Defining the intrinsic properties of amino acids which dictate the formation of helices, the most common protein secondary structure element, is an essential part of understanding protein folding. Pauling and co-workers initially predicted helical peptide folding motifs in the absence of solvent, suggesting that in vacuo studies may potentially discern the role of solvation in protein structure. Ion mobility-mass spectrometry (IMMS) combines a gas-phase ion separation based on collision cross-section (apparent surface area) with time-of-flight MS. The result is a correlation of collision cross-section with mass-to-charge, allowing detection of multiple conformations of the same ion. Most gas-phase peptide ions assume a compact, globular state that minimizes exposure to the low dielectric environment and maximizes intramolecular charge solvation. Conversely, a small number of peptides adopt a more extended (β-sheet or α-helix) conformation and exhibit a larger than predicted collision cross-section. Collision cross-sections measured using IM-MS are correlated with theoretical models generated using simulated annealing and allow for assignment of the overall ion structural motif (e.g. helix vs. chargesolvated globule). Here, two series of model peptides having known solution-phase helical propensities, namely Ac-(AAKAA)nY-NH2 (n = 3, 4, 5, 6 and 7) and Ac-Y(AEAAKA)nF-NH2 (n = 2, 3, 4, and 5), are investigated using IM-MS. Both protonated ([M + H]+) and metalcoordinated ([M + X]+ where X = Li, Na, K, Rb or Cs) species were analyzed to better understand the interplay of forces involved in gas-phase helical structure and stability. The data are analyzed using computational methods to examine the influence of peptide length, primary sequence, and number of basic (Lys, K) and acidic (Glu, E) residues on anhydrous ion structure.

This work investigates pathways of conformational transitions in ubiquitous RNA structural motifs. In our lab, we have developed multi-scale structural datamining techniques for identification of three-dimensional structural patterns in high-resolution crystal structures of globular RNA. I have applied these techniques to identify variations in the conformations of RNA double-helices and tetraloops. The datamined structural information is used to propose reaction coordinates for conformational transitions involved in double-strand helix propagation and tetraloop folding in RNA. I have also presented an algorithm to identify stacked RNA bases. In this work, experimentally derived thermodynamic evaluation of the conformations has been used to as an additional parameter to add detail to RNA structural transitions. RNA conformational transitions help control processes in small systems such as riboswitches and in large systems such as ribosomes. Adopting functional conformations by globular RNA during a folding process also involves structural transitions. RNA double-helices and tetraloops are common, ubiquitous structural motifs in globular RNA that independently fold in to a thermodynamically stable conformation. Folding models for these motifs are proposed in this work with probable intermediates ordered along the reaction coordinates. We hypothesize that frequently observed structural states in crystals structures are analogous in conformation to stable thermodynamic â on-pathwayâ folded states. Conversely, we hypothesize that conformations that are rarely observed are improbable folding intermediates, i.e., these conformational states are â off-pathwayâ states. In general on-pathway states are assumed to be thermodynamically more stable than off-pathway states, with the exception of kinetic traps. Structural datamining shows that double helices in RNA may propagate by the â stack-ratchetâ mechanism proposed here instead of the commonly accepted zipper mechanism. Mechanistic models for RNA tetraloop folding have been proposed and validated with experimentally derived thermodynamic data. The extent of stacking between bases in RNA is variable, indicating that stacking may not be a two-state phenomenon. A novel algorithm to define and identify stacked bases at atomic resolution has also been presented in this work.

Group I introns are structured RNAs that have been used extensively as model systems for RNA folding because they are experimentally tractable, yet complex enough to have folding challenges associated with larger RNAs. The Tetrahymena group I intron consists of a set of conserved core helices and a set of peripheral elements. Peripheral elements surround the core helices and form long range tertiary contacts between each other and to the core. Interestingly, a long-lived misfolded state is populated that has the same long range tertiary contacts as the native state but differs locally within the core. Our lab showed that the intact periphery is necessary to specify the correct core structure, as mutating tertiary contacts or removing the P5abc peripheral element dramatically destabilized the native ribozyme relative to the misfolded form. However, we also showed that the thermodynamic benefit peripheral structure provided is accompanied by kinetic liability in folding, apparently because native tertiary contacts formed by peripheral elements around the misfolded core must come apart to allow refolding of the misfolded RNA to the native state. In addition to peripheral elements, proteins also play a role in stabilizing the native structures of many group I introns. The CYT-18 protein, which occupies the same binding site as P5abc, stabilizes the functional structures of certain group I introns by using a set of insertions that are absent in other related bacterial and mitochondrial aminoacyl tRNA synthetases. Using the P5abc deletion variant of the Tetrahymena ribozyme, I sought to further define CYT-18 roles in RNA folding by probing its thermodynamic and kinetic effects on the native state formation relative to the misfolded state. I demonstrated that CYT-18, like P5abc, provided thermodynamic stability to the native state. However, unlike P5abc, CYT-18 had no apparent effect on the refolding kinetics, suggesting that a protein co-factor can stabilize the functional structure without acquiring the associated costs in RNA folding kinetics. Furthermore, I found that the mechanism of CYT-18 action appears to be distinct from P5abc. Disruption of the long-range contact P14, which is formed between P5c and L2 and is part of the network of peripheral contacts, dramatically weakened P5abc binding to the native ribozyme core by ~10⁸ fold. Interestingly, CYT-18 maintained specific and tight binding to these mutants, which suggests that CYT-18 does not rely on a circular network of contacts to specifically stabilize the native state. Instead, the specificity may arise from a more direct and intimate contact of CYT-18 with the ribozyme core. This study gives insight into an evolutionary advantage of protein co-factors in RNA folding; proteins may offer thermodynamic assistance without inhibiting folding kinetics. / text

The fundamental process by which an alpha-helical membrane protein attains its ultimate structure has previously been depicted as two energetically distinct stages where (1) the transmembrane (TM) segments are first threaded into the membrane bilayer as stable alpha-helices; and then (2) laterally interact to form the correct tertiary and/or quaternary structures. Central to the second stage of this model is the presence of amino acid sequence motifs in the TM segments that provide interaction-compatible surfaces through which the TM alpha-helices interact. Although these ideas have proven to be pivotal to the progress of the membrane protein folding field, a growing number of examples indicates that a variety of additional factors work together to dictate the ultimate interaction fate of TM embedded segments. In this context, we expand on these factors and explore other properties that can modulate the association of TM alpha-helices. A peptide model of myelin proteolipid protein (PLP) TM4 is capable of TM helix-helix interactions in SDS and biological membranes. Increasing the side chain volumes of two disease relevant residues (Ala242 and A248) reduces peptide self-association, indicating that these sites mediate TM helix packing through van der Waals interactions. Examination of the PLP TM2 alpha-helix shows that it is also capable of self-association and that its dimeric state depends on the presence or absence of residues at its C-terminus. Specifically, this sensitivity was attributed to changes in local hydrophobicity; a decrease in hydrophobicity likely reduces detergent-peptide interactions, which disrupts peptide alpha-helicity and the effectiveness of a nearby interaction compatible surface. We take advantage of this finding to determine the feasibility of coupling helix-helix interactions to an external factor such as pH. Our results indicate that pH can indeed modulate the dimerization state of the TM2 peptide and does so through the change in protonation state of Glu88. Increasing our knowledge of the variables contributing to TM helix-helix interactions provides valuable insights into membrane protein folding and how mutations can compromise this process. This knowledge will allow us to expand our arsenal of approaches to counter membrane protein misassembly--and ultimately human disease.

The fundamental process by which an alpha-helical membrane protein attains its ultimate structure has previously been depicted as two energetically distinct stages where (1) the transmembrane (TM) segments are first threaded into the membrane bilayer as stable alpha-helices; and then (2) laterally interact to form the correct tertiary and/or quaternary structures. Central to the second stage of this model is the presence of amino acid sequence motifs in the TM segments that provide interaction-compatible surfaces through which the TM alpha-helices interact. Although these ideas have proven to be pivotal to the progress of the membrane protein folding field, a growing number of examples indicates that a variety of additional factors work together to dictate the ultimate interaction fate of TM embedded segments. In this context, we expand on these factors and explore other properties that can modulate the association of TM alpha-helices. A peptide model of myelin proteolipid protein (PLP) TM4 is capable of TM helix-helix interactions in SDS and biological membranes. Increasing the side chain volumes of two disease relevant residues (Ala242 and A248) reduces peptide self-association, indicating that these sites mediate TM helix packing through van der Waals interactions. Examination of the PLP TM2 alpha-helix shows that it is also capable of self-association and that its dimeric state depends on the presence or absence of residues at its C-terminus. Specifically, this sensitivity was attributed to changes in local hydrophobicity; a decrease in hydrophobicity likely reduces detergent-peptide interactions, which disrupts peptide alpha-helicity and the effectiveness of a nearby interaction compatible surface. We take advantage of this finding to determine the feasibility of coupling helix-helix interactions to an external factor such as pH. Our results indicate that pH can indeed modulate the dimerization state of the TM2 peptide and does so through the change in protonation state of Glu88. Increasing our knowledge of the variables contributing to TM helix-helix interactions provides valuable insights into membrane protein folding and how mutations can compromise this process. This knowledge will allow us to expand our arsenal of approaches to counter membrane protein misassembly--and ultimately human disease.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work is designed to study helices and Bertrand curves. A circular helix is characterized by having constant curvature k 6= 0 and constant torsion t . If the ratio t k is constant, the curve is called generalized helix. A curve g : I −→R3 is called a Bertrand curve if there is another curve g : I −→R3 such that the normal lines of g and g at s ∈ I are equal. Generalized helices and Bertrand curves can be viewed as generalizations of the circular helix. In this work, we obtain important characterizations of these curves. Besides, we also study these curves from the view point of the theory of curves on ruled surfaces. / O presente trabalho destina-se a um estudo sobre hélices e curvas de Bertrand. Uma hélice circular é caracterizada por ter curvatura k 6= 0 e torção t constantes. Se a razão t k for constante, a curva é chamada hélice generalizada. Uma curva g : I −→ R3 é chamada curva de Bertrand se existe uma outra curva g : I −→ R3 tal que as retas normais de g e g em s ∈ I são iguais. Tanto a hélice generalizada como a curva de Bertrand podem ser vistas como generalizações da hélice circular. Neste trabalho, além de obtermos importantes caracterizações destas curvas, realizamos também um estudo destas do ponto de vista da teoria de curvas em superfícies regradas.

Les α-hélices sont des éléments clés de la reconnaissance biomoléculaire, comme en témoigne le fait qu'une quantité significative de complexes protéine-protéine, dans la banque de données protéiques (PDB), présentent des interfaces inter-hélices. Cependant, de courtes hélices peptidiques isolées dans le but de bloquer ces interactions ne sont généralement que faiblement présentes en milieu aqueux et sont sensibles à la dégradation protéolytique, limitant ainsi leur potentiel thérapeutique. Diverses approches chimiques ont été proposées pour augmenter la propension au repliement en hélice des α-peptides. Une stratégie consiste à pré-organiser les premières liaisons amides à l'aide d'une « capping box » ou d'un substitut de liaison hydrogène. Récemment, nous nous sommes intéressés à la possibilité d'interfacer des peptides-α et des foldamères afin d’élaborer des « foldamères à blocs » générant ainsi un nouveau type d’architecture mime de l'hélice-α. Dans notre laboratoire, nous avons développé des foldamers à base d’urées qui s’organisent pour former des structures hélicoïdales. Les similitudes du sens d’enroulement, du pas et de la polarité entre l’hélice-α peptidique et l’hélice-2.5 d'oligourée suggéraient qu'il serait possible de combiner ces deux squelettes. Au cours de cette thèse, nous avons montré que les chimères : oligourée/α-peptide, forment des structures hélicoïdales bien définies dans les solvants organiques polaires avec la propagation d'un réseau de liaisons hydrogène intramoléculaires continu couvrant la totalité de la séquence. Ces études ont suggéré que le squelette de l'oligourée qui possède une forte propension à adopter une structuration en hélice pourrait conduire au développement d’un « cap » permettant la pré-organisation des quatre premiers NHs ainsi que des quatre derniers groupements carbonyles d’une hélice-α peptidique. Nous avons donc étudié l'influence de courts fragments oligourées sur la stabilisation de séquences peptidiques modèles solubles dans l'eau en hélices-α, conduisant au développement de la « foldamer capping box ». Grâce à cette nouvelle stratégie de stabilisation des hélices peptidiques, nous avons pu concevoir des inhibiteurs de l’interaction protéine/protéine p53/MDM2. / Α-Helices are key elements of biomolecular recognition, as reflected by the fact that a large fraction of the protein-protein complexes in the Protein Data Bank (PDB) feature helical interfaces. However, short isolated peptide helices are generally only weakly populated in aqueous environment and are sensitive to proteolytic degradation, thus limiting their therapeutic potential. Various chemical approaches have been proposed to increase the helix folding propensity of α-peptides. One strategy is to pre-organize the first amide bonds through the use of a "capping box" or a hydrogen bond surrogate. Recently we became interested by the possibility to interface peptide and foldamer helical backbones in order to develop “block co-foldamers“, to generate new generations of α-helix mimics. In our laboratory, we have developed oligourea foldamers which are organized to form helical structures. The similarities in helix screw sense, pitch, and polarity between the peptide α-helix and the oligourea 2.5-helix suggested that it would be feasible to combine these two backbones. In this thesis, we have shown that the resulting oligourea/α-peptide chimeras form well-defined helical structures in polar organic solvents with the propagation of a continuous intramolecular hydrogen bonding network spanning the entire sequence. These studies provided a rationale for the use of the oligourea backbone which is strongly biased towards helix formation could lead to the development of pre-organized caps for the initial four amide NHs and the final four carbonyl groups of a peptide α-helix. We have therefore studied the influence of short oligourea fragments on the stabilization of model water-soluble peptide sequences in α-helices, leading to the development of the foldamer capping box. This strategy awas pplied for the first time to the design of potent inhibitors of protein/protein interactions (e.g. p53/MDM2).

La formation de nanostructures bien définies par autoassemblage de briques organiques a reçu de nombreuses attentions dues à leurs potentielles applications en chimie comme en biologie. Parmi toutes ces éléments organiques, les peptides et pseudopeptides font partie des plus prometteurs de par leur ressemblance aux protéines. Nous pouvons dénombrer de nombreux autoassemblages peptidiques, comme les nanotubes, les nanofibres, les vésicules, les nanosphères etc… Dans ce contexte, nous nous sommes intéressés à la synthèse et à l’étude structurale globale des hydrazinopeptides. Grâce à leur azote supplémentaire, ces pseudopeptides bis-azotés sont capables de s’auto-assembler en de nouvelles structurations. Les 1:1[α/α-Nα-Bn-hydrazino]peptides linéaires ont démontré se structurer en hydrazinoturn et γ-turn en solution. Nous mettrons en évidence la capacité des analogues 1:1[ß/α-Nα-Bn-hydrazino]peptides linéaires à s’autostructurer en hydrazinoturn en solution, n’impliquant pas le squelette du motif ß-aminoacide. De la même manière, nous démontrerons la capacité des pseudopeptides purs hydrazino à se structurer en solution en une structure très solide formées de successions d’hydrazinoturn, observable à l’état cristallin également. Dans un deuxième temps, et dans le cadre de l’élaboration de structures nanotubulaires, nous avons étudié une série de 1:1[α/α-Nα-Bn-hydrazino]peptides cycliques, et tout particulièrement les cyclotétramères. Dans ce contexte, nous avons cherché à mettre en lumière les différents paramètres pouvant influencer l’organisation nanotubulaires dans nos macrocycles, dans le but d’élaborer la meilleure stratégie afin d’obtenir cette nanostructuration en vues d’éventuelles applications. Les différents paramètres étudiés sont les suivants : la stratégie de synthèse, la chiralité, l’orientation des chaînes latérales, et enfin l’aptitude à former des gels / The formation of nanostructures with well-defined organic brick self-assembly has received much attention due to their potential applications in chemistry and biology. Among all these organic elements, peptides and pseudopeptides are among the most promising because of their similarity to proteins. We can enumerate many peptides self-assembly, such as nanotubes, nonafibres, vesicles, nanospheres etc … In this context, we are interested by the synthesis and overall structural study of hydrazinopeptides. Thanks to the additional nitrogen, these bis-nitrogen pseudopeptides are able to self-assemble into new structuring. The 1:1[α/α-Nα-Bn-hydrazino] linear peptides showed coalesce into hydrazinoturn and γ-turn in solution. We have highlighted the ability of analogues of 1:1[ß/α-Nα-Bn-hydrazino] linear peptides to be structured in hydrazinoturn only, in solution. Similarly, we have demonstrated the ability of pure α-Nα-Bn-hydrazino pseudopeptides to be structured in a very solid solution structure formed of hydrazinoturn observable also in crystal state. Secondly, and as part of the development of nanotube structures, we have studied a serie of 1:1[α/α-Nα-hydrazino] cyclic peptides, and especially cyclotetrameres. In this context, we have sought to highlight the various parameters that may affect the organization in nanotubes, in order to develop the best strategy to achieve this potential application in nanostructuring views. The various parameters studied are: the synthetic strategy, the chirality, the orientation of side chains, and finally the ability to form gels

Positive-strand RNA viruses associate with specific organelle membranes of host cells to establish viral replication complexes. The replication protein 1a of brome mosaic virus associates strongly with the nuclear endoplasmic reticulum (ER) membranes, invaginates membranes into the lumen, and recruits various host proteins to establish replication complexes termed spherules. 1a has a strong affinity towards the perinuclear ER membrane, however, the structural features in 1a that dictate its membrane associations and thereby membrane remodeling activities are unclear. This study examined the possible role of an amphipathic α-helix, helix B, in BMV 1a's membrane association. Deletion or single substitution of multiple amino acids of helix B abolished BMV 1a's localization to nuclear ER membranes. Additional reporter-based, gain-of-function assays showed that helix B is sufficient in targeting several soluble proteins to the nuclear ER membranes. Furthermore, we found that the helix B-mediated organelle targeting is a functionally conserved feature among positive-strand RNA viruses of the alphavirus-like superfamily that includes notable human viruses such as Hepatitis E virus and Rubella virus as well as plant viruses such as cucumber mosaic virus and cowpea chlorotic mottle virus. Our results demonstrate a critical role for helix B across members of the alphavirus-like superfamily in anchoring viral replication complexes to the organelle membranes. We anticipate our findings to be a starting point for the development of sophisticated models to use helix B as a novel target for the development of antivirals for positive-strand RNA viruses that belong to the alphavirus-like superfamily. / Doctor of Philosophy / Among the seven classes of viruses, the positive-strand RNA viruses dominate the domain of viral diseases of the world. Brome mosaic virus (BMV) is a positive-strand RNA virus that infects cereal crops such as wheat, barley, and rice. BMV has a simple genome organization and serves as a suitable model virus to study and characterize positive-strand RNA viruses. The replication of all positive-strand RNA viruses occurs at the organelle membranes of the host. Membrane association of the replication is one of the early steps and a crucial event in the life cycle of positive-strand RNA viruses. One of the proteins produced early on during BMV infection is the replication protein 1a, which is also the master regulator of viral replication; 1a recruits viral factors in addition to hijacking the necessary host factors at the membranous sites to initiate replication. Upon reaching the organelle membranes, 1a induces membrane rearrangements to form viral replication complexes that safeguard the recruited factors from the deleterious effects of the host cell. The structural determinants within 1a that are responsible for such membrane association are unknown. This study explored the potential roles of a short helical motif within the 1a protein for its ability to dictate such site-specific membrane associations. We show here that this helical region is necessary and sufficient for 1a's membrane-binding activity. We also discovered it to be a functionally conserved feature that is responsible for membrane associations in various viruses of the alphavirus-like superfamily that includes some of the notable human viruses such as Hepatitis E virus and Rubella virus in addition to plant viruses such as cucumber mosaic virus and cowpea chlorotic mottle virus.