Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references. Also available in electronic version.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 154-168).

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

abstract: Protein folding is essential in all cells, and misfolded proteins cause many diseases. In the Gram-negative bacterium Escherichia coli, protein folding must be carefully controlled during envelope biogenesis to maintain an effective permeability barrier between the cell and its environment. This study explores the relationship between envelope biogenesis and cell stress, and the return to homeostasis during envelope stress. A major player in envelope biogenesis and stress response is the periplasmic protease DegP. Work presented here explores the growth phenotypes of cells lacking degP, including temperature sensitivity and lowered cell viability. Intriguingly, these cells also accumulate novel cytosolic proteins in their envelope not present in wild-type. Association of novel proteins was found to be growth time- and temperature-dependent, and was reversible, suggesting a dynamic nature of the envelope stress response. Two-dimensional gel electrophoresis of envelopes followed by mass spectrometry identified numerous cytoplasmic proteins, including the elongation factor/chaperone TufA, illuminating a novel cytoplasmic response to envelope stress. A suppressor of temperature sensitivity was characterized which corrects the defect caused by the lack of degP. Through random Tn10 insertion analysis, aribitrarily-primed polymerase chain reaction and three-factor cross, the suppressor was identified as a novel duplication-truncation of rpoE, here called rpoE'. rpoE' serves to subtly increase RpoE levels in the cell, resulting in a slight elevation of the SigmaE stress response. It does so without significantly affecting steady-state levels of outer membrane proteins, but rather by increasing proteolysis in the envelope independently of DegP. A multicopy suppressor of temperature sensitivity in strains lacking degP and expressing mutant OmpC proteins, yfgC, was characterized. Bioinformatics suggests that YfgC is a metalloprotease, and mutation of conserved domains resulted in mislocalization of the protein. yfgC-null mutants displayed additive antibiotic sensitivity and growth defects when combined with null mutation in another periplasmic chaperone, surA, suggesting that the two act in separate pathways during envelope biogenesis. Overexpression of YfgC6his altered steady-state levels of mutant OmpC in the envelope, showing a direct relationship between it and a major constituent of the envelope. Curiously, purified YfgC6his showed an increased propensity for crosslinking in mutant, but not in a wild-type, OmpC background. / Dissertation/Thesis / Ph.D. Microbiology 2010

Le but de cette thèse est d’étudier profondément le repliement des protéines, au moyendes concepts d’invariance de jauge et d’universalité. La structure de jauge émerge del’équation de Frenet qui est utilisée pour décrire la forme de la chaîne principale de laprotéine. Le principe d’invariance de jauge conduit à une fonctionnelle d’énergieeffective pour une protéine, développée dans le but d’extraire les propriétésuniverselles des protéines repliées durant la phase d’effondrement, et qui estcaractérisée par la loi d’échelle du rayon de giration au niveau tertiaire de la structureprotéique. Dans cette thèse, on étudie l’existence d’une large universalité au niveausecondaire de la structure protéique. La fonctionnelle d’énergie invariante de jaugealliée à l’équation de Frenet discrète conduit à une solution solitonique, identifiéecomme un motif hélice-boucle-hélice dans la protéine. / The purpose of this thesis is to investigate protein folding, by means of the general concepts of gauge invariance and universality. The gauge structure emerges in the Frenet equation which is utilized to describe the shape of protein backbone. The gauge invariance principle leads us an effective energy functional for a protein, which bas been found to catch the universal properties of folded proteins in their collapse phase,characterized by the scaling law of gyration radius on the tertiary level of proteinstructure. In this thesis, the existence of wide universality on the secondary level of protein structure is investigated. The synthesis of the gauge-invariant energy functional with the discrete Frenet equation leads to a soliton solution, which is identified as the helix-loop-helix motif in protein.

Made available in DSpace on 2014-06-11T19:22:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-25Bitstream added on 2014-06-13T20:10:05Z : No. of bitstreams: 1 contessoto_vg_me_sjrp.pdf: 418254 bytes, checksum: 47617fd8d1433490e1d604852d699c37 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No processo de enovelamento as proteínas seguem o principio de “mínima frustração”, garantindo ao estado nativo o seu mínimo global de energia e a sua estabilidade termodinâmica. Apesar de, a proteína estar no seu estado nativo, esta condição não impede o surgimento de algumas interações energeticamente desfavoráveis, caracterizando a frustração no estado nativo. Há evidências indicando que um pequeno grau de frustração pode favorecer o processo de enovelamento. Neste estudo são investigadas as condições em que a presença de frustração, é ou não, favorável ao processo de enovelamento. São realizadas simulações computacionais de dinâmica molecular utilizando o modelo Cα (um modelo baseado em estrutura e sem frustração). É adicionado um termo ao potencial do modelo associado à presença de frustração. As simulações do modelo Cα são realizadas para um grupo de proteína com características distintas. É introduzido um critério determinando os casos em que a frustração auxilia o processo de enovelamento. São observados os efeitos da presença de frustração ao longo do processo de enovelamento e sua influência na alteração da temperatura e do tempo de enovelamento. De acordo com o critério introduzido é possível separar as proteínas estudadas em dois grupos distintos, um grupo em que a adição de frustração auxilia o processo de enovelamento e outro grupo em que a presença de frustração não é favorável. A separação das proteínas em dois grupos distintos está relacionada com a ordem de contato absoluta e com a barreira de energia livre de cada proteína / In the folding process the proteins follow the minimal frustration principle, which guarantees to the native state the minimum global energy and the thermodynamic stability. However, not even the native state can prevent the occurrence of some unfavorable interactions, characterizing the frustration in the native state. There are evidences that a low degree of frustration can favorable to the folding process. In this work it was investigated the conditions under which some frustration helps the protein folding process. Through computer simulations of molecular dynamics, using a structure-based model (non-frustrated model) and adding a parameterized nonspecific energetic frustration to the potential, were studied the kinetics and the thermodynamics properties of a large group of proteins. It is introduced a criterion to determine when the presence of frustration assists the folding process. The effects of the frustration addition are observed and its influence produces changes in the folding temperature and in the folding time. In agree with the adopted criterion, it was observed two well separated groups: one in which some frustration helps the folding process, and another in which frustration hinders it, determining when the presence of frustration is favorable. These results are correlated with the proteins absolute contact order parameter and free energy barrier

Made available in DSpace on 2014-06-11T19:22:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-01Bitstream added on 2014-06-13T20:27:26Z : No. of bitstreams: 1 lima_dt_me_sjrp.pdf: 414343 bytes, checksum: e3e88fd703395da0dc423bca56673295 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Por meio de simulações computacionais, acompanhamos o processo de enovelamento de um conjunto de proteínas. Neste estudo utilizamos a abordagem conhecida como teoria de Superfície de Energia (Energy Landscape), que trata o enovelamento por uma descrição estatística. O modelo utilizado foi um modelo baseado em estrutura. Para investigarmos os efeitos das interações não nativas no enovelamento de um grupo de proteínas, acrescentamos um termo de frustração ao potencial de energia. Os resultados obtidos a partir dos estudos das propriedades termodinâmicas e cinéticas das proteínas, mostraram que para um grupo de proteínas a frustração ajudou o enovelamento e para outro grupo a frustração atrapalhou. Analizando propriedades como ordem de contato absoluta, tamanho da proteína e barreira de energia livre, foi possível obter um limiar em que a frustração auxilia no enovelamento. Para encontrarmos um limiar, foi necessário uma quantidade grande de proteínas de diferentes tamanhos e diferentes motifs / Through computational simulations, the folding process of a set of proteins was monitored. The framework used to describe protein folding was the energy landscape theory, which is a statistical description of folding. The model used was the structural based model. A frustration term was added to the potential to investigate the non-native interactions effect. The results obtained from studies of thermodynamics and kinetics properties, showed for a one group of proteins the frustration helps the folding and for another group the frustrations hinders the folding. The properties as absolute contact order, size of protein and free energy barrier was analyzed and it was possible to obtain a threshold that the frustration helps the folding. It was necessary a large set of proteins of different sizes and motifs to find a threshold

Abstract Protein disulfide bonds are covalent links formed between the thiol groups of cysteine residues. In many proteins, they have an important role in stabilizing the three-dimensional conformation of the polypeptide chain. Usually proteins are physiologically active and functional only when they are correctly folded. Protein folding takes place very soon after the synthesis of a new polypeptide chain. Proteins which are to be secreted from the cell fold in a specialized compartment, the endoplasmic reticulum (ER). Folding and disulfide bond formation in the ER does not happen spontaneously, there are proteins which are specialized in assisting in these processes. Protein disulfide isomerase (PDI) is a multifunctional protein, which is capable of catalysing both of disulfide bond formation and folding of a protein. In addition, it has other functions: it is an essential part of two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein. C-P4H is an enzyme essential in the formation of collagens, proteins found in connective tissue. The function of C-P4H is to catalyse the hydroxylation of prolines, which is essential for the structural stability of collagens. C-P4H is a tetramer, formed of two catalytic α subunits and two β subunits, which are identical to PDI. The function of PDI in C-P4H is apparently to keep it in a soluble, functionally active conformation. In mammals there are several proteins similar to PDI, together forming a PDI family of proteins. They share both structural and functional similarities. One of these proteins is ERp57. It is specialized in assisting in the folding and disulfide bond formation of glycoproteins. PDI consists of four domains, two of which contain a catalytic site for disulfide bond formation. One domain is the main site of interaction with other proteins and one domain is of unknown function. In this study, the role of these domains in the activities of PDI was investigated. The peptide-binding domain was characterized in detail. In addition, structural similarities of PDI and ERp57 were studied by formation of hybrid proteins containing domains of both and comparing the activities of these recombinant proteins to those of PDI.

Abstract The correct three dimensional structures of proteins are essential for their ability to function properly. Proteins start to fold as soon as they are synthesized in the ribosomes from activated amino acids. Many secreted, cell-surface, secretory pathway and endoplasmic reticulum (ER) lumenal proteins have in their amino acid sequence cysteine residues which form intra- and intermolecular disulfide bridges that stabilize the overall fold of the proteins and protein complexes. The formation of correct disulfide bonds is a complex process which takes place within the ER. Protein disulfide isomerase (PDI) is the key enzyme in the formation and rearrangement of correct disulfide bonds in the ER. It is an archetypal and the best studied member of the PDI family, i.e. a group of ER proteins that resemble thioredoxin (TRX), a protein reductase, in their structure. PDI has a four domain a-b-b'-a' structure the a and a' domains having the catalytic activity and amino acid sequence similarity to TRX. In addition to its function as a thiol-disulfide oxidoreductase, PDI acts as the β subunit in two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein (MTP). The closest homologue of PDI is the multifunctional enzyme and chaperone ERp57 that functions in concert with two lectins, calnexin (CNX) and calreticulin (CRT) specifically in the folding of proteins that have sugar moieties linked to them. ERp57 is 56% similar to PDI in its amino acid sequence and has also the four-domain architecture. Despite the high similarity in their structures ERp57 cannot substitute for PDI as the β subunit of C-P4H. The minimum requirement for the C-P4H tetramer assembly is fulfilled by domains b' and a' of PDI, while domains a and b enhance this function and can be substituted in part by those of ERp57. Until very recently the structural information of any of the PDI family members, which contains the TRX active site was limited to solution structures of human PDI domains a and b. In this research the domain boundaries of the full length ERp57 were defined and the individual domains characterized. Furthermore the solution structures of the catalytically active domains a and a' of ERp57 were studied by nuclear magnetic resonance (NMR).

Abstract The triosephosphate isomerase (TIM) barrel superfamily is a broad family of proteins, most of which are enzymes. At the amino-acid-sequence level, many of the members of this family share little, if any, homology. Yet, they adopt the same three-dimensional (βα)8 fold. The TIM barrel fold seems to be a good framework for many different kinds of enzymes, providing unique possibilities for both natural and human-designed evolution, as the catalytic center and the stabilizing features are separated to different ends of the barrel. Indeed, in the light of most recent studies, it seems likely that at least most of the different TIM barrel enzymes, catalyzing a huge variety of reactions, have evolved from a common ancestor. TIM can be considered a real text-book enzyme — its catalytic properties and stucture-function relationships have been studied for decades. Still, at present, we are quite far from understanding the structural features that make TIM and other enzymes such superior catalysts in both efficiency and precision. TIM is a dimeric enzyme that consists of two identical subunits of 250 residues. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in glycolysis. The basics of this reaction are well known, but there is ongoing discussion about the details of the proton transfer steps, and three alternative pathways have been suggested. In addition, it is a fascinating question how the enzyme succeeds in abstracting a highly stable proton from a carbon atom of the substrate. This study was undertaken to shed light on some of the questions concerning the structure-function relationships in TIM. The most important findings are the elucidation of the role of Asn11 as a catalytic residue and the meaning of the flexibility of both the catalytic Glu167 side chain as well as the substrate during catalysis, and the presence of a low-barrier hydrogen bond between Glu167 and a transition-state analogue, 2-phosphoglycolate. Furthermore, significant results were obtained on the importance of a conserved salt bridge, 20 Å away from the active site and the dimer interface, for the stability and folding of TIM as well as on the factors influencing the opening of the flexible loop 6 upon product release.