Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
STAR Protoc. 2022 Jul 18;3(3):101562. doi: 10.1016/j.xpro.2022.101562. eCollection 2022 Sep 16.
Solvent accessibilities of and distances between protein residues measured by pulsed-EPR approaches provide high-resolution information on dynamic protein motions. We describe protocols for the purification and site-directed spin labeling of integral membrane proteins. In our protocol, peptide-level HDX-MS is used as a precursor to guide single-residue resolution ESEEM accessibility measurements and spin labeling strategies for EPR applications. Exploiting the pentameric MscL channel as a model, we discuss the use of cwEPR, DEER/PELDOR, and ESEEM spectroscopies to interrogate membrane protein dynamics. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).
通过脉冲 EPR 方法测量的溶剂可及性和蛋白质残基之间的距离为动态蛋白质运动提供了高分辨率信息。我们描述了用于纯化和定点自旋标记完整膜蛋白的方案。在我们的方案中，肽水平的 HDX-MS 被用作先导，以指导用于 EPR 应用的单残基分辨率 ESEEM 可及性测量和自旋标记策略。我们利用五聚体 MscL 通道作为模型，讨论了使用连续波 EPR、DEER/PELDOR 和 ESEEM 光谱学来研究膜蛋白动力学。有关此协议的使用和执行的完整详细信息，请参阅 Wang 等人。（2022 年）。
