Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US); 12 Dec 2003. / Published through the Information Bridge: DOE Scientific and Technical Information. "IS-T 2028" Haibo Cao. 12/12/2003. Report is also available in paper and microfiche from NTIS.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.

Currently, one of the main research problems in Structural Bioinformatics is associated to the study and prediction of the 3-D structure of proteins. The 1990’s GENOME projects resulted in a large increase in the number of protein sequences. However, the number of identified 3-D protein structures have not followed the same growth trend. The number of protein sequences is much higher than the number of known 3-D structures. Many computational methodologies, systems and algorithms have been proposed to address the protein structure prediction problem. However, the problem still remains challenging because of the complexity and high dimensionality of a protein conformational search space. This work presents a new computational strategy for the 3-D protein structure prediction problem. A first principle strategy which uses database information for the prediction of the 3-D structure of polypeptides was developed. The proposed technique manipulates structural information from the PDB in order to generate torsion angles intervals. Torsion angles intervals are used as input to a genetic algorithm with a local-search operator in order to search the protein conformational space and predict its 3-D structure. Results show that the 3-D structures obtained by the proposed method were topologically comparable to their correspondent experimental structure.

This Ph.D. thesis addresses the structural characterization of the unique family of tetrameric flavoproteins WrbA, the role of which in the life of cells is still largely unknown but its enzymatic activity and expression properties implicate it in the cell protection against oxidative stress. Proteins of the WrbA family were proved to carry out two-electron reductions of quinones and in this way to prevent generation of the free radicals, similarly to other flavoproteins known as quinone oxidoreductases. Crystal structures of the liganded and unliganded forms of the prototypical WrbA from Escherichia coli were determined. Comparative analyses of these structures with the related flavoproteins were intended to identify and explain the defining structural features of the WrbA family and to clarify its structural and functional relationships to the other flavoproteins.

Les protéines intrinsèquement désordonnées sont caractérisées par un manque de structure 3D stable et sont biologiquements actives dans cet état. La spectroscopie RMN est la méthode de choix pour leurs études à une résolution atomiques, car la cristallographie aux rayons X ne permet pas leur étude en raison de leur caractère hautement dynamique.Cependant, l'étude par spectroscopie RMN de ces protéines est difficiles à cause du grand nombre de recouvrement entre les signaux dans le spectre résultant de l'absence d'un réseau de liaison hydrogène qui pourrait stabiliser la structure et permettre d'obtenir une dispersion des signaux plus élevé. Un autre problème est la sensibilité expérimentale car souvent le temps de mesure est limité en raison de leur prédisposition à la dégradation protéolytique. Dans la première partie de cette thèse les protéines intrinsèquement désordonnées sont introduites. La deuxième partie porte sur la spectroscopie RMN des protéines intrinsèquement désordonnées, des expériences RMN de type BEST-TROSY sont présentées et sont montrées comme étant bien adapté pour l'étude de protéines intrinsèquement désordonnées, en particulier pour celle avec une grande étendue de structure résiduelle. Des expériences 3D BEST-TROSY sont présentées pour leur attribution, une version proline-éditée permet d'aider à l'identification de ce type d'acide aminé et enfin l'expérience HETex-BEST-TROSY qui permet une mesure rapide des taux de change de solvants. Dans la troisième partie de cette thèse ces expériences RMN sont appliquées pour l'étude de la région intrinsèquement désordonnés (domaines 2 et 3) de la protéine NS5A du virus de l'hépatite C (VHC). La structure secondaire résiduel présente dans le fragment de la protéine est analysée. La comparaison des données RMN sur trois constructions de la protéine de différentes longueurs ainsi que les données de SAXS permettent l'identification des interactions transitoires à longue portée entre les différentes régions de cette protéine. En outre, les modes de liaison de ce fragment de protéine à Bin1 domaine SH3 sont analysés. Enfin, les résultats préliminaires obtenus sur l'étude de la phosphorylation de NS5A du VHC par certaines kinases, qui ont été montrées comme biologiquement pertinents, sont présentés. / Intrinsically disordered proteins are characterized by a lack of a stable, 3D structure and fulfill their biological role as such. NMR spectroscopy is the method of choice for their atomic resolution studies, as X-ray crystallography is not amenable to them due to their highly dynamic character.However, NMR spectroscopic studies of these proteins are challenging, because of the high extent of signal overlap in the spectra, resulting from the absence of a hydrogen-bonding network that would lead to structuring and higher signal dispersion. A further problem is experimental sensitivity as often measurement time is limited due to their predisposition for proteolytic degradation. In the fist part of this thesis intrinsically disordered proteins are introduced. The second part focuses on NMR spectroscopy of IDPs, BEST-TROSY-type NMR methods are presented and are shown to be well suited for large IDPs, especially for those with high extent of residual structure. 3D BEST-TROSY experiments are presented for assignment, a proline-edited version for aiding amino acid-type identification, and the HETex-BEST-TROSY experiment that allows rapid measurement of solvent exchange rates. In the third part of this thesis NMR methods are applied for study of the entire intrinsically disordered region (domains 2 and 3) of NS5A protein of hepatitis C virus. The residual secondary structure in this protein fragment is analyzed. Comparison of NMR data on three protein constructs of different lengths together with SAXS data allows identification of transient long range interactions between different regions of this protein. Furthermore, the binding modes of this protein fragment to Bin1 SH3 domain are analyzed. Finally, the preliminary results obtained on investigation of phosphorylation of NS5A of HCV by certain kinases, reported to be biologically relevant, are presented

Les méthodes de prédiction structurale constituent une alternative relativement efficace aux approches expérimentales pour donner un premier aperçu du repliement natif d'une protéine. L'écart entre le nombre de structures et de séquences protéiques disponibles dans les bases de données ne cesse en effet de croître depuis l'arrivée des technologies de séquençage à haut débit. Cette forte croissance des informations génomiques a remis à l'ordre du jour des techniques modélisant les données capturées au cours de l'évolution. La conservation d'une fonction protéique impose de fortes contraintes sur les contacts impliqués dans le repliement et la fonction se traduisant par une trajectoire évolutive commune. Une fois détectées, ces interactions peuvent aider à modéliser la conformation d'une protéine. Les méthodes résolvant la structure tridimensionnelle des protéines à partir des données évolutives présentent encore plusieurs limitations notamment pour la détection des contacts faux positifs. Ces problèmes restent similaires à ceux rencontrés en détermination de structure par spectrométrie de Résonnance Magnétique Nucléaire où l'intégration des données est un processus clairement établit et en grande partie automatisé. Le logiciel ARIA (Ambiguous Restraints for Iterative Assignment) utilise le concept de contraintes de distances ambiguës et suit un processus itératif afin d'attribuer et d'affiner la liste des noyaux proches dans l'espace pour calculer un ensemble de modèles structuraux en accord avec les données. Ce travail a pour objectif d'adapter cette approche pour prédire de novo la structure d'une protéine en utilisant l'information évolutive. / Structural prediction methods provide a relatively effective alternative to experimental approaches to provide a first insight into native folding of a protein. The gap between the number of structures and protein sequences available in databases has steadily increased since the advent of high throughput sequencing technologies. This strong growth of genomic information helped bring to light prediction tools using coevolutionary data. Conservation of a specific function implies strong restraints on interacting residues involved in the folding and function. Once detected, these interactions can help to model the conformation of a protein. Some important aspects needs to be improved during the modelling process including the detection of false positive among the predicted contacts. Limitations in the field are similar to those encountered in nuclear magnetic resonance spectrometry structure determination where data integration is a clearly established and largely automated process. The Ambiguous Restraints for Iterative Assignment (ARIA) software uses the concept of ambiguous distance restraints and follows an iterative process to assign and refine the list of nearby nuclei in space to compute a set of structural models in accordance with the data. This work aims to adapt this approach to de novo predict the structure of a protein using evolutionary information.

Currently, one of the main research problems in Structural Bioinformatics is associated to the study and prediction of the 3-D structure of proteins. The 1990’s GENOME projects resulted in a large increase in the number of protein sequences. However, the number of identified 3-D protein structures have not followed the same growth trend. The number of protein sequences is much higher than the number of known 3-D structures. Many computational methodologies, systems and algorithms have been proposed to address the protein structure prediction problem. However, the problem still remains challenging because of the complexity and high dimensionality of a protein conformational search space. This work presents a new computational strategy for the 3-D protein structure prediction problem. A first principle strategy which uses database information for the prediction of the 3-D structure of polypeptides was developed. The proposed technique manipulates structural information from the PDB in order to generate torsion angles intervals. Torsion angles intervals are used as input to a genetic algorithm with a local-search operator in order to search the protein conformational space and predict its 3-D structure. Results show that the 3-D structures obtained by the proposed method were topologically comparable to their correspondent experimental structure.

En bioinformatique structurale, une partie des méthodes computationnelles qui calculent les structures de protéines à l'aide de données expérimentales, effectuent une optimisation de la position des atomes sous les contraintes expérimentales mesurées sur le système étudié, ainsi que sous des contraintes provenant de la connaissance générique de la stéréochimie organique. Ces méthodes d'optimisation présentent l'inconvénient de ne pas garantir la détermination de la meilleure solution. De plus, la validation de l'optimisation se fait en comparant les résultats obtenus pour des calculs répétés, et le résultat d'un calcul est accepté dans la mesure où le même résultat est obtenu plusieurs fois. Par cette approche, on rend plus difficile la détection de conformations alternatives de protéines, qui sont pourtant le sujet d'un vif intérêt dans la littérature. En effet, le développement de la sensibilité des techniques de résonance magnétique nucléaire (RMN) a permis de mettre en évidence plusieurs cas d'échange conformationnel reliés à la fonction des protéines. Dans ce projet de thèse, nous avons étudié une nouvelle approche pour le calcul de structures des protéines et l'exploration de leurs espaces conformationnels, basée sur la résolution du problème de Géométrie de Distance associé aux contraintes de distances dans une protéine par l'algorithme "interval Branch and Prune". Le logiciel implémentant cette méthode est appelée iBPprot, il incarne l'une des premières tentatives d'échantillonnage exhaustive des espaces conformationnels des protéines. Dans un premier temps, on s'est intéressé à l'application de la méthode en utilisant exclusivement des constraintes de distances exactes. Les résultats ont démontré que iBPprot était capable de reconstruire des structures références en s'appuyant seulement sur quelques contraintes à courte portée. De plus, la reconstruction a été d'une précision telle que la conformation générée présentait un RMSD de 1 Angstrom maximum avec la structure référence. L'exploration exhaustive de l'espace conformationnel a été possible pour une bonne partie des protéines cibles. Les temps de calcul pour l'exploration des espaces conformationnels ont été très variables allant de quelques secondes pour quelques protéines jusqu'à des semaines pour d'autres. L'évaluation de la qualité des structures obtenues a démontré qu'au moins 68% des valeurs de phi et psi sont localisées dans la zone 'core' du diagramme de Ramachandran. Cependant, des clash stériques ont été détectées dans plusieurs conformations mettant en jeu jusqu'à 7% d'atomes dans quelques unes de ces conformations. Dans un deuxième temps, on s'est intéressé à l'application de la méthode en incluant des intervalles de distances comme contraintes dans les calculs. Dans ce cas de figure, la méthode a réussi a reconstruire des structures références avec un RMSD inférieur à 5 Angstrom pour plus de la moitié des protéines cibles. En contre partie, le parcours complet de l'espace conformationnel n'a été possible que pour la plus petite protéine de l'ensemble des protéines étudiées. Pour la moitié des autres protéines, plus de 70% des atomes ont vu leurs positions échantillonnées. La qualité des structures obtenues a regressé en comparaison avec les simulations faites avec des distances exactes. En effet, seulement 53% des valeurs de phi et psi étaient localisées dans la zone 'core' du diagramme de Ramachandran, et le pourcentage d'atomes impliqués dans un clash stérique s'élevait jusqu'à 22% pour quelques protéines. Concernant le temps de calcul, le taux de génération de conformations a été déterminé pour chaque protéine cible, et il s'est avéré que globalement sa valeur etait compétitive par rapport aux valeurs des taux observables dans la littérature... / Structural biology has allowed us expand our knowledge of living organisms. It is defined as the investigation of the structure and function of biological systems at the molecular level. Studying a biomolecule's structure offers insight into its geometry, as angles and distances between the biomolecule's atoms are measured in order to determine the biomolecular structure. The values of these geometrical parameters may be obtained from biophysical techniques, such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. One of the most used methods to calculate protein structures from geometric restraints is simulated annealing. This method does not guarantee an exhaustive sampling of protein conformational space, which is a shortcoming as one protein may adopt multiple functional conformations, and it is important to determine them exhaustively. In this PhD project, the efficiency of a new method - derived from operations research and computational geometry - is studied in order to answer this question: How does this method explore the conformational spaces of small proteins? This method - implemented within the iBPprot software framework - treats protein structure determination as a distance geometry problem, which the interval branch-and-prune algorithm tries to solve by the full exploration of its solutions space. The results obtained by iBPprot on a set of test proteins, with sizes ranging from 24 to 120 residues and with known structures, are analyzed here. Using short-range exact distance restraints, it was possible to rebuild the structure of all protein targets, and for many of them it was possible to exhaustively explore their conformational spaces. In practice, it is not always possible to obtain exact distance restraints from experiments. Therefore, this method was then tested with interval data restraints. In these cases, iBPprot permitted the sampling of the positions of more than 70% of the atoms constituting the protein backbone for most of the targets. Furthermore, conformations whose r.m.s. deviations closer than 6 Angstrom to the target ones were obtained during the conformational space exploration. The quality of the generated structures was satisfactory with respect to Ramachandran plots, but needs improvement because of the presence of steric clashes in some conformers. The runtime for most performed calculations was competitive with existing structure determination method...

FGFs compreendem um grande família de 24 proteínas, participando de processos chaves nos mais variados tecidos, tendo funções parácrina, autócrina e intrácrina, regulando mitogênese, diferenciação celular, morfogênese e cicatrização. Mas, a relação estrutura-função dos FGFs é pobremente entendida. O membro protótipo desta família é o FGF-2, que apresenta quatro isoformas moleculares incluindo a forma de 18 kDa que é secretada e se liga aos receptores específicos (FGFRs) e dispara uma complexa sinalização. As outras isoformas, de alto peso molecular (21, 22 e 22,5 kDa) são expressas por códons alternativos (CUG) e permanecem no interior da célula interagindo com parceiros moleculares desconhecidos. Para antecipar mecanismos e parceiros do FGF-2 HMW foi realizada modelagem molecular desta isoforma que mostrou: uma estrutura do N-terminal da proteína com motivo β→α&#8594β e manutenção do barril β. A busca por parceiros intracelulares, foi realizada através da técnica do duplo hibrido de levedura, usando um biblioteca de cDNA de cérebro de rato. Foram encontrados 4 possíveis parceiros: BRD2, UBE2I, BRPF1, PC4. Todas essas interações foram confirmadas através do crescimento da levedura em meio sem histidina, produção de β-galactosidase e ensaios de \"pull-down\" com GST. Analises por FACS confirmam que FGF2 não causa apoptose em células adrenais tumorais Y1 de camundongo, mas promovem um acumulo de células na fase S com bloqueio do ciclo celular e da proliferação, configurando uma forma de senescência. Resultados com as células humanas HEK-ER:Ras permitem fazer a seguinte generalização: FGF2 induz senescência em células malignas transformadas pelos oncogenes raso A superexpressão da proteína de fusão FGF-2(18kDa):protA, mas não a da FGF-2(22,5 kDa):protA, protege a célula Y1 da senescência induzida por FGF-2. Por outro lado, a superexpressão destas mesmas isoformas de FGF-2 fusionadas à proteína A em células imortalizadas Balb3T3 não causou transformação celular e nem alterou a resposta mitogênica destas células ao FGF-2 recombinante adicionado ao meio de cultura. Células Y1 quando tratadas com FGF-2 recombinante produz ROS intracelular e libera anions superóxido no meio extracelular. Além disso, o anti-oxidante NAC protege estas células da indução de senescência induzida por FGF-2, sugerindo que ROS pode ser intermediário no disparo de senescência por FGF-2. / FGFs comprise a large fami1y of 24 proteins that play key roles in a number of tissues as local paracrine, autocrine and intracrine regulators of mitogenesis, cellular differentiation, organ morphogenesis and tissue repair. Structure-function relationship among FGFs is still poorly understood. FGF-2, the fami1y prototype member, exists as four molecular species. The 18 kDa form is released to the extracellular milieu and binds to specific receptors (FGFR), initiating a complex array of signals. Other isoforms of higher molecular weights (21, 22 and 22,5 kDa) are translated from alternative codons (CUG) and remain inside of the cell interacting with unknown partners. Aiming to anticipate mechanisms and partners, we modeled the FGF2-HMW molecule, showing that the protein displays β→α&#8594β motif in the N-terminal region and maintains the β-barrel structure common to ali FGFs. By the yeast two-hybrid method, using a cDNA rat brain library, we found four possible partners for FGF2-HMW: BRD2, UBE2I, BRP1 and PC4. Ali partners were confirmed by yeast growth without histidine, production of β-galactosidase and \"pull-down\" assays with GST. FACS analyses confirmed that FGF2 does not cause apoptosis in mouse Y1 adrenal tumor cells. But, FGF2 inhibited S phase progression blocking cell cycle and proliferation, characterizing a form of senescence. In addition, results obtained with the human HEK-ER:Ras cells support the following general statement: FGF2 triggers senescence in malignant cells transformed by ras oncogenes. Ectopic expression of the fusion protein FGF-2(18 kDa):protA, but not of FGF-2(22,s kDa):protA, protected Y1 cells senescence induced by FGF-2. On the other hand, ectopic expression of FGF-2 isoforms fusioned to protA in Balb3T3 immortalized cells did not cause transformation and neither modified the mitogenic response of this cell to recombinant FGF2. Recombinant FGF-2 stimules Y1 cells to produce intracellular ROS and to release superoxide anions into intracellular medium. Moreover, the ROS scavenger NAC protect Y1 cells from senescence induced by FGF-2, suggesting that ROS may be mediate senescence triggering induced by FGF-2.