HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

Abstract

Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Biophysics
• Cell Membrane
• Molecular Biology
• Protein Biochemistry
• Protein expression and purification
• Structural Biology