Abstract
Proteins are dynamic entities and populate ensembles of conformations. Transitions between states within a conformational ensemble occur over a broad spectrum of amplitude and time scales, and are often related to biological function. Whereas solid-state NMR (SSNMR) spectroscopy has recently been used to characterize conformational ensembles of proteins in the microcrystalline states, its applications to membrane proteins remain limited. Here we use SSNMR to study conformational dynamics of a seven-helical transmembrane (TM) protein, Anabaena Sensory Rhodopsin (ASR) reconstituted in lipids. We report on site-specific measurements of the 15N longitudinal R1 and rotating frame R1ρ relaxation rates at two fields of 600 and 800 MHz and at two temperatures of 7 and 30 °C. Quantitative analysis of the R1 and R1ρ values and of their field and temperature dependencies provides evidence of motions on at least two time scales. We modeled these motions as fast local motions and slower collective motions of TM helices and of structured loops, and used the simple model-free and extended model-free analyses to fit the data and estimate the amplitudes, time scales and activation energies. Faster picosecond (tens to hundreds of picoseconds) local motions occur throughout the protein and are dominant in the middle portions of the TM helices. In contrast, the amplitudes of the slower collective motions occurring on the nanosecond (tens to hundreds of nanoseconds) time scales, are smaller in the central parts of helices, but increase toward 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 amplitude of motions on the cytoplasmic side of the TM helices correlates with the ability of ASR to undergo large conformational changes in the process of binding/unbinding the transducer.
Highlights
While three-dimensional structures of proteins provide important basic insights into their internal organization, it has been long recognized that internal dynamics play a critical role in protein function
Anabaena Sensory Rhodopsin (ASR) remains in a trimeric state at 30 °C, and this prevents the protein from rapid axial diffusion in the bilayer
We have previously reported spectroscopic assignments of ASR at 5 °C,47,70 and they remain unchanged at 7 °C
Summary
While three-dimensional structures of proteins provide important basic insights into their internal organization, it has been long recognized that internal dynamics play a critical role in protein function. A variety of biological processes such as conformational transitions, allostery, enzymatic activity depend on the proteins’ internal plasticity[1−4] on the time scales that span many orders of magnitude.[1,5,6] Because of its functional significance, protein dynamics have attracted considerable attention in recent years. A wide range of experimental methodologies is required in order to capture the dynamic richness of proteins’ internal motions.[7−10] In particular, solution nuclear magnetic resonance (NMR) methods have been used extensively to probe dynamics of globular proteins, as described in a number of recently published review articles.[1,3,5,6,11,12].
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