AbstractsBiology & Animal Science

Studying protein-ligand binding by spectroscopy and calorimetry

by Erick Alejandro Meneses

Institution: McGill University
Department: Department of Chemistry
Degree: PhD
Year: 2015
Keywords: Biophysics - General
Record ID: 2057904
Full text PDF: http://digitool.library.mcgill.ca/thesisfile130426.pdf


Different molecular, thermodynamic, and kinetic aspects of protein-ligand binding were studied in the present thesis. This was accomplished using diverse techniques such as Nuclear Magnetic Resonance (NMR), fluorescence spectroscopy, and calorimetry. Chapter 1 introduces the basic concepts to understand the dynamics and thermodynamics of binding for systems where a ligand receives just one binding molecule.Chapter 2 gives an up-to-date technical background on NMR experimental techniques to study dynamics and thermodynamics of protein-ligand binding. These techniques were key to the research developed in chapters 3, 4 and to a lesser extent in Chapter 5.Chapters 3 and 4 focused on the interaction between the Fyn SH3 domain and several proline rich peptides. Chapter 3 describes the role of electrostatic interactions in the binding pathway of transient protein complexes formed by the SH3 domain and three proline-rich peptides. We showed that the electrostatic enhancement of binding for this weak (≥µM KD), short-lived complex with lifetimes on the order of milliseconds is much less, and basal association rates are greater than those previously observed for tight (<nM KD) long-lived systems with lifetimes on the order of minutes or longer. This suggests that electrostatics may play different roles in short-lived and long-lived protein complexes.Chapter 4 mapped the changes in volume for the association pathway for the Fyn SH3 domain and a proline rich peptide. Via pressure we were able to quantify changes in molar volume between the free, bound and transition states for this system. This is, to our knowledge, the first measurement of the activation volume for a protein-ligand binding reaction. The results agree with a protein-ligand binding pathway involving significant desolvation of the binding surfaces. We found that the volume of transition state is very close to that of the fully bound state. This suggests that the rearrangement of solvating water molecules and protein and ligand and conformational changes occur before the transition state.Chapter 5 explored different NMR experimental approaches for studying the interaction between bisphophonate inhibitors and human farnesyl pyrophosphate synthase. Different isotopic protein labeling patterns were used to study this system together with a series of NMR experiments designed to exploit these specifically labeled proteins. Dans cette thèse nous avons étudié plusieurs aspects de biologie moléculaire, de thermodynamique, et de cinétique chimique liés aux processus d'union entre une protéine et son ligand. Afin de mener ces études, diverses techniques telles que la Résonance Magnétique Nucléaire (RMN), la spectroscopie de fluorescence et la calorimétrie ont été utilisées.Le premier chapitre introduit les concepts de base afin de comprendre la dynamique et la thermodynamique d'union entre protéines. Un modèle simplifié dans lequel une protéine joint seulement un ligand sera utilisé.Le deuxième chapitre donne une mise à jour des techniques RMN récentes que l'on peut…