HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

Benjamin J. Lane, Bolin Wang, Yue Ma, Antonio N. Calabrese, Hassane El Mkami, Christos Pliotas*

*Corresponding author for this work

Research output: Contribution to journalReview articlepeer-review

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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).
Original languageEnglish
Article number101562
Number of pages28
JournalSTAR Protocols
Issue number3
Early online date18 Jul 2022
Publication statusPublished - 16 Sept 2022


  • Biophysics
  • Cell membrane
  • Molecular biology
  • Protein biochemistry
  • Protein expression and purification
  • Structural biology
  • Mass spectrometry


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