Department of Chemistry, University of Warwick , Coventry CV4 7AL, United Kingdom.
J Am Chem Soc. 2017 Jul 12;139(27):9246-9258. doi: 10.1021/jacs.7b03974. Epub 2017 Jun 30.
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 N longitudinal R and rotating frame R relaxation rates at two fields of 600 and 800 MHz and at two temperatures of 7 and 30 °C. Quantitative analysis of the R and R 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.
蛋白质是动态实体，存在于构象的集合中。构象集合内的状态之间的转变发生在广泛的幅度和时间尺度上，并且通常与生物功能有关。虽然固态 NMR（SSNMR）光谱学最近已被用于研究微晶体状态下蛋白质的构象集合，但它在膜蛋白中的应用仍然有限。在这里，我们使用 SSNMR 研究了在脂质中重建的七螺旋跨膜（TM）蛋白 Anabaena Sensory Rhodopsin（ASR）的构象动力学。我们报告了在 600 和 800 MHz 两个场和 7 和 30°C 两个温度下的 N 纵向 R 和旋转框架 R 弛豫率的位点特异性测量值。对 R 和 R 值及其场和温度依赖性的定量分析提供了证据，证明存在至少两个时间尺度的运动。我们将这些运动建模为快速局部运动和 TM 螺旋和结构化环的较慢集体运动，并使用简单的无模型和扩展无模型分析来拟合数据并估计幅度、时间尺度和活化能。更快的皮秒（几十到几百皮秒）局部运动发生在整个蛋白质中，在 TM 螺旋的中部占主导地位。相比之下，在纳秒（几十到几百纳秒）时间尺度上发生的较慢集体运动的幅度在螺旋的中心部分较小，但在其细胞质侧以及在螺旋间环中增加。ASR 在其细胞质表面与可溶性转导蛋白相互作用，其结合亲和力受光调节。TM 螺旋细胞质侧的运动幅度较大，与 ASR 在结合/解结合转导蛋白过程中经历大的构象变化的能力相关。
