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.

Metadata

Authors/Creators:

Lane, BJ
Wang, B
Ma, Y
Calabrese, AN
El Mkami, H
Pliotas, C https://orcid.org/0000-0002-4309-4858

Keywords:

Biophysics; Cell Membrane; Mass Spectrometry; Molecular Biology; Protein Biochemistry; Protein expression and purification; Structural Biology; Electron Spin Resonance Spectroscopy; Membrane Proteins; Spin Labels

Dates: