Molecular chaperones and proteases help maintain protein homeostasis in the cell. While chaperones assist in the folding of polypeptide chains to their native state, proteases degrade misfolded or unfolded proteins and also help regulate protein levels. While mapping chaperone interaction networks, we found that tig (trigger factor chaperone gene), clpP and clpX genes co-localize next to each other on the genome of most examined bacteria. This led us to hypothesize that trigger factor (TF) chaperone and ClpXP protease might interact functionally. TF is a ribosome-associated chaperone that co-translationally folds polypeptide chains. ClpXP is a proteolytic complex that degrades a wide range of substrate proteins. We observed that TF enhanced the rate of the ClpXP degradation of the λO phage protein in vitro and in vivo. TF was also found to enhance the degradation of ribosome-stalled λO thus suggesting the existence of co-translational protein degradation in E. coli.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-01Bitstream added on 2014-06-13T20:01:01Z : No. of bitstreams: 1 oliveira_rj_dr_sjrp.pdf: 3645510 bytes, checksum: 181949bcc7d9fbd2c0929fdd73a9cf0a (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A difusão desempenha um papel importante na cinética de eno-velamento de proteínas. Nessa tese, desenvolvemos métodos analíticos e computacionais para o estudo do coeficiente de difusão dependente da posi-ção e do tempo. Para estes estudos, utilizou-se sobretudo o modelo baseado na estrutura (modelo G¯o) via simulação computacional da representação em carbonos alfa. Investigou-se o efeito da difusão no enovelamento da proteína cold-shock (TmCSP). Encontrou-se que o efeito temporal da difusão leva a cinéticas não-exponenciais e a estatística não-poissônica da distribuição de tempos de enovelamento. Com relação a dependência com a posição, o coeficiente de difusão revelou ter um comportamento não-monotônico que foi compreendido pela análise dos valores- e da entropia residual no estado nativo. Para uma versão frustrada do modelo, encontrou-se que um baixo nível de frustração energética aumenta a difusão no estado nativo e torna o estado de transição mais homogêneo. Esses resultados corroboram com experimentos recentes de fluorescência de uma única molécula. Esse trabalho também propõe um método para a determinação da superfície de energia de enovelamento de proteína. A partir da caracterização da superfície de energia, definimos a quantidade (LD – Landscape Descriptor) que mostrou uma forte correlação entre a cinética e a termodinâmica de uma dezena de proteínas globulares, tornando-se um método útil para classificar proteínas / Diffusion plays an important role in protein folding kinetics. In this thesis we developed analytical and computational methods in order to study the diffusion coefficient dependent on position and time. For these studies we used mainly the structure-based model (G¯o model) via computer simulation of the alpha-carbon representation. We investigated the effect of diffusion in the folding of the cold-shock protein (TmCSP). We found that the time dependence on diffusion leads to non-exponential kinetics and non-Poisson statistics of folding time distribution. With respect to the position dependence, the diffusion coefficient reveled a non-monotonic behavior that was understood by analyzing the -values and the residual entropy in the native state. For a frustrated version of the model, we found that a low level of frustration energy stabilizes and increases the diffusion in the native state and the transition state becomes more homogeneous. These results are supported by recent single-molecule fluorescence experiments. This work also proposes a method to determine the protein folding energy landscape. With the energy landscape characterized, we defined the quantity (LD – Landscape Descriptor) which showed a strong correlation between kinetics and thermodynamics of a dozen globular proteins making it a useful method to classify proteins

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 140-148).

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Neal Zondlo, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Proper folding of a protein to its native state is critical for the protein to be fully functional under biological conditions. Understanding protein folding and protein folding evolution within the same structural family are key to understand which processes assist or hinder protein folding and how to prevent misfolding. Tm0979 from Thermotoga maritima, Mth1491 from Methanobacterium thermoautotrophicum and YchN from Escherichia coli belong to the homologous superfamily of YchN-like proteins (SCOP and CATH: 3.40.1260.10). The structures of these proteins have been solved as part of structural proteomics projects, which consist of solving protein structures on a genome wide scale. In solution, Tm0979 forms a homodimer whereas Mth1491 folds as a trimer and YchN is a homohexamer. The structures of the individual monomeric subunits of these three proteins have high structural similarity, despite very low sequence similarity. The biological roles of these proteins are not yet well defined, but seem to be involved in catalysis of sulphur redox reactions. This thesis focuses on characterisation of the Tm0979 homodimer and the Mth1491 homotrimer, as well as the determination of the folding mechanisms of these two proteins. The folding mechanisms of the proteins are compared to each other and to the mechanisms of other dimeric and trimeric proteins. The evolution and basis of oligomeric structure within the YchN family are analyzed. Mutations of Tm0979 and Mth1491 are designed as a basis for future work to investigate processes responsible for switches in oligomeric protein quaternary.structure.

Proper folding of a protein to its native state is critical for the protein to be fully functional under biological conditions. Understanding protein folding and protein folding evolution within the same structural family are key to understand which processes assist or hinder protein folding and how to prevent misfolding. Tm0979 from Thermotoga maritima, Mth1491 from Methanobacterium thermoautotrophicum and YchN from Escherichia coli belong to the homologous superfamily of YchN-like proteins (SCOP and CATH: 3.40.1260.10). The structures of these proteins have been solved as part of structural proteomics projects, which consist of solving protein structures on a genome wide scale. In solution, Tm0979 forms a homodimer whereas Mth1491 folds as a trimer and YchN is a homohexamer. The structures of the individual monomeric subunits of these three proteins have high structural similarity, despite very low sequence similarity. The biological roles of these proteins are not yet well defined, but seem to be involved in catalysis of sulphur redox reactions. This thesis focuses on characterisation of the Tm0979 homodimer and the Mth1491 homotrimer, as well as the determination of the folding mechanisms of these two proteins. The folding mechanisms of the proteins are compared to each other and to the mechanisms of other dimeric and trimeric proteins. The evolution and basis of oligomeric structure within the YchN family are analyzed. Mutations of Tm0979 and Mth1491 are designed as a basis for future work to investigate processes responsible for switches in oligomeric protein quaternary.structure.

Proteins need fold to perform their biological function. Thus, understanding how proteins fold could be the key to understanding life. In the first study, the stability and structure of several !-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G (PGB1) were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the !-turn conformation. Our results indicate that the structure and stability of this !?hairpin peptide can be modulated in numerous ways and thus contributes towards a more complete understanding of this important model !-hairpin as well as to the folding and stability of larger peptides and proteins. The second study revealed that PGB1 and its variants can form amyloid fibrils in vitro under certain conditions and these fibrils resemble those from other proteins that have been implicated in diseases. To gain a further understanding of molecular mechanism of PGB1 amyloid formation, we designed a set of variants with mutations that change the local secondary structure propensity in PGB1, but have similar global conformational stability. The kinetics of amyloid formation of all these variants have been studied and compared. Our results show that different locations of even a single mutation can have a dramatic effect on PGB1 amyloid formation, which is in sharp contrast with a previous report. Our results also suggest that the "-helix in PGB1 plays an important role in the amyloid formation process of PGB1. In the final study, we investigate the forces that contribute to protein stability in a very general manner. Based on what we have learned about the major forces that contribute to the stability of globular proteins, protein stability should increase as the size of the protein increases. This is not observed: the conformational stability of globular proteins is independent of protein size. In an effort to understand why large proteins are not more stable than small proteins, twenty single-domain globular proteins ranging in size from 35 to 470 residues have been analyzed. Our study shows that nature buries more charged groups and more non-hydrogen-bonded polar groups to destabilize large proteins.

Protein structure and motion plays an essential role in nearly all forms of life. Understanding both protein folding and protein conformational change can bring deeper insight to many biochemical processes and even into some devastating diseases thought to be the result of protein misfolding. Experimental methods are currently unable to capture detailed, large-scale motions. Traditional computational approaches (e.g., molecular dynamics and Monte Carlo simulations) are too expensive to simulate time periods long enough for anything but small peptide fragments. This research aims to model such molecular movement using a motion framework originally developed for robotic applications called the Probabilistic Roadmap Method. The Probabilistic Roadmap Method builds a graph, or roadmap, to model the connectivity of the movable object?s valid motion space. We previously applied this methodology to study protein folding and obtained promising results for several small proteins. Here, we extend our existing protein folding framework to handle larger proteins and to study a broader range of motion problems. We present a methodology for incrementally constructing roadmaps until they satisfy a set of evaluation criteria. We show the generality of this scheme by providing evaluation criteria for two types of motion problems: protein folding and protein transitions. Incremental Map Generation eliminates the burden of selecting a sampling density which in practice is highly sensitive to the protein under study and difficult to select. We also generalize the roadmap construction process to be biased towards multiple conformations of interest thereby allowing it to model transitions, i.e., motions between multiple known conformations, instead of just folding to a single known conformation. We provide evidence that this generalized motion framework models large-scale conformational change more realistically than competing methods. We use rigidity theory to increase the efficiency of roadmap construction by introducing a new sampling scheme and new distance metrics. It is only with these rigidity-based techniques that we were able to detect subtle folding differences between a set of structurally similar proteins. We also use it to study several problems related to protein motion including distinguishing secondary structure formation order, modeling hydrogen exchange, and folding core identification. We compare our results to both experimental data and other computational methods.

Thesis (Ph. D.)--University of Chicago, Department of Chemistry, 2001. / Includes bibliographical references. Also available on the Internet.