NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Utrecht University, Pandualaan 8, 3584 CH, Utrecht, The Netherlands.
Angew Chem Int Ed Engl. 2016 Oct 17;55(43):13606-13610. doi: 10.1002/anie.201606594. Epub 2016 Sep 27.
H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.
H 检测可以显著提高固态 NMR 光谱灵敏度，从而可以研究更复杂的蛋白质。然而，H 检测的常见前提条件是在其他氘代蛋白质中引入可交换的质子，这迄今为止极大地阻碍了对部分水不可及的蛋白质（例如膜蛋白）的研究。在此，我们提出了一种方法，该方法能够对水不可及的蛋白质进行高分辨率的 H 检测固态 NMR（ssNMR）研究，即使在高度复杂的环境中（例如细胞表面）也能工作。 特别是，该方法应用于研究脂质体和天然细菌细胞膜中 K 通道 KcsA。我们使用我们的数据进行了动态分析，结果表明，负责离子传导且在 K 通道中高度保守的选择性过滤器会发生明显的分子运动。我们期望这种方法为生物分子 ssNMR 开辟新途径。
