Master of Science
Deciphering protein structure and dynamics is a key prerequisite for understanding biological function. The current work aims to apply HDX-MS to improve the understanding of protein structure and dynamics for systems that remain challenging for other techniques. Following a general overview of the field (Chapter 1), Chapter 2 investigates the relationship between enzyme dynamics and catalysis. By conducting comparative HDX-MS measurements on rM1-PK during substrate turnover and in the resting state, catalytically active rM1-PK undergoes significant rigidification of the active site. However, virtually the same rigidification was seen upon exposing rM1-PK to substrate or product in the absence of turnover. These findings demonstrate that comparative experiments on enzyme dynamics by HDX-MS (and other bioanalytical techniques) should be interpreted with caution. In Chapter 3, HDX-MS is used to probe the intrinsically disordered protein Nrf2. HDX-MS is used to investigate the structure and dynamics of the full-length Nrf2 and its interaction with the Kelch domain. The data obtained demonstrate the highly-disordered nature of Nrf2. Its interaction with Kelch causes protection of the binding sites on Nrf2, while the rest of the protein becomes slightly more dynamic. This works highlights the limitations of using truncated protein constructs when investigating their structure and dynamic properties using biophysical techniques.
Fast, Courtney S., "Investigations into the Role of Conformational Dynamics in Protein Function: Insights From Hydrogen/Deuterium Exchange-Mass Spectrometry" (2017). Electronic Thesis and Dissertation Repository. 4907.
Available for download on Monday, October 01, 2018