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.
| Original language | English |
|---|---|
| Article number | 101562 |
| Number of pages | 28 |
| Journal | STAR protocols |
| Volume | 3 |
| Issue number | 3 |
| Early online date | 18 Jul 2022 |
| DOIs | |
| Publication status | Published - 16 Sept 2022 |
Bibliographical note
Copyright © The Author(s). This is an open access article distributed under the terms of the Creative Commons CC-BY (https://creativecommons.org/licenses/by/4.0/) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Data Access Statement
Data are available within the following link: http://archive.researchdata.leeds.ac.uk/777/ and via the https://doi.org/10.5518/914.This paper does not report original code. Any additional information required to reanalyze the data reported is available from the lead contact upon request.
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.