Changing shape: an investigation into allostery and protein conformational ensembles

Abstract

Allostery is the mechanism by which action at one site on a protein causes a functional change at a distant site. An allosteric change can manifest as conformational change or a change in protein dynamics. In this way, the study of allostery, protein dynamics, and structural biology are individual, yet related fields. Progress and technical advancements in one field inform and drive the others. In this thesis, four protein complexes 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), acetone carboxylase (AC), Replication protein A (RPA), and Radiation sensitive 52 (Rad52) were studied to elucidate conformational change and allostery during catalysis. A variety of orthogonal biophysical approaches were used to study these systems. To infer changes in protein dynamics and conformation, hydrogen-deuterium exchange coupled to mass spectrometry was used in three of these studies. This technique allows for the probing of the hydrogen bonding network based on ligand binding or mutation. Probing of AC, RPA and Rad52 has led to the description of conformational changes essential for function. Ion mobility coupled to native mass spectrometry was used to investigate the available conformations of 2-KPCC during catalysis and led to the discovery of residues essential for modulating those conformations. The concepts of allostery, conformational ensemble, and protein dynamics have evolved since they were first described. Utilizing mass spectrometry-based techniques, my work helped expand the knowledge of several protein systems that contain allosteric networks which are necessary for function. The studies presented in this thesis increase the understanding not only for these protein systems, but also of protein function on a deeper level.