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Allostery is a regulatory process whereby a perturbation by an effector at one discrete locus creates a conformational change that stimulates a functional change at another. The two sites communicate through networks of interacting residues that respond in a concerted manner to the allosteric perturbation. These allosteric networks are traditionally mapped with high resolution structure determination techniques to understand the conformational changes that regulate protein function as well as its modulation by allosteric ligands and its dysfunction caused by disease-related mutations (DRMs). However, high resolution structural determination techniques, such as X-ray crystallography, cryo-electron microscopy and nuclear Overhauser effect NMR spectroscopy are not always amenable for systems plagued by poor solubility and line broadening caused by μs-ms dynamics or systems where allostery relies primarily on dynamical rather than structural changes. This dissertation discusses methodologies to map the allosteric sites and pathways for such challenging systems. The foundation of this approach is to model allosteric pathways in the context of their respective thermodynamic cycles. In chapter 2, the thermodynamic cycle of a DRM in the hyperpolarization-activated cyclic nucleotide-gated ion channel 4 (HCN4) is analyzed with respect to structure, dynamics and kinetics, revealing how the DRM remodels the free energy landscape of HCN4 and results in a loss-of-function disease phenotype. In chapter 3, the mechanism of action of an uncompetitive inhibitor for the exchange protein activated by cAMP is elucidated by characterizing its selectivity for distinct conformations within the thermodynamic cycle that are trapped using a combination of mutations and ligand analogs. In chapter 4, we discuss two new protocols for the chemical shift covariance analysis (CHESCA). The CHESCA is an approach that identifies allosteric signaling pathways by measuring concerted residue responses to a library of chemical perturbations that stabilize conformational equilibria at different positions. Overall, the approaches discussed in this dissertation are widely applicable for mapping the mechanisms of allosteric perturbations that arise from ligand binding, post-translational modifications and mutations, even in systems where traditional structure determination techniques remain challenging to implement. / Thesis / Doctor of Philosophy (PhD) / Allostery is a regulatory mechanism for proteins, which controls functional properties of one distinct site through the perturbation of another distinct, and often distant, site. The two sites are connected via a series of residues that undergo conformational changes once perturbed by the allosteric effector. Mapping these communication pathways reveals mechanisms of protein regulation, which are invaluable for developing pharmacological modulators to target these pathways or for understanding the mechanisms of disease mutations that disrupt these pathways. Allosteric pathways have been traditionally determined using structure determination approaches that provide a static snapshot of the protein’s structure. However, these approaches are typically not effective when allostery relies extensive changes in dynamics. The goal of this thesis was to develop methods to characterize systems that are dynamic or otherwise unsuitable for traditional structure determination. Herein, we utilize NMR spectroscopy to analyze the allosteric mechanisms of three cAMP-binding proteins involved in cardiovascular health.

O trocador de Na+/Ca2+ (NCX) constitui um dos principais mecanismos de extrusão de Ca2+ intracelular em células excitáveis. Foi demonstrado que alterações no funcionamento do NCX estão relacionadas a diversas situações patológicas. Por este motivo, o entendimento do mecanismo molecular da manutenção da concentração de Ca2+ intracelular via NCX é importante para a compreensão do funcionamento do trocador, bem como para o desenvolvimento de fármacos. Além de transportar Na+/Ca2+, o NCX também é regulado por esses íons. Este trocador é composto por dois domínios transmembranares, cada um deles contendo 5 α-hélices (TM), e uma grande alça intracelular que conecta as hélices TM5 e TM6. O domínio transmembranar é responsável por catalisar o transporte de Na+/Ca2+ através da bicamada lipídica, enquanto que a alça citoplasmática está envolvida com a regulação do trocador. Esta alça contém dois domínios sensores de Ca2+ adjacentes, denominados CBD1 e CBD2. Apesar da importância fisiológica do NCX, o mecanismo de regulação alostérica do trocador por Ca2+ intracelular permanece desconhecido. Neste trabalho, a espectroscopia de ressonância magnética nuclear (RMN) de alta resolução foi utilizada para investigar a conformação e a dinâmica de CBD1 e CBD2 do trocador de Na+/Ca2+ de Drosophila melanogaster (CALX), isolados ou conectados covalentemente em uma construção denominada CBD12. Um total de 98% das ressonâncias da cadeia principal de CBD1 isolado na presença de Ca2+ foi assinalado, enquanto que na ausência de Ca2+, assinalamentos para apenas uma parte da cadeia principal puderam ser obtidos. Os assinalamentos adquiridos para CBD12 foram baseados na análise de um conjunto de espectros de RMN tridimensional heteronuclear e por comparação com os espectros dos domínios isolados. Uma análise preliminar dos deslocamentos químicos e dos parâmetros de relaxação de 15N obtidos para CBD1 indicou que este domínio é flexível na ausência de Ca2+, mas torna-se rígido após a adição deste íon. As medidas das velocidades de relaxação de 15N e de acoplamentos dipolares residuais (RDCs) de 1H-15N realizadas para CBD12 nas formas apo e holo indicaram que a ligação de Ca2+ em CBD1 estabiliza uma orientação rígida entre os domínios. A análise dos RDCs de 1H-15N mostrou ainda que a orientação média entre CBD1 e CBD2 é praticamente linear na ausência de Ca2+, enquanto que um ângulo menor é assumido após a adição deste íon. Os dados descritos nesta tese suportam um modelo de regulação alostérica em que a modulação da plasticidade de CBD12 pela ligação de Ca2+ no domínio CBD1 controla a abertura e o fechamento do trocador. / The Na+/Ca2+ exchanger (NCX) is a major mechanism for the extrusion of intracellular Ca2+ in excitable cells. It was demonstrated that altered functioning of this protein is related to various pathological situations. Therefore, the understanding of the molecular mechanism for maintaining the intracellular Ca2+ concentration by means of the NCX is important to understand the functioning of the exchanger and to develop drug-based therapies. Besides transporting Na+/Ca2+, the exchanger is also regulated by these ions. The NCX is composed of two transmembrane domains, each of them containing 5 transmembrane alpha-helices (TM), and a very large cytosolic loop that connects TM5 to TM6. The transmembrane domains are responsible for catalyzing the transport of Na+ and Ca2+ ions across the lipid bilayer, while the cytosolic loop is involved in regulation of the exchanger activity. It contains two regulatory Ca2+- binding domains, called CBD1 and CBD2, that appear in tandem. Despite the physiological importance of the NCX, the mechanism of allosteric regulation of the exchanger by intracellular calcium remains unclear. In this work we used high-resolution NMR spectroscopy to study the conformation and the dynamics of the two Ca2+-binding regulatory domains of Drosophila\'s Na+/Ca2+ exchanger (CALX), CBD1 and CBD2, in isolation as well as in a covalent construct called CBD12. Complete backbone NMR resonance assignments were obtained for the isolated CBD1 domain in the Ca2+-bound state, while partial assignments were obtained for CBD1 in the free state. Partial backbone NMR resonance assignments were obtained for the CBD12 construct through the analysis of a standard set of triple resonance NMR spectra. Additional assignments were obtained by comparison with the isolated CBD1 and CBD2 domains. A preliminary analysis of NMR chemical shifts and 15N relaxation data obtained for CBD1 indicates that this domain displays considerable amount of flexibility in the free state, but becomes more rigid upon Ca2+-binding. NMR 15N relaxation rates and 1H-15N residual dipolar couplings (RDCs) obtained for the Apo and Ca2+-bound states of the CBD12 domain indicate that calcium binding stabilizes a rigid inter-domain orientation. Analysis of 1H-15N RDCs further shows that Drosophila\'s CBD12 domain assumes an almost linear inter-domain orientation in the absence of Ca2+, while a smaller inter-domain angle was found in its presence. These findings support a model in which modulation of CBD12 plasticity by the binding of Ca2+ to the CBD1 domain controls the opening and closing of the exchanger.

Um mit ihrer Umgebung zu kommunizieren verfügen lebende Zellen über Rezeptoren, welche die umschließende Membran überbrücken. Die vorherrschende G-Protein-gekoppelte Rezeptoren (GPCR) erhalten Informationen von Außerhalb durch Bindung eines Liganden, wodurch der Rezeptor aktiviert wird. Während der Aktivierung bildet sich innerzellulär ein offener Spalt, in den ein G-Protein (Gαβγ, G) mit seinem C-terminalen Ende koppeln kann. Die Bindung an einen GPCR führt in der Gα-Untereinheit vom Gαβγ zu einen GDP/GTP-Austausch, welcher für die weitere Signalübertragung ins Zellinnere notwendig ist. Die Kopplung von Rezeptor und Gαβγ umfasst eine Reihe von dynamischen strukturellen Änderungen, die Geschwindigkeit und Spezifität der Interaktion regeln. Hier haben wir MD-Simulationen (Molekulardynamik) verwendet, um die molekularen Details der GPCR Gαβγ Kopplung vor und während der GPCR-Gαβγ-Komplexbildung bis hin zum GDP/GTP-Austausch zu untersuchen. / To communicate with their environment, living cells feature receptors that provide a bridge across the enclosing membrane. The prevalent G protein-coupled receptors (GPCR) receive outside information through the binding of a ligand, which activates the receptor. During activation, an open intracellular crevice forms, to which a G protein (Gαβγ, G) can couple with its Gα C-terminus. Binding to GPCRs triggers GDP/GTP exchange in the Gα subunit of Gαβγ, necessary for further signal transfer within the cell. The coupling between receptor and Gαβγ involves a series of dynamic structural changes that govern speed and specificity of the interaction. Here we used molecular dynamics (MD) simulations to elucidate molecular details of the GPCR Gαβγ coupling process before and during GPCR Gαβγ complex formation up to the GDP/GTP exchange.

O trocador de Na+/Ca2+ (NCX) constitui um dos principais mecanismos de extrusão de Ca2+ intracelular em células excitáveis. Foi demonstrado que alterações no funcionamento do NCX estão relacionadas a diversas situações patológicas. Por este motivo, o entendimento do mecanismo molecular da manutenção da concentração de Ca2+ intracelular via NCX é importante para a compreensão do funcionamento do trocador, bem como para o desenvolvimento de fármacos. Além de transportar Na+/Ca2+, o NCX também é regulado por esses íons. Este trocador é composto por dois domínios transmembranares, cada um deles contendo 5 α-hélices (TM), e uma grande alça intracelular que conecta as hélices TM5 e TM6. O domínio transmembranar é responsável por catalisar o transporte de Na+/Ca2+ através da bicamada lipídica, enquanto que a alça citoplasmática está envolvida com a regulação do trocador. Esta alça contém dois domínios sensores de Ca2+ adjacentes, denominados CBD1 e CBD2. Apesar da importância fisiológica do NCX, o mecanismo de regulação alostérica do trocador por Ca2+ intracelular permanece desconhecido. Neste trabalho, a espectroscopia de ressonância magnética nuclear (RMN) de alta resolução foi utilizada para investigar a conformação e a dinâmica de CBD1 e CBD2 do trocador de Na+/Ca2+ de Drosophila melanogaster (CALX), isolados ou conectados covalentemente em uma construção denominada CBD12. Um total de 98% das ressonâncias da cadeia principal de CBD1 isolado na presença de Ca2+ foi assinalado, enquanto que na ausência de Ca2+, assinalamentos para apenas uma parte da cadeia principal puderam ser obtidos. Os assinalamentos adquiridos para CBD12 foram baseados na análise de um conjunto de espectros de RMN tridimensional heteronuclear e por comparação com os espectros dos domínios isolados. Uma análise preliminar dos deslocamentos químicos e dos parâmetros de relaxação de 15N obtidos para CBD1 indicou que este domínio é flexível na ausência de Ca2+, mas torna-se rígido após a adição deste íon. As medidas das velocidades de relaxação de 15N e de acoplamentos dipolares residuais (RDCs) de 1H-15N realizadas para CBD12 nas formas apo e holo indicaram que a ligação de Ca2+ em CBD1 estabiliza uma orientação rígida entre os domínios. A análise dos RDCs de 1H-15N mostrou ainda que a orientação média entre CBD1 e CBD2 é praticamente linear na ausência de Ca2+, enquanto que um ângulo menor é assumido após a adição deste íon. Os dados descritos nesta tese suportam um modelo de regulação alostérica em que a modulação da plasticidade de CBD12 pela ligação de Ca2+ no domínio CBD1 controla a abertura e o fechamento do trocador. / The Na+/Ca2+ exchanger (NCX) is a major mechanism for the extrusion of intracellular Ca2+ in excitable cells. It was demonstrated that altered functioning of this protein is related to various pathological situations. Therefore, the understanding of the molecular mechanism for maintaining the intracellular Ca2+ concentration by means of the NCX is important to understand the functioning of the exchanger and to develop drug-based therapies. Besides transporting Na+/Ca2+, the exchanger is also regulated by these ions. The NCX is composed of two transmembrane domains, each of them containing 5 transmembrane alpha-helices (TM), and a very large cytosolic loop that connects TM5 to TM6. The transmembrane domains are responsible for catalyzing the transport of Na+ and Ca2+ ions across the lipid bilayer, while the cytosolic loop is involved in regulation of the exchanger activity. It contains two regulatory Ca2+- binding domains, called CBD1 and CBD2, that appear in tandem. Despite the physiological importance of the NCX, the mechanism of allosteric regulation of the exchanger by intracellular calcium remains unclear. In this work we used high-resolution NMR spectroscopy to study the conformation and the dynamics of the two Ca2+-binding regulatory domains of Drosophila\'s Na+/Ca2+ exchanger (CALX), CBD1 and CBD2, in isolation as well as in a covalent construct called CBD12. Complete backbone NMR resonance assignments were obtained for the isolated CBD1 domain in the Ca2+-bound state, while partial assignments were obtained for CBD1 in the free state. Partial backbone NMR resonance assignments were obtained for the CBD12 construct through the analysis of a standard set of triple resonance NMR spectra. Additional assignments were obtained by comparison with the isolated CBD1 and CBD2 domains. A preliminary analysis of NMR chemical shifts and 15N relaxation data obtained for CBD1 indicates that this domain displays considerable amount of flexibility in the free state, but becomes more rigid upon Ca2+-binding. NMR 15N relaxation rates and 1H-15N residual dipolar couplings (RDCs) obtained for the Apo and Ca2+-bound states of the CBD12 domain indicate that calcium binding stabilizes a rigid inter-domain orientation. Analysis of 1H-15N RDCs further shows that Drosophila\'s CBD12 domain assumes an almost linear inter-domain orientation in the absence of Ca2+, while a smaller inter-domain angle was found in its presence. These findings support a model in which modulation of CBD12 plasticity by the binding of Ca2+ to the CBD1 domain controls the opening and closing of the exchanger.

Près de 40% des protéines présentes dans les cellules sont prédites partiellement ou complètement désordonnées. Ces protéines dépourvues de structure tridimensionnelle à l'état natif sont impliquées dans de nombreux mécanismes biologiques, la flexibilité jouant un rôle moteur dans les mécanismes de reconnaissance moléculaire. La prise en considération de l'existence de flexibilité au sein des protéines et des interactions protéines-protéines a nécessité le renouvellement de nos connaissances, de notre appréhension des fonctions biologiques ainsi que des approches pour étudier et interpréter ces phénomènes. La méthode retenue pour étudier ces transitions conformationnelles est la spectroscopie par résonance magnétique nucléaire. Elle dispose d'une sensibilité unique, d'une résolution à l'échelle atomique et permet par diverses expériences d'accéder à l'ensemble des échelles de temps définissant les mouvements de ces protéines. Nous combinons ces mesures expérimentales à un modèle statistique représentant l'ensemble du paysage énergétique des protéines désordonnées : la description par ensemble explicite de structures. Ce modèle est une représentation discrète des différents états échantillonnés par ces protéines. Il permet, combinant les déplacements chimiques, les couplages dipolaires et la relaxation paramagnétique, de développer une description moléculaire de l'état déplié en caractérisant à la fois l'information locale et l'information à longue portée présente dans les protéines intrinsèquement désordonnées. / Around 40% of the human genome does not fold into stable three-dimensional structures but are either unfolded, or contain unfolded regions of significant length. The inherent flexibility of this class of proteins is essential for their function in a vast range of biomolecular process such as molecular recognition. In order to take into account the specificity of these interactions, it has been necessary to invent new approaches to study and interpret their behaviour. Nuclear magnetic resonance spectroscopy is a unique atomic resolution probe which is sensitive to a very large range of time scales. We combine experimental NMR data with a statistical model describing the energy landscape of unfolded state : the explicit ensemble description. This model is a discrete representation of the different states of theses proteins. Combining chemical shifts, residual dipolar couplings and paramagnetic relaxation enhancement, it is then possible to develop a molecular description of the unfolded state caracterising both the local and long-range information of intrinsically disordered proteins.

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The selection of the forcefield is a crucial issue in any MD related work and there is no clear indication as to which of the many available forcefields is the best for protein analysis. Many recent literature surveys indicate that MD work may be hindered by two limitations, namely conformational sampling and forcefields used (inaccuracies in the potential energy function may bias the simulation toward incorrect conformations). However, the advances in computing infrastructures, theoretical and computing aspects of MD have paved the way to carry out a sampling on a sufficiently longtime scale, putting a need for the accuracies in the forcefield. Because there are established differences in MD results when using forcefields, we have sought to ask how we could assess common mobility segments from a protein by analysis of trajectories using three forcefields in a similar environment. This is important because, disparate fluctuations appear to be more at flexible regions compared to stiff regions; in particular, flexible regions are more relevant to functional activities of the protein molecule. Therefore, we have tried to assess the similarity in the dynamics using three well-known forcefields ENCAD, CHARMM27 and AMBERFF99SB for 61 monomeric proteins and identify the properties of dynamic residues, which may be important for function. The comparison of popular forcefields with different parameterization philosophy may give hints to improve some of the currently existing agnostics in forcefields and characterization of mobile regions based on dynamics of proteins with diverse folds. These may also give some signature on the proteins at the level of dynamics in relation to function, which can be used in protein engineering studies. Nanosecond level MD simulation(30ns) on 61 monomeric proteins were carried out using CHARMM and AMBER forcefields and the trajectories with ENCAD forcefield obtained from Dynameomics database. The trajectories were first analyzed to check whether structural and dynamic properties from the three forcefields similar choosing few parameters in each case. The gross dynamic properties calculated (root mean square deviation (RMSD), TM-score derived RMSD, radius of gyration and accessible surface area) indicated similarity in many proteins. Flexibility index analysis on 17 proteins, which showed a notable difference in the flexibility, indicated that tertiary interactions (fraction of nonnative stable hydrogen bonds and salt bridges) might be responsible for the difference in the flexibility index. The normalized subspace overlap and shape overlap score taken based on the covariance matrices derived from trajectories indicated that majority of the proteins show a range between 0.3-0.5 indicating that the first principal components from these proteins in different combinations may not match well. These results indicate that although dynamic properties in general are similar in many proteins. However, flexibility index and normalized subspace overlap score indicate that subspaces on the first principal component in many proteins may not match completely. The number of proteins showing a better correlation is higher in CHARMM-AMBER combinations than the other two. The structural features from trajectories have been computed in terms of fraction of secondary structure, hydrogen bonds, salt bridges and native contacts. Although secondary structures and native contacts are well preserved during the simulations, the tertiary interactions (hydrogen bonds) are lost in many proteins and may be responsible for the difference in the some of properties among forcefields. Comparison of simulation results to experimental structures in terms of Root mean square fluctuations, Accessible surface area and radius of gyration indicates that the simulations results are on par with the ones derived from experimental structures. We have tried to assess the flexibility in the proteins using normalized Root mean square fluctuations (nRMSF), which for a residue is the ratio of RMSF from simulation to that of crystal structure. We have selected a threshold for this nRMSF to indicate the mobile regions in a protein based on secondary structure analysis. Based on the threshold of nRMSF and conformational properties (deviation in the dihedral angles), we have classified the residue and evaluated the properties of rigid hinge residues and corresponding mobile residues in terms of residue propensity, secondary structure preference and accessible surface area ranges. Since the rigid dynamic residues represent the inherent mobility, they might be important for function. Therefore, we have tried to assess the functional relevance considering the dynamic mobile residues from each protein from each forcefield simulation with the residues important for the function (taken from literature and databases). It is observed that some residues found to be mobile from the simulation are found to match with the experimental ones, although in many cases the number of these mobile residues is higher compared to the experimental ones. In summary, an analysis of protein simulation trajectories using three forcefields on a set of monomeric protein has shown that the gross structural properties and secondary structures from many proteins remain similar, but there are differences as may be seen from flexibility index. However correlation in parameters from CHARMM and AMBER force field is better compared to other two combinations. The differences seen in some of structural properties may arise mainly due to the loss of few tertiary interactions as indicated by the fraction of native hydrogen bonds and salt bridges. Based on the nRMSF, mobile segments obtained from the simulations were identified, and some of the mobile segments are found to match the functionally important residues from the experimental ones. Our work indicates that there are still some differences in the properties from the simulations, which indicates that care must be exercised when choosing a forcefield, especially assessing the functionally relevant residues from the simulations.