PhD Candidate

Daryl Good

Abstract

Proteins are known to populate ensembles of conformations at room temperature. Transitions between conformational states within an ensemble occur over a broad range of amplitudes and time scales. In membrane proteins, which reside in a highly anisotropic environment of a lipid bilayer, understanding how the protein’s interactions with solvent, lipids and other proteins in the surrounding environment affect the energetics of the internal dynamics is a major challenge. Here we use solid-state NMR (SSNMR) spectroscopy to perform two studies of the internal dynamics of two seven transmembrane helical (7TM) microbial rhodopsins embedded in a native like lipid environment.

First, we study the conformational dynamics of Anabaena Sensory Rhodopsin (ASR), a light sensing protein. Quantitative analysis of site-specific measurements of the ¹⁵N longitudinal R₁ and rotating frame R_1ρ relaxation rates at two magnetic fields and at two temperatures provides evidence of motions on at least two timescales. Model-free analyses showed that faster picosecond local motions occur throughout the protein while the amplitudes of slower nanosecond collective motions are much larger in the interhelical loop regions and the cytoplasmic sides of TM helices than most of the TM helical regions. The location of larger amplitude collective motions on the cytoplasmic side of the TM helices correlates with the location where ASR to known to undergo large conformational changes in the process of binding/unbinding a soluble transducer.

Second, we measure temperature-dependent nuclear spin relaxation rates of the bulk protein in the light-driven proton pump green proteorhodopsin (GPR). In two samples with different lipid compositions we measured a total of six relaxation rates over the temperature range from 104 K – 289 K. Using model free analysis, we directly determine activation energies of motional modes representing sidechain rotations, low activation energy local backbone and sidechain fluctuation as well as high activation energy collective backbone and side chain motions. We were able to relate differences in these motions between the two samples to the different dynamics of the solvent and lipids in the samples, thereby highlighting the influence of the surrounding environment on protein motions.

Examination Committee 

• Dr. Hermann Eberl, Chair
• Dr. Vladimir Ladizhansky, Advisor
• Dr. Steffen Graether, Advisory Committee
• Dr. James Davis, Graduate Faculty
• Dr. Ayyalusamy Ramamoorthy, External Examiner (University of Michigan)