PhD Thesis Presentation

Candidate: Meaghan Elizabeth Ward

Abstract 

Membrane proteins account for 30% of all proteins and perform many important roles in the cell, yet are difficult to study due to the necessity to maintain their lipidic environment during all stages of purification and characterization. Often during these processes the lipid environment is drastically altered, and high resolution studies of membrane protein structure commonly require the use of membrane-mimetic environments. Solid-state NMR (SSNMR) spectroscopy using magic angle spinning (MAS) is a rapidly developing and promising technique to study such proteins in their native, lipid-associated environment. However, these experiments are generally limited by sensitivity. This thesis centers upon the development and application of novel SSNMR approaches to study large, seven transmembrane (7TM) α-helical proteins – a class of proteins to which microbial rhodopsins, which we use as model systems for these experiments, belong. First, the sensitivity available on such samples under ultrafast MAS conditions (> 50 kHz) was investigated. As smaller sample volumes are necessary at these spinning frequencies, low power decoupling and paramagnetic enhancement of the signal relaxation time were implemented to facilitate a condensed data collection scheme. Under these conditions, it was found that the paramagnetic relaxation enhancement was uniformly distributed throughout the proteins and that sensitivity comparable to that available in larger rotors was obtainable under proton detection. Next, the implementation of proton detection to specifically detect the mobile regions of proteins was developed. Using these methods it was found that for the mobiles regions, an ~10x increase in sensitivity was available and that both the loop regions and tightly-bound lipid and carbohydrates of these proteins could be studied. Finally, methods with which to characterize these proteins in the native E. coli membrane environment were developed and implemented. Small, site-specific perturbations in protein structure, which occur as the local membrane milieu changes, indicate that the protein can subtly adapt to its environment without large structural rearrangement. In summary, this work has advanced our ability to use MAS-SSNMR spectroscopy as a structural probe for large and oligomeric membrane proteins.

Examination Committee:

Dr. Michele Oliver, Chair (Department of Mathematics and Statistics)

Dr. Vladimir Ladizhansky, Advisor (Department of Physics)

Dr. Leonid Brown, Co-Advisor (Department of Physics)

Dr. Steffen Graether, Examination Committee (Department of Molecular & Cellular Biology)

Dr. Simon Sharpe, External Examiner (The Hospital for Sick Children, Toronto, ON)