HDX-guided EPR spectroscopy to interrogate membrane protein dynamics
Abstract
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).
Citation
Lane , B J , Wang , B , Ma , Y , Calabrese , A N , El Mkami , H & Pliotas , C 2022 , ' HDX-guided EPR spectroscopy to interrogate membrane protein dynamics ' , STAR Protocols , vol. 3 , no. 3 , 101562 . https://doi.org/10.1016/j.xpro.2022.101562
Publication
STAR Protocols
Status
Peer reviewed
ISSN
2666-1667Type
Journal item
Description
Funding: This project was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/S018069/1) to C.P., who also acknowledges support from the Wellcome Trust (WT) (219999/Z/19/Z) and the Chinese Scholarship Council (CSC) in the form of studentships for B.J.L. and B.W., respectively. A.N.C. is a Sir Henry Dale Fellow jointly funded by the WT and the Royal Society (220628/Z/20/Z). Funding from the BBSRC (BB/M012573/1) enabled the purchase of mass spectrometry equipment.Collections
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