Using hydrogen/deuterium exchange mass spectrometry to understand bacterial membrane efflux proteins
Using hydrogen/deuterium exchange mass spectrometry to understand bacterial membrane efflux proteins
Bacterial multidrug efflux pumps play major roles in antibiotic and multidrug resistance as well as fulfilling many important physiological functions. These molecular machines are highly dynamic, with structural malleability at the core of their action, and elaborating their conformational features can provide insight into the mechanisms underpinning their activities and responses to transport substrates and inhibitors. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a structural biology assay used to monitor protein backbone dynamics and is capable of elucidating conformational signatures that define function and fold. In recent years, HDX-MS methods have advanced, making its use more amenable to the study of membrane proteins, yet experimental challenges remain. In this chapter, we provide background on HDX-MS, its limitations, and discussions around experimental design and optimization for studying efflux pump proteins, including its application to study membrane proteins in lipid environments. To demonstrate its utility, we provide an original case study on AcrB inhibition by an efflux pump inhibitor (MBX-3756), employing membrane-scaffold protein lipid nanodiscs, revealing perturbation of backbone motions distinguishing of hydrophobic-trap targeting inhibitory action. This chapter can serve as a guide for designing HDX-MS investigations on efflux pump proteins and membrane proteins in general.
Efflux pump inhibitors, Efflux pumps, Hydrogen/deuterium exchange, Mass spectrometry, Membrane proteins, Protein dynamics, Structural biology
Russell Lewis, Benjamin
6052ce96-6af8-4c50-a49c-594782e76493
Hammerschmid, Dietmar
6f12ca9a-3ec4-499e-94b8-9aff279f23a4
Sys, Jakub
6c2415a1-1e3c-4570-abe1-57be7cdaf826
Reading, Eamonn
62fed933-f867-4c72-89e7-83aea573a836
Russell Lewis, Benjamin
6052ce96-6af8-4c50-a49c-594782e76493
Hammerschmid, Dietmar
6f12ca9a-3ec4-499e-94b8-9aff279f23a4
Sys, Jakub
6c2415a1-1e3c-4570-abe1-57be7cdaf826
Reading, Eamonn
62fed933-f867-4c72-89e7-83aea573a836
Russell Lewis, Benjamin, Hammerschmid, Dietmar, Sys, Jakub and Reading, Eamonn
(2025)
Using hydrogen/deuterium exchange mass spectrometry to understand bacterial membrane efflux proteins.
Methods in Enzymology.
(doi:10.1016/bs.mie.2025.08.026).
Abstract
Bacterial multidrug efflux pumps play major roles in antibiotic and multidrug resistance as well as fulfilling many important physiological functions. These molecular machines are highly dynamic, with structural malleability at the core of their action, and elaborating their conformational features can provide insight into the mechanisms underpinning their activities and responses to transport substrates and inhibitors. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a structural biology assay used to monitor protein backbone dynamics and is capable of elucidating conformational signatures that define function and fold. In recent years, HDX-MS methods have advanced, making its use more amenable to the study of membrane proteins, yet experimental challenges remain. In this chapter, we provide background on HDX-MS, its limitations, and discussions around experimental design and optimization for studying efflux pump proteins, including its application to study membrane proteins in lipid environments. To demonstrate its utility, we provide an original case study on AcrB inhibition by an efflux pump inhibitor (MBX-3756), employing membrane-scaffold protein lipid nanodiscs, revealing perturbation of backbone motions distinguishing of hydrophobic-trap targeting inhibitory action. This chapter can serve as a guide for designing HDX-MS investigations on efflux pump proteins and membrane proteins in general.
Text
CH0005_Reading_Accepted manuscript
- Accepted Manuscript
More information
e-pub ahead of print date: 18 September 2025
Keywords:
Efflux pump inhibitors, Efflux pumps, Hydrogen/deuterium exchange, Mass spectrometry, Membrane proteins, Protein dynamics, Structural biology
Identifiers
Local EPrints ID: 506256
URI: http://eprints.soton.ac.uk/id/eprint/506256
ISSN: 0076-6879
PURE UUID: 39e6c8ad-edc7-42cd-bc16-9ca46cae5c4c
Catalogue record
Date deposited: 31 Oct 2025 17:37
Last modified: 01 Nov 2025 03:06
Export record
Altmetrics
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics
