HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

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...

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Detalles Bibliográficos
Autores principales: Lane, Benjamin J., Wang, Bolin, Ma, Yue, Calabrese, Antonio N., El Mkami, Hassane, Pliotas, Christos
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9304679/
https://www.ncbi.nlm.nih.gov/pubmed/35874470
http://dx.doi.org/10.1016/j.xpro.2022.101562
Descripción
Sumario: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).

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