Doctor of Philosophy
Hydrogen deuterium exchange (HDX) coupled with mass spectrometry is widely used for probing protein structure and dynamics. Protein-ligand interactions usually induce a reduction in the measured HDX rates an effect that may be ascribed to stabilization of the protein structure. This work aims to improve the general understanding of the changes in HDX patterns associated with ligand binding.
We initially applied HDX for studying differences between oxy-hemoglobin (Oxy-Hb) and aquomet-hemoglobin (Chapter 2). The results show that the α and β subunits respond differently to the oxy to aquomet transition with the heme binding pocket being destabilized in both cases. The results suggest that enhanced structural dynamics in the heme binding pocket may have adverse effects on heme-protein interactions.
Chapter 3 focuses on the different scenarios that can be encountered in an HDX experiment upon ligand binding. Myoglobin and hemoglobin were used as model systems, focusing on the oxy and deoxy states of both proteins. Our results demonstrate that ligand binding can be stabilizing or destabilizing, leading to decreased or increased HDX rates respectively.
In Chapters 4 HDX was used to probe the changes in structural dynamics of caseinolytic protease P (ClpP), an antibiotic drug target, after binding ADEP antibiotics. The mechanism of ADEP binding and the N-terminal structure of ClpP is not well understood with conflicting x-ray structures reported in literature. Our findings demonstrate that the N-terminus of ClpP remains quite unstructured after ADEP binding, while belt region undergoes tightening.
Pin 1, a peptidyl prolyl isomerase, binding to a cyclic peptide inhibitor was studied in Chapter 5. Characterization of Pin1-CRYPEVEIC interactions by other techniques has been iv difficult. This study demonstrates that binding of the inhibitor triggers an overall stabilization of Pin 1. We identify a loop that interacts with basic sites of the ligand and that becomes destabilized upon ligand binding. This destabilization is ascribed to steric clashes between the peptide inhibitor and the protein
Sowole, Modupeola A., "Hydrogen Exchange Mass Spectrometry for Studying Protein-Ligand Interactions" (2015). Electronic Thesis and Dissertation Repository. 2942.