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Integrating hydrogen–deuterium exchange mass spectrometry with molecular dynamics simulations to probe lipid-modulated conformational changes in membrane proteins


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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Fig. 1: Four-stage workflow to identify lipid–protein interactions involved in conformational transitions of membrane proteins.
Fig. 2: Select strategies to produce conformationally locked membrane proteins.
Fig. 3: Workflow for identification of lipids modulating conformational dynamics.
Fig. 4: Workflow for MD simulations of membrane protein in mixed bilayers.
Fig. 5: General protocol for MD simulations of membrane proteins in a bilayer.
Fig. 6: Experimental validation of a molecular hypothesis suggested by MD simulations.
Fig. 7: Using Deuteros to visualize and filter peptides displaying significant differences in deuterium uptake.
Fig. 8: MD simulations in mixed bilayers predict direct interactions between the phospholipid PE and the transporters XylE and LacY.

Data availability

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 and


  1. 1.

    Laganowsky, A. et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Martens, C. et al. Direct protein-lipid interactions shape the conformational landscape of secondary transporters. Nat. Commun. 9, 4151 (2018).

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Pliotas, C. et al. The role of lipids in mechanosensation. Nat. Struct. Mol. Biol. 22, 991–998 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Yin, H. & Flynn, A. D. Drugging membrane protein interactions. Annu. Rev. Biomed. Eng. 18, 51–76 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Gao, Y., Cao, E., Julius, D. & Cheng, Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534, 347–351 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Zhou, M. et al. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334, 380–385 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Allison, T. M. et al. Quantifying the stabilizing effects of protein-ligand interactions in the gas phase. Nat. Commun. 6, 8551 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Landreh, M. et al. Integrating mass spectrometry with MD simulations reveals the role of lipids in Na(+)/H(+) antiporters. Nat. Commun. 8, 13993 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Gupta, K. et al. Identifying key membrane protein lipid interactions using mass spectrometry. Nat. Protoc. 13, 1106–1120 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Pyle, E. et al. Structural lipids enable the formation of functional oligomers of the eukaryotic purine symporter UapA. Cell Chem. Biol. 25, 840–848.e4 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Sanders, M. R., Findlay, H. E. & Booth, P. J. Lipid bilayer composition modulates the unfolding free energy of a knotted alpha-helical membrane protein. Proc. Natl. Acad. Sci. USA 115, E1799–E1808 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Alam, A., Kowal, J., Broude, E., Roninson, I. & Locher, K. P. Structural insight into substrate and inhibitor discrimination by human P-glycoprotein. Science 363, 753–756 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Hunte, C. & Richers, S. Lipids and membrane protein structures. Curr. Opin. Struct. Biol. 18, 406–411 (2008).

    CAS  PubMed  Google Scholar 

  14. 14.

    Majumdar, D. S. et al. Single-molecule FRET reveals sugar-induced conformational dynamics in LacY. Proc. Natl. Acad. Sci. USA 104, 12640–12645 (2007).

    CAS  PubMed  Google Scholar 

  15. 15.

    Claxton, D. P., Kazmier, K., Mishra, S. & McHaourab, H. S. Navigating membrane protein structure, dynamics, and energy landscapes using spin labeling and EPR spectroscopy. Methods Enzymol. 564, 349–387 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Liang, B. & Tamm, L. K. NMR as a tool to investigate the structure, dynamics and function of membrane proteins. Nat. Struct. Mol. Biol. 23, 468–474 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Konermann, L., Stocks, B. B., Pan, Y. & Tong, X. Mass spectrometry combined with oxidative labeling for exploring protein structure and folding. Mass Spectrom. Rev. 29, 651–667 (2010).

    CAS  PubMed  Google Scholar 

  18. 18.

    Konijnenberg, A. et al. Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry. Proc. Natl. Acad. Sci. USA 111, 17170–17175 (2014).

    CAS  PubMed  Google Scholar 

  19. 19.

    Marcoux, J. et al. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc. Natl. Acad. Sci. USA 110, 9704–9709 (2013).

    CAS  PubMed  Google Scholar 

  20. 20.

    Vahidi, S., Bi, Y., Dunn, S. D. & Konermann, L. Load-dependent destabilization of the gamma-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry. Proc. Natl. Acad. Sci. USA 113, 2412–2417 (2016).

    CAS  PubMed  Google Scholar 

  21. 21.

    Li, S., Lee, S. Y. & Chung, K. Y. Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry. Methods Enzymol. 557, 261–278 (2015).

    CAS  PubMed  Google Scholar 

  22. 22.

    Mehmood, S., Domene, C., Forest, E. & Jault, J. M. Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations. Proc. Natl. Acad. Sci. USA 109, 10832–10836 (2012).

    CAS  PubMed  Google Scholar 

  23. 23.

    Zhou, M. et al. Ion mobility-mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility. Nat. Chem. 6, 208–215 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Eisinger, M. L., Dorrbaum, A. R., Michel, H., Padan, E. & Langer, J. D. Ligand-induced conformational dynamics of the Escherichia coli Na(+)/H(+) antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry. Proc. Natl. Acad. Sci. USA 114, 11691–11696 (2017).

    CAS  PubMed  Google Scholar 

  25. 25.

    Merkle, P. S. et al. Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT. Sci. Adv. 4, eaar6179 (2018).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Vadas, O. & Burke, J. E. Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange-MS (HDX-MS). Biochem. Soc. Trans. 43, 773–786 (2015).

    CAS  PubMed  Google Scholar 

  27. 27.

    Harrison, R. A. & Engen, J. R. Conformational insight into multi-protein signaling assemblies by hydrogen-deuterium exchange mass spectrometry. Curr. Opin. Struct. Biol. 41, 187–193 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Trabjerg, E., Nazari, Z. E. & Rand, K. D. Conformational analysis of complex protein states by hydrogen/deuterium exchange mass spectrometry (HDX-MS): challenges and emerging solutions. Trends Anal. Chem. 106, 125–138 (2018).

    CAS  Google Scholar 

  29. 29.

    Reading, E. et al. Interrogating membrane protein conformational dynamics within native lipid compositions. Angew. Chem. 56, 15654–15657 (2017).

    CAS  Google Scholar 

  30. 30.

    Hebling, C. M. et al. Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry. Anal. Chem. 82, 5415–5419 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Adhikary, S. et al. Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer. Proc. Natl. Acad. Sci. USA 114, E1786–E1795 (2017).

    CAS  PubMed  Google Scholar 

  32. 32.

    Duc, N. M. et al. Effective application of bicelles for conformational analysis of G protein-coupled receptors by hydrogen/deuterium exchange mass spectrometry. J. Am. Soc. Mass Spectrom. 26, 808–817 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Schuler, M. A., Denisov, I. G. & Sligar, S. G. Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol. Biol. 974, 415–433 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Keener, J. E. et al. Chemical additives enable native mass spectrometry measurement of membrane protein oligomeric state within intact nanodiscs. J. Am. Chem. Soc. 141, 1054–1061 (2018).

    Google Scholar 

  35. 35.

    Marty, M. T., Hoi, K. K., Gault, J. & Robinson, C. V. Probing the lipid annular belt by gas-phase dissociation of membrane proteins in nanodiscs. Angew. Chem. 55, 550–554 (2016).

    CAS  Google Scholar 

  36. 36.

    Murcia Rios, A., Vahidi, S., Dunn, S. D. & Konermann, L. Evidence for a partially stalled gamma rotor in F1-ATPase from hydrogen-deuterium exchange experiments and molecular dynamics simulations. J. Am. Chem. Soc. 140, 14860–14869 (2018).

    CAS  PubMed  Google Scholar 

  37. 37.

    Skinner, J. J. et al. Benchmarking all-atom simulations using hydrogen exchange. Proc. Natl. Acad. Sci. USA 111, 15975–15980 (2014).

    CAS  PubMed  Google Scholar 

  38. 38.

    Persson, F. & Halle, B. How amide hydrogens exchange in native proteins. Proc. Natl. Acad. Sci. USA 112, 10383–10388 (2015).

    CAS  PubMed  Google Scholar 

  39. 39.

    Marrink, S. J. et al. Computational modeling of realistic cell membranes. Chem. Rev. 119, 6184–6226 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Wen, P. C. et al. Microscopic view of lipids and their diverse biological functions. Curr. Opin. Struct. Biol. 51, 177–186 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Contreras, F. X. et al. Molecular recognition of a single sphingolipid species by a protein’s transmembrane domain. Nature 481, 525–529 (2012).

    CAS  PubMed  Google Scholar 

  42. 42.

    Zeppelin, T., Ladefoged, L. K., Sinning, S., Periole, X. & Schiott, B. A direct interaction of cholesterol with the dopamine transporter prevents its out-to-inward transition. PLoS Comput. Biol. 14, e1005907 (2018).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Hedger, G. & Sansom, M. S. P. Lipid interaction sites on channels, transporters and receptors: recent insights from molecular dynamics simulations. Biochim. Biophys. Acta 1858, 2390–2400 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Salas-Estrada, L. A., Leioatts, N., Romo, T. D. & Grossfield, A. Lipids alter rhodopsin function via ligand-like and solvent-like interactions. Biophys. J. 114, 355–367 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Frauenfeld, J. et al. A saposin-lipoprotein nanoparticle system for membrane proteins. Nat. Methods 13, 345–351 (2016).

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Eswar, N. et al. Comparative protein structure modeling using Modeller. Curr. Protoc. Bioinform. 15, 5.6.1–5.6.30 (2006).

    Google Scholar 

  47. 47.

    Sun, L. et al. Crystal structure of a bacterial homologue of glucose transporters GLUT1-4. Nature 490, 361–366 (2012).

    CAS  PubMed  Google Scholar 

  48. 48.

    Abramson, J. et al. Structure and mechanism of the lactose permease of Escherichia coli. Science 301, 610–615 (2003).

    CAS  PubMed  Google Scholar 

  49. 49.

    West, G. M. et al. Ligand-dependent perturbation of the conformational ensemble for the GPCR beta2 adrenergic receptor revealed by HDX. Structure 19, 1424–1432 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Yellen, G. The voltage-gated potassium channels and their relatives. Nature 419, 35–42 (2002).

    CAS  PubMed  Google Scholar 

  51. 51.

    Gurevich, V. V. & Gurevich, E. V. Molecular mechanisms of GPCR signaling: a structural perspective. Int. J. Mol. Sci. 18, E2519 (2017).

    PubMed  Google Scholar 

  52. 52.

    Drew, D. & Boudker, O. Shared molecular mechanisms of membrane transporters. Annu. Rev. Biochem. 85, 543–572 (2016).

    CAS  PubMed  Google Scholar 

  53. 53.

    Denisov, I. G. & Sligar, S. G. Nanodiscs in membrane biochemistry and biophysics. Chem. Rev. 117, 4669–4713 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Lau, A. M. C., Ahdash, Z., Martens, C. & Politis, A. Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry. Bioinformatics 35, 3171–3173 (2019).

    PubMed  PubMed Central  Google Scholar 

  55. 55.

    Manna, M. et al. Mechanism of allosteric regulation of beta2-adrenergic receptor by cholesterol. eLife 5, e18432 (2016).

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Guixa-Gonzalez, R. et al. Membrane cholesterol access into a G-protein-coupled receptor. Nat. Commun. 8, 14505 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

    CAS  PubMed  Google Scholar 

  60. 60.

    Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    CAS  PubMed  Google Scholar 

  61. 61.

    Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).

    CAS  PubMed  Google Scholar 

  62. 62.

    Kandt, C., Ash, W. L. & Tieleman, D. P. Setting up and running molecular dynamics simulations of membrane proteins. Methods 41, 475–488 (2007).

    CAS  PubMed  Google Scholar 

  63. 63.

    Forest, E. & Man, P. Conformational dynamics and interactions of membrane proteins by hydrogen/deuterium mass spectrometry. Methods Mol. Biol. 1432, 269–279 (2016).

    CAS  PubMed  Google Scholar 

  64. 64.

    Denisov, I. G. & Sligar, S. G. Nanodiscs for structural and functional studies of membrane proteins. Nat. Struct. Mol. Biol. 23, 481–486 (2016).

    CAS  PubMed  Google Scholar 

  65. 65.

    Bayburt, T. H. & Sligar, S. G. Membrane protein assembly into nanodiscs. FEBS Lett. 584, 1721–1727 (2010).

    CAS  PubMed  Google Scholar 

  66. 66.

    Ritchie, T. K. et al. Chapter 11—Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Denisov, I. G., Grinkova, Y. V., Lazarides, A. A. & Sligar, S. G. Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004).

    CAS  PubMed  Google Scholar 

  68. 68.

    Distler, U., Kuharev, J. & Tenzer, S. Biomedical applications of ion mobility-enhanced data-independent acquisition-based label-free quantitative proteomics. Expert Rev. Proteom. 11, 675–684 (2014).

    CAS  Google Scholar 

  69. 69.

    Martens, C. et al. Lipids modulate the conformational dynamics of a secondary multidrug transporter. Nat. Struct. Mol. Biol. 23, 744–751 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Sohlenkamp, C. & Geiger, O. Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiol. Rev. 40, 133–59 (2015).

    PubMed  Google Scholar 

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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.).

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C.M. and A.P. designed the experiments. C.M. performed the mutagenesis, expression, purification, reconstitution and HDX-MS experiments. M.S. and E.T. performed and analyzed the MD simulations. C.M. and A.M.L. analyzed the HDX-MS data. C.M. and A.P. wrote the paper with input from all authors.

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Correspondence to Emad Tajkhorshid or Argyris Politis.

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The authors declare no competing interests.

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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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Martens, C. et al. Nat. Commun. 9, 4151 (2018)

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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).

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