UC San Diego

Allostery is a critical process that allows for the regulation of proteins : binding of a molecule at one distal, functional site in a protein can cause changes to a different functional site. Early on, allostery was described as a change in protein structure upon the binding of another molecule. However, a dynamic model of protein allostery is emerging, which more completely describes allosteric phenomena. This work investigates protein dynamics under the lens of allosteric regulation for two different protein systems : (1) IkappaBalpha and (2) thrombin and its cofactor, thrombomodulin. In Chapter II, we present results from NMR studies which probe the roles of ankyrin repeat consensus mutations in IkappaBalpha. These results reveal that consensus mutations cause long-range, allosteric effects throughout the ankyrin repeat domains, including the ordering of the C-terminal PEST sequence on the ps-ns timescale, which is a region critical for proteosomal degradation. In Chapter III, a new construct of thrombomodulin, named TM456m, is characterized using kinetic protein C activation assays to determine whether the protein is suitable for use in future NMR experiments bound to thrombin. Here, we show that TM456m has tighter binding than the previously studied TM45 and higher stability than full-length TM456. Furthermore, we present the first HSQC of TMbound thrombin and observe allosteric changes across the thrombin molecule. Chapter IV presents a hydrogen-deuterium exchange mass spectrometry study that we performed in order to investigate where changes in solvent accessibility occur in the thrombin molecule when an active site substrate (PPACK) or an effector molecule (TM456m) is bound. In both cases, allosteric changes occurred across the thrombin molecule. This study reveals that N-terminus of the heavy chain of thrombin is not buried in the Ile cleft, contrary to all crystal structures of thrombin to date, and that substrate and TM binding allosterically pull the N-terminus into the Ile cleft. In Chapter V, we assign the amide peaks of apo- thrombin and measure its NMR backbone dynamics. Comparison of PPACK-thrombin chemical shifts and backbone dynamics with those of apo-thrombin reveals that allosteric pathways are inherently built into apo-thrombin and suggests that effector-binding might work via a conformational selection mechanism from a broad selection of conformational states