Transmembrane Protein Activation Refined by Site-Specific Hydration Dynamics
Transmembrane Protein Activation Refined by Site-Specific Hydration Dynamics
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DOI：
10.1002/anie.201206147
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Han, Songi
中科院分区：
文献类型：
--
作者：
Hussain, Sunyia;Franck, John M.;Han, Songi
跨膜蛋白是细胞通道的守门人，允许离子和分子进入或离开，或者是对周围环境做出反应以改变细胞内条件的受体。膜蛋白活化的构象动力学是理解其功能细节的关键，[1-4]但由于获得大型疏水蛋白复合物的动力学信息所涉及的实验挑战，这些信息已被证明难以获得。各种磁共振技术，包括魔角旋转固态NMR（ssNMR）光谱，[5] NMR弛豫测量，[6]和电子顺磁共振（EPR）光谱，[1，3]是捕捉膜蛋白结构和动力学难以捉摸的细节的前沿。在这里，我们展示了使用一种新的方法组合，提出了一个动态的基础上的图片，涉及膜蛋白片段的结构，它支持的功能运动。这是通过观察周围的水化水来实现的，水化水与活化后发生的蛋白质构象变化同时重排。我们获得了网站特定的水化动力学信息，通过使用NMR信号增强技术奥弗豪泽动态核极化（ODNP），这是最近开发的探测局部水扩散系数内约10（2-4水层）的氮氧自由基自旋标记的氨基酸残基。[7-12]该技术与EPR线形分析相结合，捕获自旋标记周围的蛋白质片段迁移率，提供了蛋白质以及溶剂化水所经历的不同动态变化的快照。此外，一个新开发的分析允许分离的两个重要模式的水动力学检测ODNP。这种新的方法分离的贡献1）自由翻译的“水合水”，这经历了快速，皮秒级，运动，从2）纳秒时间尺度的波动，由于“绑定”水分子的自旋相互作用与较慢的动力学或不稳定的酰胺质子。7 TM形成了广泛的蛋白质类别，包括许多生理学相关的受体和大约40%的药物靶标。[13]我们澄清了蛋白质位点特异性水动力学的变化，在一个关键的PR段激活后，无论是在皮秒和纳秒的时间尺度。跨生物界面的水合动力学的定量描述仍然是一个具有挑战性的目标。目前用于研究膜蛋白系统中的水的基于EPR的技术需要低温[14，15]或引入可溶性化学试剂，[16]而ODNP可以在环境条件下直接评估自旋标记周围的水运动，正如最近已经证明的蛋白质折叠[12]和膜转运蛋白的构象变化。[17]水合水被假设为球状蛋白功能的重要决定因素，[18-20]但其在跨膜蛋白力学中的作用因周围脂质的额外作用而特别复杂，脂质被认为是更有影响力的溶剂。[21然而，最近关于牛视紫红质（Rh）[23，24]和细菌视紫红质（BR）[25，26]的实验和理论工作表明，内部蛋白质水合作用确实影响功能，并且确实在蛋白质活化时发生改变。最近的ssNMR工作补充了全球水合性质的中子散射研究...
Transmembrane proteins are the gatekeepers of the cell—channels that allow ions and molecules to enter or exit, or receptors that respond to their surroundings to change conditions inside the cell. The conformational dynamics of membrane protein activation are key to understanding the details of their function,[1–4] and yet such information has proven to be difficult to obtain because of the experimental challenges involved in obtaining dynamics information for large hydrophobic protein complexes. Various magnetic resonance techniques, including magic-angle-spinning solidstate NMR (ssNMR) spectroscopy,[5] NMR relaxation measurements,[6] and electron paramagnetic resonance (EPR) spectroscopy,[1, 3] are at the frontier for capturing elusive details of membrane protein structure and dynamics. Here, we demonstrate the use of a novel combination of methods to present a dynamics-based picture that relates the structure of a membrane protein segment to the functional movement it supports. This is achieved by observing the surrounding hydration water, which rearranges simultaneously with protein conformational changes incurred upon activation. We obtained site-specific hydration dynamics information by using the NMR-signal-enhancement technique Overhauser dynamic nuclear polarization (ODNP), which was recently developed for probing local water diffusivity within approximately 10 (2–4 water layers) of a nitroxide radical spin-labeled amino acid residue.[7–12] This technique, combined with EPR lineshape analysis, which captures protein segment mobility around the spin label, offers a snapshot of distinct dynamic changes experienced by the protein as well as the solvating water. In addition, a newly developed analysis allows the separation of two important modes of water dynamics detected by ODNP. This new method isolates the contribution of 1) freely translating “hydration water”, which experiences fast, picosecond scale, motion, from 2) nanosecond timescale fluctuations in the spin interactions due to “bound” water molecules with slower dynamics or labile amide protons.In this study, ODNP is used to observe the photoactivation of a seven-helical transmembrane (7TM) proton pump, proteorhodopsin (PR). 7TMs form a broad class of proteins, including many physiologically relevant receptors and approximately 40% of drug targets.[13] We clarify changes in protein site-specific water dynamics around a critical PR segment upon activation, both on the picosecond and nanosecond timescales. The quantitative description of hydration dynamics across a biological interface still remains a challenging objective. Current EPR-based techniques used to study water in membrane protein systems either require cryogenic temperatures [14, 15] or the introduction of a soluble chemical agent,[16] whereas ODNP can directly evaluate water motion around the spin label under ambient conditions, as has recently been demonstrated for protein folding [12] and the conformational change of a membrane transporter.[17] Hydration water has been hypothesized to be an important determinant for the function of globular proteins,[18–20] but its role in the mechanics of transmembrane proteins is particularly complicated by the additional effect of the surrounding lipids, which have been thought to be the more influential solvent.[21, 22] However, recent experimental and theoretical work on bovine rhodopsin (Rh)[23, 24] and bacteriorhodopsin (BR)[25, 26] suggest that internal protein hydration does influence function and is indeed altered upon protein activation. Recent ssNMR work complements neutron-scattering studies of global hydration properties …