HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

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

doi:10.1016/j.xpro.2022.101562

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

School of Physics and Astronomy

Research output: Contribution to journal › Review article › peer-review

3 Citations (Scopus)

12 Downloads (Pure)

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. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).

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	https://doi.org/10.1016/j.xpro.2022.101562
Publication status	Published - 16 Sept 2022

Keywords

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

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Lane_2022_STAR-protocols_HDX-guided-EPR-spectroscopy_CC
Copyright 2022 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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title = "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. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).",

keywords = "Biophysics, Cell membrane, Molecular biology, Protein biochemistry, Protein expression and purification, Structural biology, Mass spectrometry",

author = "Lane, {Benjamin J.} and Bolin Wang and Yue Ma and Calabrese, {Antonio N.} and {El Mkami}, Hassane and Christos Pliotas",

note = "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. ",

year = "2022",

month = sep,

day = "16",

doi = "10.1016/j.xpro.2022.101562",

language = "English",

volume = "3",

journal = "STAR Protocols",

issn = "2666-1667",

publisher = "Cell Press",

number = "3",

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TY - JOUR

T1 - HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

AU - Lane, Benjamin J.

AU - Wang, Bolin

AU - Ma, Yue

AU - Calabrese, Antonio N.

AU - El Mkami, Hassane

AU - Pliotas, Christos

N1 - 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.

PY - 2022/9/16

Y1 - 2022/9/16

N2 - 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).

AB - 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).

KW - Biophysics

KW - Cell membrane

KW - Molecular biology

KW - Protein biochemistry

KW - Protein expression and purification

KW - Structural biology

KW - Mass spectrometry

U2 - 10.1016/j.xpro.2022.101562

DO - 10.1016/j.xpro.2022.101562

M3 - Review article

SN - 2666-1667

VL - 3

JO - STAR Protocols

JF - STAR Protocols

IS - 3

M1 - 101562

ER -

HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

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