Magic angle spinning solid-state NMR shows great potential for studying structure and dynamics of membrane proteins, which are not readily amenable to X-ray crystallography and solution NMR. In this abstract we describe our recent efforts towards elucidation of membrane protein structure and dynamics by solid state NMR. In the first part we discuss conformational dynamics in a seven-helical transmembrane photoreceptor Anabaena Sensory Rhodopsin (ASR). Relaxation and order parameter measurements performed at two temperatures and two magnetic fields provide evidence for internal motions occurring on at least two time scales. Faster picosecond local motions occur throughout the protein and are dominant in the middle parts of helices. In contrast, the amplitudes of the slower collective motions occurring on a slower nanosecond timescale, are smaller in the central parts of helices, but increase towards their cytoplasmic sides as well as in the interhelical loops. ASR interacts with a soluble transducer protein on its cytoplasmic surface, and its binding affinity is modulated by light. The larger amplitudes of motions on the cytoplasmic sides of the TM helices is likely to correlate with the ability of ASR to undergo large conformational changes in the process of binding/unbinding its soluble transducer.<br/>In the second part, we will discuss our progress towards structural characterization of a human water channel Aquaporin 1 (hAQP1), focusing on the conformation of extracellular loops. Solid-state NMR chemical shift analysis of secondary structure agrees with the crystallographic data for transmembrane regions, but reveals pronounced differences in the extracellular loops A and C. Loop C appears to be structured and more rigid than suggested by the B-factors in the crystallographic structures. Site-specific hydrogen deuterium exchange measurements identified a number of protected backbone amide groups and nitrogen-bearing side chains that are likely involved in stabilizing the loops. To gain additional insight into a structural organization of these loops, we performed molecular dynamics simulations with NMR-derived restraints and filtering based on solvent accessibility. The simulations suggested loop-stabilizing interactions that alter the extracellular surface of hAQP1, with possible implications for water transport regulation through the channel.

Insights into structure and dynamics of membrane proteins

254th National Meeting (2017)

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