Yes / Molecular dynamics (MD) simulations contain considerable information with regard to the motions and fluctuations of a protein, the magnitude of which can be used to estimate conformational entropy. Here we survey conformational entropy across protein fold space using the Dynameomics database, which represents the largest existing dataset of protein MD simulations for representatives of essentially all known protein folds. We provide an overview of MD-derived entropies accounting for all possible degrees of dihedral freedom on an unprecedented scale. Although different side chains might be expected to impose varying restrictions on the conformational space that the backbone can sample, we found that the backbone entropy and side chain size are not strictly coupled. An outcome of these analyses is the Dynameomics Entropy Dictionary, the contents of which have been compared with entropies derived by other theoretical approaches and experiment. As might be expected, the conformational entropies scale linearly with the number of residues, demonstrating that conformational entropy is an extensive property of proteins. The calculated conformational entropies of folding agree well with previous estimates. Detailed analysis of specific cases identify deviations in conformational entropy from the average values that highlight how conformational entropy varies with sequence, secondary structure, and tertiary fold. Notably, alpha-helices have lower entropy on average than do beta-sheets, and both are lower than coil regions. / National Institutes of Health, US Department of Energy Office of Biological Research, National Energy Research Scientific Computing Center, Swedish Research Council, Knut and Alic Wallenberg Foundation

Chapter 1 gives an introduction on the physical and electronic properties of fluorine and the C-F bond. The application of fluorine in organic chemistry, which is mainly attributed to the electronic properties of fluorine is described. The role of fluorine in neuropsychiatric drug development and for influencing the conformational study of bioactive amines is also illustrated. Chapter 2 of the thesis describes the synthesis of the two fluorinated stereoisomers (2R, 3S) and (2S, 3S) 3-fluoro N-methyl–D-aspartate (NMDA). These were prepared as analogues to study the binding conformation of NMDA on the glutameric NMDA receptor. The (2S, 3S)-3-fluoro NMDA D-72 was successfully prepared from diethyl D-tartrate. The (2S,3R)- stereoisomer was prepared by separation of diastereoisomers generated by reaction of a meso- epoxide with an enantiomerically pure amine, followed by fluorination. Both the (2S,3R)- and (2R,3S)- enantiomers were prepared separately, however assignment of the absolute configuration to each enantiomer could not be unambiguously proven. The fluorinated 3F-NMDA stereoisomers were assessed by dose response analysis and TEVC analysis in the rat glutamate receptor. The biological results show that the (2S, 3S)-3F NMDA D-72 is a good agonist, whereas (2R, 3S)- and (2S, 3R)-3-fluoro NMDA are inactive stereoisomers. The result of this study indicates that (2S, 3S)-3F NMDA D-72 is the only relevant agonist that can access a conformation for binding to NMDA receptor. Chapter 3 describes the preparation of fluorinated analogues of the calcium receptor agonist Cinacalcet. The (2R,1’R)-123 and (2S,1’R)-124 fluoro Cinacalcet diastereoisomers were prepared from 3’-(trifluoromethyl)cinnamic acid and 3’’-SF₅-137 Cinacalcet was synthesized from pentafluorosulfanyl benzyl alcohol. The biological assessment in the calcium receptor (CaR) revealed that both (2R,1’R)-123 and (2S,1’R)-124 fluoro Cinacalcet is slightly lower in potency compared to the non-fluorinated Cinacalcet 117. This suggests that the Cinacalcet 117 adopts an extended conformation when bound to the receptor. The 3’’-SF₅-137 Cinacalcet possesses equipotent activity with Cinacalcet 117.

The interaction mediated by integrin alphaLbeta2 and its ligand plays major role in many immune responses by regulating leukocyte adhesion. This study investigated the conformational regulation of alphaLbeta2 and the effects of conformational change on the ligand binding of alphaLbeta2. Micropipette adhesion frequency assay was used to measure the two-dimensional binding affinity and kinetics of alphaLbeta2 on K562 cells and neutrophils. The conformations of alphaLbeta2 were regulated by mutations, antibodies, small molecule antagonists, as well as divalent cations. Our results indicated that the change in binding affinity and off-rate was mostly due to the alphaL I domain conformational change. Without affecting the I domain conformation, the extension of alphaLbeta2 only increases the on-rate for several fold by providing a better orientation and accessibility of the molecule on cell surface. The binding characteristics of divalent cations to I domain MIDAS and other metal ion binding sites in alphaLbeta2 are determined by the nature of divalent cations, Mn2+ has higher binding affinity to the metal ion binding sites than Mg2+. The conformation of I domain also affected the binding of divalent cations. Open and intermediate I domains have higher binding affinity for Mn2+ and Mg2+ than WT and closed I domains. Divalent cations dissociate from I domain MIDAS very slowly but from those metal ion binding sites that important for conformational change of alphaLbeta2 rapidly. One of the most important biological processes mediated by alphaLbeta2 and other beta2 integrins is the recruitment and migration of neutrophils during inflammation. The activation of beta2 integrins by E-selectin binding to neutrophils in this process was also investigated. The binding of E-selectin, but not P- or L-selectin, activates beta2 integrins in a timescale of ~ 5 seconds and the activation may require the crosslink of E-selectin ligands. These results provide insights into the relationship between the conformational change and the function of alphaLbeta2 and most importantly would contribute to the understanding of integrin regulation mechanisms.

No description available.

<p>Ligand binding can modulate function of proteins and nucleic acids by changing both the populations of functionally distinct conformational states and the timescales on which they interconvert. For this reason, both thermodynamic and kinetic details of coupling can be important to proper function. How tightly does ligand bind to the different conformational states? What effect does ligand binding have on the conformational equilibrium and conformational kinetics? On what timescales and in what order do binding and conformational change occur? Using a combination of stopped-flow kinetics, isothermal titration calorimetry, and x-ray crystallography, we determine the mechanisms of coupled binding and conformational change in protein (Bacillus subtilis RNase P protein) and RNA (DP17 biosensor) systems. </p><p>The results demonstrate that rigorous kinetic analysis can be used to estimate the equilibrium and rate constants for conformational changes, as well as the affinities of ligands for different conformational states. A single ligand can bind to different conformational states of the same protein or nucleic acid with affinities that differ by orders of magnitude. This binding shifts the conformational equilibrium towards the higher affinity state through a combination of increasing rate constants for the forward conformational change and decreasing rate constants for the reverse conformational change. Using a flux-based analysis of the mechanisms we show that molecular recognition is kinetically partitioned between a number of pathways that differ by the order in which binding and conformational change occur. The absolute and relative flux through these pathways varies with ligand concentration, the affinities of the ligand for the various conformational states, and the ability of ligand to accelerate the conformational change. Together, the results give insights into how biological function depends on the kinetic and thermodynamic details of coupled binding and conformational change.</p> / Dissertation

This thesis concerns the construction, use and modulation of various host–guest systems, from small bispidines for binding of inorganic ions to bisporphyrin clips for supramolecular systems. Small flexible molecules undergo fast conformational movements when in solution. These conformational movements generate time-averaged population-weighted chemical shifts, coupling constants and NOEs when analysed by NMR spectroscopy. A bisporphyrin clip was designed to be used as a host for restriction of conformational movements of small flexible molecules by ditopic metal-ligand binding. Based on conformational analysis in combination with NMR analysis of molecular flexibility in solution (NAMFIS), the relative stereochemistry of flexible alditol-derived diamines containing three or four consecutive stereocentres could be determined. To further explore the idea of conformational deconvolution via host–guest binding, two flexible molecular tweezers with photoswitchable moieties were developed. Upon photoswitching cis/trans isomerisation facilitates the opening and closing of these bisporphyrin hosts. A guest molecule could then be exposed to a “catch and stretch” or “catch and release” effect. Preliminary studies have shown that photoisomerisation of the constructed systems is possible without photodecomposition and preliminary binding studies have been conducted. Controlled modulation of molecular conformations is of interest especially if the conformational steering activates a unit working as a nucleator in a larger structure or facilitates a reaction. The protonation-triggered modulation of bispidine conformations has been investigated. In addition to previously reported conformations we have observed that upon diprotonation a bispidine derivative can be driven into the unusual boat-boat conformation. Finally, the unexpected formation of persistent organic radicals with a cyclophane motif from the reaction of N,N´-diphenyl-1-5-diazacyclooctane and AgBF4 is described. Interestingly, these diradicals exhibit features such as intramolecular π-stacking without lateral displacement and also intramolecular spin pairing.

Better understanding of protein folding process can provide physical insights on the function of proteins and makes it possible to benefit from genetic information accumulated so far. Protein folding process normally takes place in less than seconds but even seconds are beyond reach of current computational power for simulations on a system of all-atom detail. Hence, to model and explore protein folding process it is crucial to construct a proper model that can adequately describe the physical process and mechanism for the relevant time scale. We discuss the reduced off-lattice model that can express <em>&alpha;</em>-helix and <em>&beta;</em>-hairpin conformations defined solely by a given sequence in order to investigate a protein folding mechanism of conformations such as a <em>&beta;</em>-hairpin and also to investigate conformational conversions in proteins. The first two chapters introduce and review essential concepts in protein folding modelling physical interaction in proteins, various simple models, and also review computational methods, in particular, the Metropolis Monte Carlo method, its dynamic interpretation and thermodynamic Monte Carlo algorithms. Chapter 3 describes the minimalist model that represents both <em>&alpha;</em>-helix and <em>&beta;</em>-sheet conformations using simple potentials. The native conformation can be specified by the sequence without particular conformational biases to a reference state. In Chapter 4, the model is used to investigate the folding mechanism of <em>&beta;</em>-hairpins exhaustively using the dynamic Monte Carlo and a thermodynamic Monte Carlo method an effcient combination of the multicanonical Monte Carlo and the weighted histogram analysis method. We show that the major folding pathways and folding rate depend on the location of a hydrophobic. The conformational conversions between <em>&alpha;</em>-helix and <em>&beta;</em>-sheet conformations are examined in Chapter 5 and 6. First, the conformational conversion due to mutation in a non-hydrophobic system and then the conformational conversion due to mutation with a hydrophobic pair at a different position at various temperatures are examined.

Gas-phase analytical techniques provide very valuable tools for tackling the structural complexity of macromolecular structures such as those encountered in biological systems. Conformational dynamics of polypeptides and polypeptide assemblies underlie most biological functionalities, yet great difficulties arise when investigating such phenomena with the well-established techniques of X-ray crystallography and NMR. In areas such as these ion mobility interfaced with mass spectrometry (IMMS) and molecular modelling can make a significant contribution. During an IMMS experiment analyte ions drift in a chamber filled with an inert gas; measurement of the transport properties of analyte ions under the influence of a weak electric field can lead to determination of the orientationally-averaged collision cross-section of all resolved ionic species. A comparison with cross-sections estimated for model molecular geometries can lead to structural assignments. Thus IMMS can be used effectively to separate gas-phase ions based on their conformation. The drift tube employed in the experiments described herein is thermally regulated, which also enables the determination of collision cross-sections over a range of temperatures, and can provide a view of temperature-dependent conformational dynamics over the experimental (low microsecond) timescale. Studies described herein employ IMMS and a gamut of other MS-based techniques, solution spectroscopy and – importantly – molecular mechanics simulations to assess a) conformational stability of isolated peptide ions, with a focus on small model peptides and proteins, especially the Trp cage miniprotein; and b) structural characteristics of oligomeric aggregates of an amyloidogenic peptide. The results obtained serve to clarify the factors which dominate the intrinsic stability of non-covalent structure in isolated peptides and peptide assemblies. Strong electrostatic interactions are found to play a pivotal role in determining the conformations of isolated proteins. Secondary structures held together by hydrogen bonding, such as helices, are stable in the absence of solvent, however gas-phase protein structures display loss of their hydrophobic cores. The absence of a polar solvent, “self-solvation” is by far the most potent force influencing the gas-phase configuration of these systems. Geometries that are more compact than the folded state observed in solution are routinely detected, indicating the existence of intrinsically stable compact non-native states in globular proteins, illuminating the nature of proteins’ ‘unfolded’ states.

L'une des principales cibles pour la thérapie visant le virus de l'hépatite C (VHC) est l'ARN polymérase dépendante de l'ARN NS5B indispensable à la réplication du génome virale. NS5B est l'une des enzymes clefs du cycle virale de VHC et son activation met en jeu aussi bien des interactions intramoléculaires que des interactions avec des cofacteurs viraux et cellulaires au sein du complexe de réplication. Nous avons développé une nouvelle stratégie d'inhibition de NS5B basée sur l'élaboration de peptides courts dérivés de motifs exposés à la surface de l'enzyme dans le but de cibler les nombreuses interactions impliquées dans l'activation de cette protéine. En associant une analyse fine de la structure cristallographique de NS5B avec de la modélisation moléculaire, nous avons élaboré des peptides courts mimant les motifs « hotspot » de la protéine. Ces peptides ont été évalués sur système réplicon de génotype 1b et nous avons ainsi identifié un peptide leader Moon1 de 15 résidus correspondant à un motif hautement conservé du domaine "thumb". Dans ce travail, nous avons étudié en détail la structure et le mécanisme moléculaire de ce nouvel inhibiteur de NS5B. Moon1 inhibe l'activité polymérase de la forme sauvage de NS5B ainsi que celle de mutants résistants au inhibiteurs nucléosidiques et non nucléosidiques. Nous avons démontré que la fixation de Moon1 entraine un changement de conformation de NS5B et se fait préférentiellement avec NS5B dans une conformation fermée. Ce peptide inhibe spécifiquement l'interaction entre NS5B et l'ARN double brin, indépendamment de la présence d'ions métalliques et de manière dose-dépendante. Moon1 bloque la transition entre l'étape d'initiation de novo de la synthèse d'ARN et l'extension du primer. Nous avons démontré que les résidus essentiels à l'activité de Moon1 sont hautement conservés à travers les différents génotypes et sous-types de VHC. De plus, nous avons établi une séquence minimale pour l'activité de Moon1. Nos travaux permettent de valider l'intérêt d'une stratégie interfaciale ciblant une enzyme clef du cycle du VHC et les interactions intra et intermoléculaires nécessaires à son activation. / The non-structural protein RNA-dependent RNA polymerase (RdRp) NS5B plays a key role in hepatitis C virus (HCV) replication and is currently considered as one of the most relevant target to develop safe anti-HCV agents. Although many small molecules have been identified as inhibitors of NS5B, very few are active in clinic. The structure and function of NS5B have been well characterized and as other polymerases, NS5B adopts a typical “right-hand” conformation containing the characteristic fingers, palm and thumb subdomains. The activation of NS5B requires conformational changes involving intramolecular contacts as well interactions with viral proteins and host factors in the replication complex. We developed a new strategy for NS5B inhibition based on short interfacial peptides derived from NS5B surface accessible motifs that target protein-protein interfaces or essential motifs involved in NS5B-activation. Combining the NS5B crystallogaphic structure and molecular modelling, we have designed short peptides derived from NS5B surface “hotspots” that were screened using HCV genotype 1b replicon cell system. We have identified Moon1, a short 15-residu peptide, derived from a well-conserved motif located in the NS5B thumb domain that inhibits HCV replication in the low nanomolar range. Moon1 tightly binds NS5B in a conformational-dependent manner and induces NS5B conformational changes. This peptide specifically inhibits double-stranded RNA/NS5B interactions in a dose-dependent and metal ions-independent manner. Moon1 blocks the transition between RNA de novo initiation and primer-extension. We showed that residues required for Moon-1 anti-polymerase activity are well-conserved among HCV genotypes and subtypes and a minimal Moon1 active motif was established. Taken together, these results demonstrate that NS5B structural dynamics constitute an attractive target for HCV chemotherapeutics and for the design of more specific new antiviral drugs.

Mediante a técnica de espalhamento de raio-X a baixos ângulos por macromoléculas em solução, estudou-se as variações de forma e tamanho da crotamina, proteína básica e neurotóxica do veneno das cascavéis (crotalus durissus terrificus), em diferentes condições de pH e uma concentração de 10% visando a determinação dos valores do raio de giro. Este raio, RG, é definido como RG = (&#8747vr2 &#961 r&#963; dv&#963;) 1 &#8260 2 onde v é o volume da macromolécula, r o módulo do vetor posição r&#963 com origem no centro de massa, &#961 (r&#963) a densidade eletrônica e n o número de elétrons da macromolécula. Este parâmetro não conduz obviamente ao conhecimento completo da estrutura da macromolécula, mas a sua determinação possibilita estudo de variações conformacionais de forma relativamente simples em sistemas com condições semelhantes às biológicas. Os valores do raio de giro foram determinados mediante gráficos de Guinier (Log JN(s) vs s2, onde s é o módulo do vetor posição do espaço recíproco e JN(s) a intensidade normalizada e livre de espalhamento parasita). Preliminarmente, foi feito um estudo com oxi-hemoglobina, Insulina e hemoglobina de caramujo, por apresentarem características semelhantes às da crotamina, isto é, serem do tipo globular e de forma aproximadamente esférica com o objetivo de familiarizar-se com a técnica experimental e testar a aparelhagem. Os valores de raio de giro deduzidos para a oxi-hemoglobina RGOX= 23.9 &#177 0.7 (A &#176), Insulina RGI= 18.4 &#177 0.5 (A &#176), e hemoglobina de caramujo RGhc= 92 &#177 2 (A&#176), concordam satisfatoriamente com resultados previamente encontrados na literatura. Os valores de raio de giro medido para as diferentes soluções de crotamina encontram-se em bom acordo com a teoria e com o valor do raio de giro esperado de crotamina REC= 9.7 A&#176 / The small angle x-ray scattering technique was used in order to determine the radius of gyration of crotamine, a basic and neurotoxic protein from rattle snake venom, in 10% solutions at different PHs. The radius of gryration, RG is defined as: RG = (&#8747vr2 &#961 r&#963; e dv&#963;) 1 &#8260 2 Where: v is the volume of the macromolecule, r is the modulus of the position vector r with origin at the center of mass, &#961 (r&#963) is the electronic density and n the number of electrons of the macromolecule. This parameter does not give a complete information about the structure of the macromolecule but it is possible to get information about conformation to close to the biological ones. The values of the radius of gyration were fdetermined by means of the Guinier graphics (Log JN(s) vs s2, where s is the magnitude of the scattering vetor and JN(s) is the normalized intensity. In order to get familiar wi th the experimental technique and to test the equipment the study of oxihaemoglobin, Insulin and sea-mail hemoglobin was done. This proteins are very similar to crotamine in the sense that they are globular type and almost of the same spherical shape. The values of the radius of gyration obtained agree very well with the reported ones: Oxihaemoglobin RGOX= 23.9 &#177 0.7 (A &#176), Insulin RGI= 18.4 &#177 0.5 (A &#176), and sea-mail hemoglobin RGhc= 92 &#177 2 (A&#176). The values od the radius of gyration measured for the different solution of crotamine are in good agreement with the calculated and with the expected value REC= 9.7 A&#176