Biological membranes define the boundaries of cells and are composed primarily of phospholipids and membrane proteins. It has become increasingly evident that direct interactions of membrane proteins with their surrounding lipids play key roles in regulating both protein conformations and function. However, the exact nature and structural consequences of these interactions remain difficult to track at the molecular level. Here, we present a protocol that specifically addresses this challenge. First, hydrogen–deuterium exchange mass spectrometry (HDX-MS) of membrane proteins incorporated into nanodiscs of controlled lipid composition is used to obtain information on the lipid species that are involved in modulating the conformational changes in the membrane protein. Then molecular dynamics (MD) simulations in lipid bilayers are used to pinpoint likely lipid–protein interactions, which can be tested experimentally using HDX-MS. By bringing together the MD predictions with the conformational readouts from HDX-MS, we have uncovered key lipid–protein interactions implicated in stabilizing important functional conformations. This protocol can be applied to virtually any integral membrane protein amenable to classic biophysical studies and for which a near-atomic-resolution structure or homology model is available. This protocol takes ~4 d to complete, excluding the time for data analysis and MD simulations, which depends on the size of the protein under investigation.
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Data supporting the findings of this article are available from the corresponding author upon reasonable request. All the deuterium uptake plots and uptake datasets of the experiments presented in this work are available on the figshare data repository at https://figshare.com/articles/XylE_HDX-MS_uptake_plots_and_uptake_data/7072988 and https://figshare.com/articles/LacY_HDX-MS_uptake_plots_and_uptake_datasets/7073072
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We thank O. Boudker and X. Wang (Weill Cornell Medical College) for providing the GltPh samples. This work was supported by the Wellcome Trust (109854/Z/15/Z) and a King’s Health Partners R&D Challenge Fund through the MRC (MC_PC_15031) to A.P. C.M. was a research fellow from the FRS-FNRS (grant no, 1.B.261.19F). The research was supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under awards U54-GM087519, P41-GM104601 and R01-GM123455 to E.T. The London Interdisciplinary Biosciences Consortium (LIDo) BBSRC Doctoral Training Partnership (BB/M009513/1) supported A.M.L. We also acknowledge computing resources provided by Blue Waters at the National Center for Supercomputing Applications and the Extreme Science and Engineering Discovery Environment (grant TG-MCA06N060 to E.T.).
The authors declare no competing interests.
Journal peer review information Nature Protocols thanks Kasper Rand and other anonymous reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Martens, C. et al. Nat. Commun. 9, 4151 (2018) https://www.nature.com/articles/s41467-018-06704-1
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Martens, C., Shekhar, M., Lau, A.M. et al. Integrating hydrogen–deuterium exchange mass spectrometry with molecular dynamics simulations to probe lipid-modulated conformational changes in membrane proteins. Nat Protoc 14, 3183–3204 (2019). https://doi.org/10.1038/s41596-019-0219-6