Investigations of Membrane Protein Dynamics using Solid State NMR
Proteins populate ensembles of conformations at ambient temperature. Protein dynamics result from transitions between conformational states and occur over a broad range of time scales. Although dynamics play important roles in protein function, understanding how the proteins’ interactions with the surrounding environment, e.g., with solvent, lipids and other proteins, affect the energetics of internal motions is a major challenge. In this Thesis, we focus on the analysis of motions in membrane proteins. They reside in a highly anisotropic environment of a lipid bilayer, and are not easily studied by commonly used techniques, such as x-ray crystallography, electron microscopy, or solution NMR. We use a novel technique of solid-state NMR (SSNMR) spectroscopy to investigate internal motions in two alpha-helical transmembrane microbial rhodopsins. First, we combine multidimensional SSNMR relaxation and order parameter measurements to characterize conformational dynamics of Anabaena Sensory Rhodopsin (ASR), a light sensing protein. We demonstrate that both fast picosecond and slow nanosecond motions occur in ASR; the amplitudes of the slower motions are small in the well-structured TM regions and increase towards the cytoplasmic ends of helices and the interhelical loop regions. Larger amplitudes of nanosecond motions on the cytoplasmic side of the TM helices correlates with the location of the binding sites where ASR is likely to undergo large conformational changes during the binding/unbinding of a soluble transducer. Second, we measure SSNMR bulk relaxation rates over the temperature range from 104 K - 289 K in the light-driven proton pump green proteorhodopsin (GPR). Using model-free analysis, we directly determine the activation energies of the local backbone and sidechain fluctuations as well as of the sidechain rotations and collective backbone and side chain motions. We relate these motions to the dynamics of the solvent and the lipids, thus highlighting the influence of the surrounding environment on membrane protein dynamics. From a structural perspective, ASR and GPR share their alpha-helical bundle architecture with other membrane proteins and in particular, with G-protein coupled receptors. Understanding the nature of motions in ASR and GPR provides a general framework to understanding the factors determining stability and motions in other membrane proteins.