Afin de lever le mystère centenaire qui entoure l'origine du changement de volume associé au dépliement des protéines globulaires, nous avons constitué une collection de 10 mutants du variant hyperstable de la SNase, ∆+PHS. Chacune de ces mutations a été conçue pour créer une nouvelle cavité ou agrandir une cavité existante au sein de la protéine. Dans un premier temps, nous avons analysé comment l'environnement structural local conditionne l'adaptation à la mutation. Les expériences de fluorescence sous haute pression ont montré un accroissement systématique de la différence de volume entre les états replié et déplié pour les 10 variants par rapport à ∆+PHS. Ce résultat majeur, qui s'ajoute à une étude récente démontrant que les effets d'hydratation ne contribuent pas de manière significative au changement de volume, démontre sans ambiguïté que la différence de volume entre les états replié et déplié est principalement due à la présence de cavités internes dans la structure native des protéines. Les mesures par RMN des changements de volume ont permis d'établir une cartographie des effets de la pression à l'échelle du résidu et d'identifier les intermédiaires de repliement peuplant le paysage énergétique. En analysant les cinétiques de dépliement, nous avons finalement pu caractériser les conséquences de ces mutations sur les états de transitions de la SNase. / To solve the century-old question of the origin of the volume change upon unfolding for globular proteins, we built up a collection of 10 mutants of the hyperstable variant of SNase, ∆+PHS. Each of these mutations was designed to create a cavity or to enlarge a naturally occurring cavity in the protein core. We first analyzed how the local structural environment determines the adaptation to the mutation. The high-pressure fluorescence experiments showed a systematic increase of the volume difference between the folded and unfolded states for the 10 variants, compared to ∆+PHS. This major result, in addition to a recent study demonstrating that the hydration effects do not provide any significant contribution to the volume changes, clearly demonstrates that the volume difference between the folded and unfolded states is predominantly due to the presence of internal cavities in the native structure. NMR measurements of the volume change allowed a mapping of the pressure effects on a residue scale and the identification of the folding intermediates populating the free-energy landscape. By analyzing the unfolding kinetics, we finally characterized the consequences of these mutations on the transition state ensembles of SNase.

L' objet d'étude de cette thèse est l' origami d' ADN. Le nombre important d'applications utilisant cette technique, et leurs diversités, sont des preuves que ces structures présentées en 2007 sont une avancée importante pour la technologie ADN. Dans cette thèse nous présentons dans un premier temps les résultats de simulations sur les propriétés mécaniques de ces nouvelles structures. Les résultats d'expériences et de simulations sur le processus de formations pour de petites structures d'ADN et sur des origamis d'ADN sont présentés dans une deuxième partie. Finalement une application de ces structures à la détection SERS est proposée, et les résultats expérimentaux concernant la réalisation de cette structure sont discutés. / DNA origami are new nanostructures (2006) whose physical properties are still to be understood. In this work we were first interested in their mechanical properties. The first approach of this study was through the use of polymer physics, as it is the classical way to study DNA. We then used computer simulations to model the system in a more detailed manner and to extract general rules on the mechanical behaviour of DNA constructs. The other aspect that we studied is the process of formation of DNA origamis. We first realised an experimental study of the process of formation of the simplest origami that we could envision. This study was intended to investigate basic principles on the process of formation of DNA structures. A coarse grain model is then developed to have a first insight onto the formation process. Then an experimental study on large origamis follows with a modeling of the annealing and melting curves based on the principles determined from the study of the simplest origami. We also worked on the development of a SERS platform.

Doctor of Philosophy / Department of Biochemistry / Michal Zolkiewski / ClpB is a bacterial heat-shock protein that disaggregates and reactivates strongly aggregated proteins in cooperation with the DnaK chaperone system. ClpB contains two ATP-binding AAA+ modules, a linker coiled-coil domain, and a highly mobile N-terminal domain. It forms ring-shaped hexamers in a nucleotide-dependent manner. The unique aggregation reversing chaperone activity of ClpB involves ATP-dependent translocation of substrates through the central channel in the ClpB ring. The initial events of aggregate recognition and the events preceding the translocation step are poorly understood. In addition to the full-length ClpB95, a truncated isoform ClpB80, that is missing the whole N-terminal domain, is also produced in vivo. Various aspects of the structure and function of ClpB were addressed in this work. The thermodynamic stability of ClpB in its monomeric and oligomeric forms, as well as the nucleotide-induced conformational changes in ClpB were investigated by fluorescence spectroscopy. Equilibrium urea-induced unfolding showed that two structural domains-the small domain of the C-terminal AAA+ module and the coiled-coil domain-were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation or interactions during formation of the ClpB rings. Several locations of Trp-fluorescence probes were also found to respond to nucleotide binding. The biological role of the two naturally-occurring ClpB isoforms was also investigated. We discovered that ClpB achieves optimum chaperone activity by synergistic cooperation of the two isoforms that form hetero-oligomers. We found that ClpB95/ClpB80 hetero-oligomers form preferentially at low protein concentration with higher affinity than homo-oligomers of ClpB95. Moreover, hetero-oligomers bind to aggregated substrates with a similar efficiency as homo-oligomers of ClpB95, do not show enhanced ATPase activity over that of the homo-oligomers, but display a strongly stimulated chaperone activity during the reactivation of aggregated proteins. We propose that extraction of single polypeptides from aggregates and their delivery to the ClpB channel for translocation is the rate-limiting step in aggregate reactivation and that step is supported by the mobility of the N-terminal domain of ClpB. We conclude that the enhancement of the chaperone activity of the hetero-oligomers is linked to an enhancement of mobility of the N-terminal domains.

<p>Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.</p><p>In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.</p><p>The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.</p> / Dissertation

ABSTRACT Equilibrium unfolding, single- and double-jump kinetic studies were conducted to determine the unfolding and refolding pathway of glutaredoxin 2. Structural changes for wild-type glutaredoxin 2 were monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence for equilibrium unfolding and intrinsic tryptophan fluorescence for single- and double-jump kinetics studies. Glutaredoxin 2 possesses two tryptophan residues in domain 2. In order to monitor changes in domain 1, cysteine 9 at the active site cysteines, situated in domain 1, was labelled with an extrinsic fluorophore, AEDANS, and a mutant was created (Y58W glutaredoxin 2). The AEDANS labelled protein displayed decreased alpha-helical secondary structure and conformational stability. A high degree of cooperativity and similar conformational stability was observed during the two-state transition of the urea-induced equilibrium unfolding of both the wild-type and Y58W glutaredoxin 2 proteins therefore Y58W glutaredoxin 2 could be used to assess structural changes in the local environment of domain 1 during unfolding and refolding. Two phases of unfolding, the fast and slow phase, occurred for both the wild-type and Y58W proteins. The slow phase involves structural rearrangements that expose small amounts of surface area while the fast phase represents gross structural unfolding exposing large amounts of surface area. The isomerization of the Val48-Pro49 peptide bond to the trans conformation occurs during the slow phase and this isomerization is coupled to conformational unfolding of the protein. The structural separation of these phases could be represented by two structural units (unit x and unit y), these units do not represent domain 1 and 2. The units could also result in parallel refolding pathways with the folding of the x unit involving the fast and slow refolding phases and the folding of the y unit of structure is represented by the medium phase of refolding. The fast and slow phases are further separated as the fast phase represents the gross structural folding of glutaredoxin 2 for species with the Val48-Pro49 peptide bond in the native cis conformation. The development of the slow phase after extended unfolding delay periods during double-jump refolding studies, as well as the acceleration of the rate of the phase by the peptidyl prolyl isomerase hFKBP-12 proved that the phase involves a proline peptide bond iv isomerization. This phase represents a slow isomerization coupled with conformational folding similar to the slow unfolding phase. Complex unfolding and refolding kinetics indicated the involvement of kinetic intermediates during (un)folding.

Thesis advisor: Welkin E. Johnson / Thesis advisor: Peter G. Clote / Synthetic biology currently is a rapidly emerging discipline, where innovative and interdisciplinary work has led to promising results. Synthetic design of RNA requires novel methods to study and analyze known functional molecules, as well as to generate design candidates that have a high likelihood of being functional. This thesis is primarily focused on the development of novel algorithms for the design of synthetic RNAs. Previous strategies, such as RNAinverse, NUPACK-DESIGN, etc. use heuristic methods, such as adaptive walk, ensemble defect optimization (a form of simulated annealing), genetic algorithms, etc. to generate sequences that minimize specific measures (probability of the target structure, ensemble defect). In contrast, our approach is to generate a large number of sequences whose minimum free energy structure is identical to the target design structure, and subsequently filter with respect to different criteria in order to select the most promising candidates for biochemical validation. In addition, our software must be made accessible and user-friendly, thus allowing researchers from different backgrounds to use our software in their work. Therefore, the work presented in this thesis concerns three areas: Create a potent, versatile and user friendly RNA inverse folding algorithm suitable for the specific requirements of each project, implement tools to analyze the properties that differentiate known functional RNA structures, and use these methods for synthetic design of de-novo functional RNA molecules. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Este estudo tem como finalidade principal a análise termodinâmica e estatística de proteínas através de modelos mínimos. Uma proteína é um polímero de aminoácidos, cuja função está essencialmente relacionada às conformações espaciais que ela adota em solução aquosa. Na forma funcional (dita nativa), essas conformações flutuam levemente em torno de um mínimo de energia-livre. O processo pelo qual uma cadeia protéica transita de estados não-nativos para a estrutura nativa é chamado de enovelamento, ou dobramento. Uma questão em aberto no campo de estudo de proteínas consiste justamente em entender a fundo o processo de enovelamento, cujo avanço tem um vasto potencial de aplicação, desde a predição de estruturas a partir de sequências de aminoácidos até o planejamento de fármacos e moléculas bioativas. Nossa investigação teórica procura abordar aspectos do enovelamento expressos através de grandezas termodinâmicas (energia média, calor específico, número de ligações de hidrogênio, entre outras) derivadas de modelos estatísticos na rede. Assim, num primeiro momento, analisamos o chamado modelo HP, ora por meio de enumeração exata, para cadeias curtas, ora por simulações de Monte Carlo, para cadeias maiores. No primeiro caso, propusemos a existência de uma relação entre a ocorrência de um segundo pico no calor específico associado na literatura à transição de congelamento com uma drástica redução no número de configurações entre os primeiros estados excitados e aqueles de menor energia. Observamos, também, que esse pico pode aparecer tanto para homopolímeros quanto para heteropolímeros, em ambas as redes quadrada e triangular. Num segundo momento, nosso enfoque se voltou para a inclusão de um solvente aquoso (dado pelo modelo de Bell-Lavis) ao sistema inicial. Isso nos possibilitou verificar, usando exclusivamente simulações de Monte Carlo e o algoritmo de Metropolis, o comportamento e a competição das ligações de hidrogênio água-água, água-proteína, proteína-proteína e na primeira camada de solvatação. O modelo acoplado exibiu algumas características do enovelamento, como o colapso hidrofóbico e a separação de monômeros (apolares no núcleo e polares na superfície), embora não capture a desnaturação fria. No apêndice, adicionamos algumas propostas para realização do cálculo numérico da pressão no ensemble canônico, desenvolvidas em paralelo ao projeto principal desta dissertação, mas que, numa primeira análise, verificamos serem consistentes e passíveis de futuros desdobramentos. / The finality of this study is to analyse proteins thermodynamics and statistics through minimal models. A protein is a polymer of amino acids, whose spatial conformations in aqueous solution determine its function. In the functional form (said native), those conformations fluctuates slightly around a free-energy minimum. The process by which a protein chain passes from non-native states to a stable native structure is called protein folding. An open question in the field of protein studies is to understand more deeply the folding process, whose advance can find a wide range of potential applications, since ab initio structure prediction from the amino acids sequence to biomolecules design. The theoretical approaches used here focus on aspects of protein folding given by some thermodynamic quantities (as mean energy, specific heat, number of hydrogen bonds and so on) obtained from statistical lattice models. Initially, we analyse the so-called HP model, at first using exact enumeration for short chains, then by Monte Carlo simulations for longer chains. In the first case, we propose a correlation between the occurrence of a second peak in the specific heat associated in the literature with a freezing transition and a sharp reduction on the number of configurations from the first excited states to the lowest energy states. In addition, we observe that this peak may appear to both homopolymers and heteropolymers on square and triangular lattices. At a second moment, our focus turned to the introduction of a water-like solvent (Bell-Lavis model) to the initial system. This allowed us to verify, exclusively by means of Monte Carlo simulations with Metropolis algorithm, the behavior and competition of hydrogen bonds between water-water molecules, water-protein, and protein-protein monomers and at the first hydration layer. The combined model showed some classical folding properties, as hydrophobic collapse and monomers segregation (apolar residues at the core and polar residues at the surface), although it did not capture cold denaturation. We have included in the appendix some proposals to perform numerical calculations of the canonical pressure, which were developed alongside the main subject of this thesis and a first analysis has proved to be consistent and susceptible to further developments.

Les structures pliées intéressent architectes et ingénieurs pour leurs qualités structurelles autant que spatiales et plastiques. En prenant comme point de départ la bande rectiligne pliée, ce travail de recherche aborde non pas la conception de surfaces plissées mais celle de structures réticulées. La conception de structures “à trous” à partir de bandes pliées constitue un angle d’approche original. La notion de continuité induite par le pli transforme la manière de concevoir la structure réticulée, habituellement caractérisée par des nœuds et barres dissociés. L’emploi de bandes continues rectilignes est une approche possible pour rationaliser la conception de formes complexes en minimisant la quantité de chutes de matière. Cette recherche explore de nouvelles méthodes de conception des formes courbes. Les objectifs de cette recherche sont :- la classification des différentes approches possibles dans la conception à partir de bandes rectilignes pliées- l'étude d'éléments modulaires pliés dont l’assemblage permet la génération de surfaces courbes variables: la géométrie de différentes configurations de bandes pliées sera décrite ainsi que les assemblages autorisés / Folded structures are attractive to architects and engineers for their structural qualities as well as spatial and plastic qualities. Taking as a starting point a linear folded stripe, this research does not address the design of pleated surfaces but that of reticular structures. The design of structures with "holes" from folded stripes is an original angle of approach. From a conceptual point of view, the continuity caused by folds transforms the usual conception by separated nodes and bars of reticular structures. The use of continuous linear stripes is one possible approach to make more efficient the design of complex shapes by minimizing the produced offcuts. This research investigates new methods of designing curved shapes. The aims of this research are:- A classification of different possible approaches encountered when conceiving from folded stripes.- The study a modular folded elements which assembly allows the generation of various curved surfaces : the geometry of different configurations of folded stripes and the assemblies allowed will be described

The mitochondrion is the powerhouse of the cell. Therefore, it is critical to the homeostasis of the cell that populations of mitochondria that are damaged or in excess are degraded. The process of targeted elimination of damaged or excess mitochondria by autophagy is called mitophagy. In this report, analysis of the mitophagy regulators PINK1/PARKIN and BNIP3L and their roles are assessed in the lens. PARKIN, an E3 ubiquitin ligase, has been shown to play a role in directing damaged mitochondria for degradation. While BNIP3L, an outer mitochondrial membrane protein, increases in expression in response to excess mitochondria and organelle degradation during cellular differentiation. We have shown that PARKIN is both induced and translocates from the cytoplasm to the mitochondria in human epithelial lens cells upon oxidative stress exposure. In addition, our findings also show that overexpression of BNIP3L causes premature clearance of mitochondria and other organelles, while loss of BNIP3L results in lack of clearance. Prior to this work, PARKIN mediated mitophagy had not been shown to act as a protective cellular response to oxidative stress in the lens. This project also resulted in the novel finding that BNIP3L-mediated mitophagy mechanisms are required for targeted organelle degradation in the lens. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Os estudos sobre o mecanismo de enovelamento das proteínas é o resultado de um estudo intenso utilizando métodos bioquímicos, biofísicos e teóricos. \"In vitro\", o estado inicial deste estudo é a proteína desnaturada. Neste trabalho, temos estudado o reenovelamento, após desnaturação térmica, de uma glicoproteína denominada frutalina; da família das lectinas. A característica principal desta classe de proteínas é sua habilidade para interagir com carboidratos e, portanto, combinar-se com glicocomponentes da superfície da célula, induzindo suas propriedades biológicas. A frutalina é uma lectina tetramérica extraída das sementes de Artocarpus incisa. Ela é ligante de D-galactose e o espectro de CD (dicroísmo circular) de sua estrutura nativa foi identificado como sendo dominado por folhas ?. A desnaturação térmica e as etapas do reenovelamento foram monitoradas por espectroscopia de CD, fluorescência e também pela perda da atividade hemaglutinante. As condições de desnaturação utilizadas foram aquecimento à 60°C por 30 a 60 minutos, dependendo do tempo de estocagem (a -18°C) da proteína na forma nativa. Os resultados indicaram que o reenovelamento é promovido por um processo de congelamento na presença de PBS contendo 0,l M de D-galactose seguida por centrífugoconcentração em Centriprep 3. A hemaglutinação positiva ocorreu tanto para a fração nativa quanto para a fração reenovelada. O reenovelamento da frutalina desnaturada também ocorreu com PBS contendo 0,1 M de solução de D-glicose. Quando a forma desnaturada foi concentrada antes do congelamento em PBS sem D-galactose ou em PBS contendo xilose, o reenovelamento não ocorreu. Estes resultados mostraram que o reenovelamento da frutalina foi dependente da ligação com a D-galactose ou D-glicose, bem como a importância do congelamento para obter a forma biologicamente ativa. A análise da estrutura secundária utilizando o programa CCA forneceu um resultado importante: para a forma nativa da frutalina obtivemos 85% de folhas ? paralelas e antiparalelas, incluindo voltas ?, enquanto que para a forma reenovelada obtivemos 73%, mostrando que a estrutura reenovelada, a nível secundário, se aproximou satisfatoriamente da nativa, concordando com os resultados obtidos nos testes de hemaglutinação. / Our current understanding of the protein folding mechanism is the result of intense study employing biophysical, biochemical and theoretical methods. \"In vitro\", the initial state of the protein in this puzzle is its unfolded form. In the present work we have studied the refolding, after thermal denaturation, of the glycoprotein frutalin, a member of the lectin class. The main characteristic of these proteins is their ability to interact with carbohydrates and thus combine with glycocomponents of the cell surface, leading to their biological properties. Frutalin is a tetrameric lectin extracted from the seeds of Artocarpus incisa. It is D-galactose specific and its native CD spectrum was identified as being dominated by ? -sheet. The thermal unfolding and refolding steps were measured by CD and fluorescence spectroscopies together with the loss of hemagglutinating activity. The unfolding conditions used were 60°C for 30 to 60 minutes, depending on the protein storage time. The results indicate that refolding is promoted by the freezing process in the presence of 0,1 M D-galactose-PBS followed by three-fold concentration in a Centriprep 3. Positive hemagglutination occurred for both the native and refolded forms. Refolding of denatured frutalin also occurred with PBS containing 0,1 M D-glucose. When the unfolded form was concentrated before freezing in PBS without D-galactose or in PBS containing xylose, refolding did not occur. These results show that the refolding of frutalin is dependent on the binding of D-galactose or D-glucose, and demonstrate the importance of freezing in order to obtain the biologically activity form. An analysis of secondary structure using the CCA program showed an important result: the native form, presented 85% ? -sheet/ ?-turns, while in the refolded form, this content fell to 73%. These results show that the refolded form is very similar to the native protein, which is in agreement with the hemagglutination results.