Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Lars Konermann

Abstract

Deciphering properties of proteins are essential for human health and aiding in the development of new pharmaceuticals. This dissertation uses hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and molecular dynamics (MD) simulations to study protein dynamics, for improving the understanding of protein folding/unfolding mechanisms, and ligand binding and allosteric regulation.

Chapter 2 uses HDX-MS for probing the conformational dynamics of myoglobin in the presence of N2 bubbles. We propose a dynamic model that reproduces the observed data: “semi-unfolded” “native” “globally unfolded” -> “aggregated”. Chapter 3 focuses on osteoprotegerin (OPG), which hinders bone resorption by inhibiting RANK/RANKL interactions. The dimerization of OPG is regulated by heparan sulfate (HS). Basing on HDX data, a mechanism is proposed for the formation of the RANKL/OPG/HS ternary complex, according to which HS-mediated C-terminal contacts on OPG lower the entropic penalty for RANKL binding.

Chapter 4 represents the centerpiece of this thesis. It explores the allosteric regulation of S100A11, a dimeric EF-hand protein with two hydrophobic target binding sites. Both HDX/MS and MD data showed the metalation sites become more dynamic after Ca2+ loss. However, these enhanced dynamics do not represent the trigger of the allosteric cascade. Instead, a labile salt bridge acts as an active “agitator” that destabilizes the packing of adjacent residues, causing a domino chain of events that culminates in target binding site closure. Overall, this thesis highlights how the combination of HDX/MS and computational techniques can provide detailed insights into protein conformational fluctuations and their implications for protein function.

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