Molecular recognition in gas phase: theoretical and experimental study of non-covalent protein-ligand complexes by mass-spectrometry

• Autores: Andrey Dyachenko
• Directores de la Tesis: Ernest Giralt Lledó (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2013
• Idioma: inglés
• Tribunal Calificador de la Tesis: Albert Virgili (presid.), Ernesto Nicolás Galindo (secret.), Eduard Sabidó Aguadé (voc.)
• Materias:
  • Química
    • Química analítica
      • Análisis bioquímico
      • Espectroscopia de resonancia magnética
      • Espectroscopia de masas
  • Ciencias de la vida
    • Biofísica
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • 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.