In the present thesis we have explored different factors that impede accurate quantitative description of non-covalent protein-protein and protein-ligand interactions and design of new potent and specific binders from the scratch. Firstly, we addressed the role of solvent in the mechanism of non-covalent interactions. Secondly, we tackled the question about the intrinsic conformational flexibility of the protein molecules and the part it plays in weak interactions between proteins. In the first part of the thesis we studied the interactions of vascular endothelial growth factor (VEGF) protein with five cyclic peptides in solution and gas phase. The results showed that affinities of five ligands to VEGF in solution and gas phase are ranked in inversed order. That is, the that has the highest affinity in solution (as shown by chemical shift perturbation NMR and isothermal titration calorimetry) forms the weakest complex with VEGF in gas phase, and vice versa. We compared gas-phase and solution binding affinities of of five peptides and made qualitative conclusions about the role of the solvent in protein-ligand interactions. In order to obtain more quantitative information about the gas-phase behavior of non-covalent complexes we have developed a combined experimental/theoretical approach to study the energetics of collisional activation of the ion prior to dissociation. We applied developed strategy to model CID in traveling wave ion guide (TWIG) collision cell. We validated the model on the CID of leu-enkephalin peptide and then applied developed strategy to five non-covalent protein-peptide complexes and found activation energies of their dissociation reactions. Next we applied ESI native MS to study the allosteric interactions between the molecular chaperonin GroEL and ATP. The obtained data allowed to construct a scheme of conformational transition of GroEL upon binding of ATP and distinguish between two different cooperativity models, providing strong arguments in favor of Monod-Wyman-Changeux (MWC) model. Finally, be studied the backbone dynamics of VEGF with a combination of NMR relaxation and all-atom force-field based normal mode analysis (NMA). We showed that combination of experimental and computational approach allows to identify flexible zones with higher level of confidence. We also found out that residues, that are involved VEGF-receptor interactions, reside in or close to the flexible zones, suggesting the critical role conformational plasticity plays in the non-covalent protein-protein interactions. / Las biomoléculas de los organismos vivos realizan sus funciones principalmente a través de interacciones débiles reversibles entre ellas. La transducción de señal, la replicación de ADN/ARN, otros procesos enzimáticos y, virtualmente, cualquier otro proceso involucrado en las funciones vitales de cualquier organismo vivo (de las simples amebas, al complejo ser humano), requiere que las moléculas “hablen” entre ellas. Dicho lenguaje se basa en interacciones no covalentes. La flexibilidad conformacional es una propiedad esencial de las grandes biomoléculas, y muchas de las funciones desempeñadas por proteínas se basan en su capacidad para cambiar de conformación en respuesta a un factor externo. Geométricamente hablando, la presencia de flexibilidad en una proteína obstaculiza el diseño racional de medicamentos porque posibilita la existencia de un número muy elevado de conformaciones de dicha proteína. Por este motivo, cualquier información sobre la flexibilidad de una proteína es sumamente valiosa para la comprensión de PPI y PLI y para el diseño racional de medicamentos. Los capítulos 1-3 de la presente tesis versan sobre la solvatación, mientras que la flexibilidad se estudiara en el capitulo 4.

L’any 2002 tres grups de recerca, entre ells el nostre grup, van demostrar teòricament que la interacció entre anions i anells aromàtics electrodeficients, anomenada interacció anió–, era favorable. Des de llavors s’ha dut a terme un intens estudi de la seva naturalesa física fins la total comprensió. Aquesta tesi es basa amb l’estudi de la interacció anió– des de tres punts de vista. Primerament, la investigació es basa en el disseny teòric de motius estructurals per donar lloc a un receptor on la interacció anió– siga molt favorable, per posteriorment avaluar la força de la interacció experimentalment en dissolució. A continuació, es va analitzar la interrelació entre un gran nombre de combinacions d’interaccions no covalents. A partir d’aquest estudi es defineixen nous conceptes i es proposen diferents formules per calcular efectes de cooperativitat. Finalment, hem anat un pas més enllà en l’estudi de la interacció analitzant: 1) l’impacte de la interacció anió– a sistemes biològics; 2) la influència de modificacions a l’anió sobre la naturalesa física de la interacció. / In 2002 three research groups, among them our research group, theoretically demonstrated that the interaction between anions and electron-deficient aromatic rings, named anion– interaction, was favourable. Since then, an intense study of its physical nature has been performed to understand it completely. This thesis is based on the study of the anion– interaction from three points of view. Firstly, theoretical design of binding units to build a receptor and to obtain the most favourable binding based on anion– interactions. The binding properties of these receptors have been experimentally assessed in solution. Secondly, we have studied the interplay between a great combination of noncovalent interactions. From this study, new concepts and formula to calculate cooperativity effects have been described. Finally, we have study one step further the anion– interaction analysing: 1) the impact of anion– interaction in biological systems; 2) how the modifications in the anion influence the physical nature of the interaction.