determined by its sequence of amino acids. A variation of a single nucleotide in the DNA during transcription (nSNV) can alter the amino acid sequence (i.e., a mutation in the protein sequence), which can adversely impact protein function and sometimes cause disease. These mutations are the most prevalent form of variations in humans, and each individual genome harbors tens of thousands of nSNVs that can be benign (neutral) or lead to disease. The primary way to assess the impact of nSNVs on function is through evolutionary approaches based on positional amino acid conservation. These approaches are largely inadequate in the regime where positions evolve at a fast rate. We developed a method called dynamic flexibility index (DFI) that measures site-specific conformational dynamics of a protein, which is paramount in exploring mechanisms of the impact of nSNVs on function. In this thesis, we demonstrate that DFI can distinguish the disease-associated and neutral nSNVs, particularly for fast evolving positions where evolutionary approaches lack predictive power. We also describe an additional dynamics-based metric, dynamic coupling index (DCI), which measures the dynamic allosteric residue coupling of distal sites on the protein with the functionally critical (i.e., active) sites. Through DCI, we analyzed 200 disease mutations of a specific enzyme called GCase, and a proteome-wide analysis of 75 human enzymes containing 323 neutral and 362 disease mutations. In both cases we observed that sites with high dynamic allosteric residue coupling with the functional sites (i.e., DARC spots) have an increased susceptibility to harboring disease nSNVs. Overall, our comprehensive proteome-wide analysis suggests that incorporating these novel position-specific conformational dynamics based metrics into genomics can complement current approaches to increase the accuracy of diagnosing disease nSNVs. Furthermore, they provide mechanistic insights about disease development. Lastly, we introduce a new, purely sequence-based model that can estimate the dynamics profile of a protein by only utilizing coevolution information, eliminating the requirement of the 3D structure for determining dynamics. / Dissertation/Thesis / Doctoral Dissertation Physics 2016

Made available in DSpace on 2015-05-14T13:21:23Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2208268 bytes, checksum: a713e84bf72b1325038dab0816e190d4 (MD5) Previous issue date: 2009-11-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In this work the origin of the π−π-staking conformation stability of 5S in the 8-acrylate phenylmenthol 5 was revisiting. The geometries of 5S and 5T conformers were optimized from HF method and the MPW1B95 functional; and the geometries of the 11S and 11T conformers have been optimized from HF, MP2 methods and MPW1B95 functional. All calculations were performed on 6-311 + + G (2d, 2p) 6d as level of calculation. Using the optimized geometries of 5S, 5T, 11S and 11T (from MPW1B95/6-311 + + G (2d, 2p) 6d), were performed 15 energy single-point calculations, investigating various computational methods (ab initio, DFT and semiempirical). We also described the spectroscopic (1H NMR) and theoretical (DFT and ab initio) studies of 3-naphytylpropylcrotonate (12) and 3- naphytylpropylsorbate (21). The geometries of 12S and 12T conformers have been optimized from MP2, HF methods and B3LYP, LSDA and MPW1B95 functionals using 6-311+G(d,p) as calculation level. Finally, we describe the synthesis of 12 and 21 (the unpublished) in 3 steps of each synthesis (19% and 22% respectively of total yields) and the trans geometries of 12T and 21T were characterized as the most stable in the conformational equilibrium of 12 and 21. / Neste trabalho a origem do motivo para a maior estabilidade da conformação de 5S no acrilato de 8-fenilmentol 5 foi investigada. As geometrias dos confôrmeros 5S e 5T foram otimizadas nos métodos HF e no funcional MPW1B95; e as geometrias dos confôrmeros 11S e 11T foram otimizadas nos métodos HF, MP2 e o funcional MPW1B95. Todos os cálculos usaram 6-311++G(2d,2p)6d como nível de cálculo. Usando as geometrias otimizadas de 5S, 5T, 11S e 11T (por MPW1B95/6-311++G(2d,2p)6d), foram efetuados 15 cálculos de energia no ponto, investigando metodologias computacionais variadas (ab initio, DFT e semiempirico). Descrevemos também o estudo espectroscópico (RMN 1H) e teórico (DFT e ab initio) do crotonato de 3-naftilpropila (12) e o sorbato de 3-naftilpropila (21). As geometrias dos confôrmeros de 12S e 12T foram otimizadas com os métodos MP2, HF e os Funcionais B3LYP, LSDA e MPW1B95 usando 6- 311+G(d,p) como nível de cálculo. Finalmente, descrevemos a síntese total de 12 e 21 (inéditas na literatura) em 3 etapas de síntese cada (19% e 22% respectivamente de rendimentos globais) e caracterizamos por 1H RMN a geometria trans 12T e 21T como a mais estável no equilíbrio conformacional de 12 e 21.

abstract: This thesis explores a wide array of topics related to the protein folding problem, ranging from the folding mechanism, ab initio structure prediction and protein design, to the mechanism of protein functional evolution, using multi-scale approaches. To investigate the role of native topology on folding mechanism, the native topology is dissected into non-local and local contacts. The number of non-local contacts and non-local contact orders are both negatively correlated with folding rates, suggesting that the non-local contacts dominate the barrier-crossing process. However, local contact orders show positive correlation with folding rates, indicating the role of a diffusive search in the denatured basin. Additionally, the folding rate distribution of E. coli and Yeast proteomes are predicted from native topology. The distribution is fitted well by a diffusion-drift population model and also directly compared with experimentally measured half life. The results indicate that proteome folding kinetics is limited by protein half life. The crucial role of local contacts in protein folding is further explored by the simulations of WW domains using Zipping and Assembly Method. The correct formation of N-terminal β-turn turns out important for the folding of WW domains. A classification model based on contact probabilities of five critical local contacts is constructed to predict the foldability of WW domains with 81% accuracy. By introducing mutations to stabilize those critical local contacts, a new protein design approach is developed to re-design the unfoldable WW domains and make them foldable. After folding, proteins exhibit inherent conformational dynamics to be functional. Using molecular dynamics simulations in conjunction with Perturbation Response Scanning, it is demonstrated that the divergence of functions can occur through the modification of conformational dynamics within existing fold for β-lactmases and GFP-like proteins: i) the modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate, while the resurrected ancient lactamases indicate enhanced active-site flexibility, which likely allows for the binding and subsequent degradation of different antibiotic molecules; ii) the chromophore and attached peptides of photocoversion-competent GFP-like protein exhibits higher flexibility than the photocoversion-incompetent one, consistent with the evolution of photocoversion capacity. / Dissertation/Thesis / Ph.D. Physics 2014

A influência da formação de ligações de hidrogênio (LHs) em sistemas moleculares é investigada analisando-se as propriedades de aglomerados próton-ligados e de moléculas em solventes próticos. As estruturas supermoleculares dos aglomerados são obtidas por otimização de geometria com diferentes métodos quânticos ab initio. Por sua vez, as estruturas supermoleculares dos solventes dependem de condições termodinâmicas e, portanto, são obtidas utilizando-se técnicas de simulação Monte Carlo, em que as interações intermoleculares são parametrizadas pelos potenciais de Lennard-Jones e de Coulomb. Os efeitos decorrentes da formação de LHs nos aglomerados moleculares são analisados a partir de suas prioridades: estabilidades, espectros vibracionais, constante rotacionais e dipolos elétricos. Estas propriedades são calculadas usando-se métodos perturbativos (MBPT/CC) e teoria do funcional da densidade(DFT). Para as moléculas solvatadas em meio líquido, os efeitos das LHs são analisadas na interação soluto-solvente duranto o processo de equilíbrio conformacional de fuufural em solventes próticos. A contribuição da interação do soluto com o meio também é investigada no espectro de absorção UV-vis do sistema furfural/água. Os resultados destes estudos mostram que a formação de LHs é importante para explicar o comportamento de algumas propriedades de sistemas moleculares próton-ligados, principalmente de aglomerados. No caso de sistemas em fase líquida, as LHs dependem, basicamente, do caráter prótico e da polaridade do solvente, propriedades que concorrem para a estabilização de possíveis formas conformacionais da molécula do soluto. / The influence of hydrogen bond formation in molecular systems is investigated analyzing the properties of both hydrogen-bonded clusters and molecules in protic solvents. The super-molecular structures of the cluster are obtained by optimizing the geometries with different ab initio quantum methods. On the other hand, the super-molecular structures of the solvents depend on thermodynamic conditions and, so, are obtained by using Monte Carlo simulation techniques, where the intermolecular interactions are accounted for the Lennard-Jones and Coulomb pair-potentials. The effects due to H-bond formation in the molecular clusters are analyzed from their properties: stabilities, vibrational spectra, rotational constants, and electric dipoles. These properties are calculated using perturbation methods (MBPT/CC) and density functional theory (DFT). For the molecules in liquid solvents, the H-bond effects are analyzed in the solute-solvent interaction during the conformational equilibrium process of furfural in protic solvents. The contribution of the interaction of the solute with the medium is also investigated in the UV-vis absorption spectrum of the furfural/water system. The results from these studies show that the H-bonding process is important to account for the behavior of some properties of H-bonded molecular systems, mainly of clusters. In the case of liquid-phase systems, the H-bonds depend basically on both the protic nature and polarity of the solvent, properties that come together to stabilize possible conformational forms of the solute molecules.

A presente tese relata a síntese e a análise conformacional das: N-metóxi-N-metil- 2-feniltiopropanamidas 4\'-substituídas (I), N-metóxi-N-metil-2- fenilssulfinilpropanamidas 4\'-substituídas (II) e N-metóxi-N-metil-2- fenilssulfonilpropanamidas 4\'-substituídas (III), através da espectroscopia no Infravermelho na região da banda da carbonila (vco), apoiada por cálculos teóricos. A análise conformacional de (I) indicou, através de dados de DFT/cc-pVDZ a existência de um equilíbrio entre duas conformações gauche devido à ocorrência de uma forte interação orbitalar estabilizante π*co/σc-s nas mesmas. A série dos derivados sulfinilados (II) apresentou-se como um par de diastereoisômeros. A análise conformacional para o diast1 (par enantiomérico CRSS/CSSR), através do cálculo HF/6-31G** e IV, indicou a existência de um equilíbrio conformacional entre dois confôrmeros quasi-cis (q-c1 e q-c2) e outro gauche (g), sendo os confôrmeros quasi-cis (q-c1 e q-c2) mais estáveis devido à interação de transferência de carga Oσ-(CO)→Sσ+SO mais intensa do que a interação Oσ-(SO)→Cσ+CO presente no confôrmero gauche (g). A análise para o diast2 utilizando-se o cálculo HF/6-31G** indicou a presença dos confôrmeros quasi-gauche (q-g), e quasi-cis (q-c3) sendo o primeiro mais estável no estado gasoso por apresentar uma interação transferência de carga cruzada Oσ-(CO)→Sσ+SO e Oσ-(SO)→Cσ+CO. Os dados de cálculo HF/6-31G** para os derivados sulfonilados (III) indicaram um equilíbrio conformacional entre duas conformações gauche (g1 e g2) decorrente de uma interação Coulômbica e de transferência de carga cruzada Oδ-(SO2)→Cδ+CO e Oδ-(CO)→Sδ+SO2. A presente Tese também relata a obtenção de compostos ß-lactâmicos acilados no carbono C3 e C4. As N-metóxi-N-metil-2-feniltiopropanamidas-4\'-substituídas (I) agem como acilantes do anel ß-lactâmico (N-fenil-2-azetidinona) para obtenção dos derivados ß-lactâmico C3 acilados. A reação foi realizada com sucesso, contudo, a purificação dos compostos alvo não se mostrou eficaz obtendo-se apenas as 3-[2- (4\'-feniltio)propanoil]-N-fenil- e 3-[2-(4\'- clorofeniltio)propanoil]-N-fenil-2-azetidinonas através de um processo de cristalização. Para a obtenção dos derivados ß-lactâmicos C4 acilados utilizou-se a N-metóxi-N-metil-4-oxo-1-fenilazetidina-2- carboxamida como acilante dos carbânions derivados de 4\'-fenilmetilssulfóxidos [4\'-YPhS(O)CH3 Y= OMe, Me, H]. Esta reação forneceu as 1-fenil- (4-fenilssulfinilacetil- 3 4\'-substituídos)-2-azetidinonas correspondentes com bons rendimentos. A redução destas azetidinonas-sulfóxidos forneceu as azetidinonas-sulfetos correspondentes também com bons rendimentos / This thesis reports the synthesis and the conformational analysis of: N-methoxy-N- methyl-2-[(4\'-substituted)-phenylthio]-propanamides (I), N-methoxy-N-methyl-2-[(4\'- substituted)-phenylsulfinyl]-propanamides (II) and N-methoxy- N-methyl-2-[(4\'- substituted)-phenylsulfonyl]-propanamides (III) by Infrared spectroscopy through the analysis of the carbonyl stretching band supported by theoretical calculations. The conformational analysis of (I) through DFT computations have indicated the equilibrium of two gauche conformations due to a strong stabilizing π*co/σc-s orbital interaction. The group of sulfinyl derivatives (II) exist as a diastereomeric pair. The conformational analysis of diast1 (enantiomeric pair CRSS/CSSR) through IR and HF/6-31G** has shown the existence of an equilibrium between two quasi-cis (q-c1 and q-c2) and one gauche (g) conformers, being the quasi-cis conformers the more stable ones due to the Oσ-(CO)→Sσ+SO charge transfer interaction which in turn is stronger than the Oσ-(SO)→Cσ+CO orbital interaction which takes place in the gauche (g) conformer. As for diast2 the HF/6-31G** computations along IR data has indicated 2 the occurrence of the quasi-gauche (q-g) and quasi-cis (q-c ) conformers, being the former the more stable one in gaseous phase due to the Oσ-(CO)→Sσ+SO e Oσ-(SO)→Sσ+CO crossed charge transfer. The HF/6-31G** and vco data for the sulfonylated derivatives (III) have shown the existence of the equilibrium between two gauche (g1 and g2) conformations which are stabilized through Oδ-(SO2)→Cδ+CO and Oδ-(CO)→Sδ+SO2 electrostatic and charge transfer interactions. This Thesis also deals with the preparation of some C3 and C4 acylated ß-lactamic compounds. The N-methoxy- N-methyl-2-[(4\'-substituted)-phenylthio-propanamides (I) act as acylating agents of the ß-lactamic ring (N-phenyl-2-azetidinone) in order to obtain the C-3 acylated ß-lactamic derivatives. These reactions gave the target products contaminated with starting material in good yields except for the 3-[2- (4\'phenylthio)propanoyl]-N-phenyl- and 3-[2-(4\'-chlorophenylthio)propanoyl]-N- phenyl-2-azetidinones which were obtained in pure form after crystallization. As for the C-3 acylated ß-lactamic derivatives they were obtained from the reaction of N-methoxy-N-methyl-4-oxo-1-phenylazetidine-2-carboxamide and the appropriate carbanions of 4\'-substituted phenylmethylsulfoxides. This reaction gave the corresponding 1-phenyl- (4-substituted-phenylsulfinylacetyl)-2-azetidinones in good yields. The corresponding azetidinone-sulfides were obtained from the reduction of the azetidinone-sulfoxides.

Orientador: Iris Concepcion Kinares de Torriani / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-08T07:14:14Z (GMT). No. of bitstreams: 1 Silva_JulioCesarda_M.pdf: 3602925 bytes, checksum: ebc541d863211845fb7a5a80d72ed5b5 (MD5) Previous issue date: 2007 / Resumo: Durante as últimas décadas, o estudo de mudanças conformacionais de macromoléculas biológicas tem se tornado um grande desafio para os cientistas, além de ser um tema de interesse biotecnológico e de engenharia de proteínas. O processo de enovelamento (desenovelamento) de proteínas tem sido intensivamente estudado, pois isso pode contribuir para o conhecimento do processo de síntese de proteínas, além de ajudar a entender o desenvolvimento de algumas doenças associadas ao mau enovelamento ou agregação de certas proteínas. Nesse contexto, o Espalhamento de Raios-X a Baixo Ângulo (SAXS) aparece como uma técnica valiosa para esse estudo, pois ela permite obter informações estruturais da molécula em solução. Além de permitir estudos dinâmicos, as experiências de SAXS possibilitam a observação das moléculas em condições fisiológicas. Neste trabalho, a potencialidade da técnica de SAXS foi evidenciada no estudo de mudanças conformacionais de biomoléculas. O processo de desnaturação da proteína lisozima em solução foi estudado através de experiências em equilíbrio. Mudanças conformacionais foram observadas durante o processo de desnaturação por ação de uréia na solução e por altas temperaturas. Os resultados mostraram que a lisozima é uma proteína com certa resistência para se desenovelar completamente, mesmo em condições extremas de concentração de uréia e de altas temperaturas. A molécula tende a não perder totalmente sua compacidade. Além disso, foram observados somente dois estados conformacionais (enovelado e desenovelado). Um estado intermediário reportado na literatura, mas contestado por vários autores, não foi observado. Isso mostra a alta cooperatividade dessa proteína no processo de desnaturação. Outro processo estudado foi a oligomerização da proteína -Lactoglobulina sob ação de irradiação com radiação gama. A proteína foi estudada na forma sólida, com diferentes atividades de água, e em solução, em diferentes concentrações. As amostras foram irradiadas com radiação gama em diferentes doses e as mudanças foram registradas através de experiências de SAXS. Os dados experimentais foram usados para o cálculo de modelos dos oligômeros formados por ação da radiação. Concluindo, este estudo mostrou que a técnica de SAXS é uma ferramenta versátil e muito útil para o estudo de processos de mudanças nas estruturas terciária e quaternária de proteínas em solução / Abstract: During the last decades, the study of conformational changes in biological macromolecules has been a great challenge for the scientists, and continues to be an important subject of biotechnological interest and protein engineering. The process of folding (unfolding) of protein molecules has been intensively studied, because this investigation can contribute to the knowledge of the process of protein synthesis, thus helping to understand the development of some illnesses associated with misfolding or aggregation processes of certain proteins. In this context, the technique of Small Angle X-ray Scattering (SAXS) appears as a valuable technique, because it provides structural information of the molecules in solution. This technique allows dynamical studies and makes possible the study of the protein in physiological conditions. In this work the potentiality of the SAXS technique was evidenced in the study of conformational changes of biological molecules. The process of denaturation of the protein lysozyme in solution was studied using SAXS measurements in equilibrium conditions. Conformational changes were observed during the process of denaturation by the action of urea in the solution and for high temperatures. The results showed that lysozyme is a protein with certain resistance to unfold completely. Even in extreme conditions of high concentration of urea and high temperatures, this protein does not totally lose its compactness. Moreover, only two conformational states (folded and unfolded) were observed. An intermediate state was not observed. This study showed the high cooperativity of the unfolding process of this protein during its denaturation process. Another process studied was the oligomerization of the protein -Lactoglobulin under the effect of gamma irradiation. The protein was studied in the solid form, in different water activities, and in solution, in different concentrations. The samples were exposed to several doses of -radiation. The SAXS technique was used to obtain dimensional parameters of the proteins and models were calculated from the experimental scattering data. Finally, this study showed that the SAXS technique as a versatile and very useful tool for the study of changes in the tertiary and quaternary structures of proteins in solution / Mestrado / Física / Mestre em Física