Doctor of Philosophy
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.
Xiao, Yiming, "Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics Simulations for Studying Protein Structure and Dynamics" (2018). Electronic Thesis and Dissertation Repository. 5713.