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Hydrogen/Deuterium Exchange Mass Spectrometry for the Structural Analysis of Detergent-Solubilized Membrane Proteins

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Expression, Purification, and Structural Biology of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2127))

Abstract

Integral membrane proteins are involved in numerous biological functions and represent important drug targets. Despite their abundance in the human proteome, the number of integral membrane protein structures is largely underrepresented in the Protein Data Bank. The challenges associated with the biophysical characterization of such biological systems are well known. Most structural approaches, including X-ray crystallography, SAXS, or mass spectrometry (MS), require the complete solubilization of membrane proteins in aqueous solutions. Detergents are frequently used for this task, but may interfere with the analysis, as is the case with MS. The use of “MS-friendly” detergents, such as non-ionic alkyl glycoside detergents, has greatly facilitated the analysis of detergent-solubilized membrane proteins. Here, we describe a protocol, which we have successfully implemented in our laboratory to study the structure and dynamics of detergent-solubilized integral membrane proteins by Hydrogen/Deuterium eXchange and Mass Spectrometry (HDX-MS). The procedure does not require detergent removal prior to MS analysis, instead taking advantage of the ultra-high pressure chromatographic system to separate deuterated peptides from “MS-friendly” detergents.

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References

  1. Jaswal SS (2013) Biological insights from hydrogen exchange mass spectrometry. Biochim Biophys Acta 1834(6):1188–1201. https://doi.org/10.1016/j.bbapap.2012.10.011

    Article  CAS  PubMed  Google Scholar 

  2. Konermann L, Pan J, Liu YH (2011) Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem Soc Rev 40(3):1224–1234. https://doi.org/10.1039/c0cs00113a

    Article  CAS  PubMed  Google Scholar 

  3. Oganesyan I, Lento C, Wilson DJ (2018) Contemporary hydrogen deuterium exchange mass spectrometry. Methods 144:27–42. https://doi.org/10.1016/j.ymeth.2018.04.023

    Article  CAS  PubMed  Google Scholar 

  4. Tsutsui Y, Wintrode PL (2007) Hydrogen/deuterium exchange-mass spectrometry: a powerful tool for probing protein structure, dynamics and interactions. Curr Med Chem 14(22):2344–2358

    Article  CAS  Google Scholar 

  5. Wales TE, Engen JR (2006) Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom Rev 25(1):158–170. https://doi.org/10.1002/mas.20064

    Article  CAS  PubMed  Google Scholar 

  6. Brier S, Engen JR (2008) Hydrogen exchange mass spectrometry: principles and capabilities. In: Chance M (ed) Mass spectrometry analysis for protein-protein interactions and dynamics. Wiley, New York, pp 11–43

    Google Scholar 

  7. Brier S, Fagnocchi L, Donnarumma D, Scarselli M, Rappuoli R, Nissum M, Delany I, Norais N (2012) Structural insight into the mechanism of DNA-binding attenuation of the Neisserial adhesin repressor NadR by the small natural ligand 4-hydroxyphenylacetic acid. Biochemistry 51(34):6738–6752. https://doi.org/10.1021/bi300656w

    Article  CAS  PubMed  Google Scholar 

  8. Faleri A, Santini L, Brier S, Pansegrau W, Lo Surdo P, Scarselli M, Buricchi F, Volpini G, Genovese A, van der Veen S, Lea S, Tang CM, Savino S, Pizza M, Finco O, Norais N, Masignani V (2014) Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties. FASEB J 28(4):1644–1653. https://doi.org/10.1096/fj.13-239012

    Article  CAS  PubMed  Google Scholar 

  9. Georgescauld F, Popova K, Gupta AJ, Bracher A, Engen JR, Hayer-Hartl M, Hartl FU (2014) GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding. Cell 157(4):922–934. https://doi.org/10.1016/j.cell.2014.03.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pirrone GF, Emert-Sedlak LA, Wales TE, Smithgall TE, Kent MS, Engen JR (2015) Membrane-associated conformation of HIV-1 Nef investigated with hydrogen exchange mass spectrometry at a Langmuir monolayer. Anal Chem 87(14):7030–7035. https://doi.org/10.1021/acs.analchem.5b01725

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Trelle MB, Dupont DM, Madsen JB, Andreasen PA, Jorgensen TJ (2014) Dissecting the effect of RNA aptamer binding on the dynamics of plasminogen activator inhibitor 1 using hydrogen/deuterium exchange mass spectrometry. ACS Chem Biol 9(1):174–182. https://doi.org/10.1021/cb400619v

    Article  CAS  PubMed  Google Scholar 

  12. Canul-Tec JC, Assal R, Cirri E, Legrand P, Brier S, Chamot-Rooke J, Reyes N (2017) Structure and allosteric inhibition of excitatory amino acid transporter 1. Nature 544(7651):446–451. https://doi.org/10.1038/nature22064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Chung KY, Rasmussen SG, Liu T, Li S, DeVree BT, Chae PS, Calinski D, Kobilka BK, Woods VL Jr, Sunahara RK (2011) Conformational changes in the G protein Gs induced by the beta2 adrenergic receptor. Nature 477(7366):611–615. https://doi.org/10.1038/nature10488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Malito E, Faleri A, Lo Surdo P, Veggi D, Maruggi G, Grassi E, Cartocci E, Bertoldi I, Genovese A, Santini L, Romagnoli G, Borgogni E, Brier S, Lo Passo C, Domina M, Castellino F, Felici F, van der Veen S, Johnson S, Lea SM, Tang CM, Pizza M, Savino S, Norais N, Rappuoli R, Bottomley MJ, Masignani V (2013) Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen. Proc Natl Acad Sci U S A 110(9):3304–3309. https://doi.org/10.1073/pnas.1222845110

    Article  PubMed  PubMed Central  Google Scholar 

  15. O'Brien DP, Brier S, Ladant D, Durand D, Chenal A, Vachette P (2018) SEC-SAXS and HDX-MS: a powerful combination. The case of the calcium-binding domain of a bacterial toxin. Biotechnol Appl Biochem 65(1):62–68. https://doi.org/10.1002/bab.1577

    Article  CAS  PubMed  Google Scholar 

  16. O'Brien DP, Durand D, Voegele A, Hourdel V, Davi M, Chamot-Rooke J, Vachette P, Brier S, Ladant D, Chenal A (2017) Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis. PLoS Biol 15(12):e2004486. https://doi.org/10.1371/journal.pbio.2004486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. O'Brien DP, Hernandez B, Durand D, Hourdel V, Sotomayor-Perez AC, Vachette P, Ghomi M, Chamot-Rooke J, Ladant D, Brier S, Chenal A (2015) Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion. Sci Rep 5:14223. https://doi.org/10.1038/srep14223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Cavaliere P, Brier S, Filipenko P, Sizun C, Raynal B, Bonnete F, Levi-Acobas F, Bellalou J, England P, Chamot-Rooke J, Mayer C, Norel F (2018) The stress sigma factor of RNA polymerase RpoS/sigma(S) is a solvent-exposed open molecule in solution. Biochem J 475(1):341–354. https://doi.org/10.1042/BCJ20170768

    Article  CAS  PubMed  Google Scholar 

  19. Hamdi K, Salladini E, O'Brien DP, Brier S, Chenal A, Yacoubi I, Longhi S (2017) Structural disorder and induced folding within two cereal, ABA stress and ripening (ASR) proteins. Sci Rep 7(1):15544. https://doi.org/10.1038/s41598-017-15299-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wales TE, Fadgen KE, Gerhardt GC, Engen JR (2008) High-speed and high-resolution UPLC separation at zero degrees Celsius. Anal Chem 80(17):6815–6820. https://doi.org/10.1021/ac8008862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hourdel V, Volant S, O'Brien DP, Chenal A, Chamot-Rooke J, Dillies MA, Brier S (2016) MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets. Bioinformatics 32(22):3413–3419. https://doi.org/10.1093/bioinformatics/btw420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Miller DE, Prasannan CB, Villar MT, Fenton AW, Artigues A (2012) HDXFinder: automated analysis and data reporting of deuterium/hydrogen exchange mass spectrometry. J Am Soc Mass Spectrom 23(2):425–429. https://doi.org/10.1007/s13361-011-0234-5

    Article  CAS  PubMed  Google Scholar 

  23. Pascal BD, Chalmers MJ, Busby SA, Mader CC, Southern MR, Tsinoremas NF, Griffin PR (2007) The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data. BMC Bioinformatics 8:156. https://doi.org/10.1186/1471-2105-8-156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pascal BD, Willis S, Lauer JL, Landgraf RR, West GM, Marciano D, Novick S, Goswami D, Chalmers MJ, Griffin PR (2012) HDX workbench: software for the analysis of H/D exchange MS data. J Am Soc Mass Spectrom 23(9):1512–1521. https://doi.org/10.1007/s13361-012-0419-6

    Article  CAS  PubMed  Google Scholar 

  25. Lim XX, Chandramohan A, Lim XY, Bag N, Sharma KK, Wirawan M, Wohland T, Lok SM, Anand GS (2017) Conformational changes in intact dengue virus reveal serotype-specific expansion. Nat Commun 8:14339. https://doi.org/10.1038/ncomms14339

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Giladi M, Almagor L, van Dijk L, Hiller R, Man P, Forest E, Khananshvili D (2016) Asymmetric preorganization of inverted pair residues in the sodium-calcium exchanger. Sci Rep 6:20753. https://doi.org/10.1038/srep20753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Giladi M, van Dijk L, Refaeli B, Almagor L, Hiller R, Man P, Forest E, Khananshvili D (2017) Dynamic distinctions in the Na(+)/Ca(2+) exchanger adopting the inward- and outward-facing conformational states. J Biol Chem 292(29):12311–12323. https://doi.org/10.1074/jbc.M117.787168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mehmood S, Domene C, Forest E, Jault JM (2012) Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations. Proc Natl Acad Sci U S A 109(27):10832–10836. https://doi.org/10.1073/pnas.1204067109

    Article  PubMed  PubMed Central  Google Scholar 

  29. West GM, Chien EY, Katritch V, Gatchalian J, Chalmers MJ, Stevens RC, Griffin PR (2011) Ligand-dependent perturbation of the conformational ensemble for the GPCR beta2 adrenergic receptor revealed by HDX. Structure 19(10):1424–1432. https://doi.org/10.1016/j.str.2011.08.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Yang L, Yang D, de Graaf C, Moeller A, West GM, Dharmarajan V, Wang C, Siu FY, Song G, Reedtz-Runge S, Pascal BD, Wu B, Potter CS, Zhou H, Griffin PR, Carragher B, Yang H, Wang MW, Stevens RC, Jiang H (2015) Conformational states of the full-length glucagon receptor. Nat Commun 6:7859. https://doi.org/10.1038/ncomms8859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang X, Chien EY, Chalmers MJ, Pascal BD, Gatchalian J, Stevens RC, Griffin PR (2010) Dynamics of the beta2-adrenergic G-protein coupled receptor revealed by hydrogen-deuterium exchange. Anal Chem 82(3):1100–1108. https://doi.org/10.1021/ac902484p

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Antharavally BS, Mallia KA, Rosenblatt MM, Salunkhe AM, Rogers JC, Haney P, Haghdoost N (2011) Efficient removal of detergents from proteins and peptides in a spin column format. Anal Biochem 416(1):39–44. https://doi.org/10.1016/j.ab.2011.05.013

    Article  CAS  PubMed  Google Scholar 

  33. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362. https://doi.org/10.1038/nmeth.1322

    Article  CAS  PubMed  Google Scholar 

  34. Cirri E, Brier S, Assal R, Canul-Tec JC, Chamot-Rooke J, Reyes N (2018) Consensus designs and thermal stability determinants of a human glutamate transporter. eLife 7. doi:https://doi.org/10.7554/eLife.40110

  35. Hamuro Y, Coales SJ (2018) Optimization of feasibility stage for hydrogen/deuterium exchange mass spectrometry. J Am Soc Mass Spectrom 29(3):623–629. https://doi.org/10.1007/s13361-017-1860-3

    Article  CAS  PubMed  Google Scholar 

  36. Guttman M, Wales TE, Whittington D, Engen JR, Brown JM, Lee KK (2016) Tuning a high transmission ion guide to prevent gas-phase proton exchange during H/D exchange MS analysis. J Am Soc Mass Spectrom 27(4):662–668. https://doi.org/10.1007/s13361-015-1330-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Adhikary S, Deredge DJ, Nagarajan A, Forrest LR, Wintrode PL, Singh SK (2017) Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer. Proc Natl Acad Sci U S A 114(10):E1786–E1795. https://doi.org/10.1073/pnas.1613293114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hebling CM, Morgan CR, Stafford DW, Jorgenson JW, Rand KD, Engen JR (2010) Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry. Anal Chem 82(13):5415–5419. https://doi.org/10.1021/ac100962c

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Morgan CR, Hebling CM, Rand KD, Stafford DW, Jorgenson JW, Engen JR (2011) Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs. Mol Cell Proteomics 10(9):M111.010876. https://doi.org/10.1074/mcp.M111.010876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Parker CH, Morgan CR, Rand KD, Engen JR, Jorgenson JW, Stafford DW (2014) A conformational investigation of propeptide binding to the integral membrane protein gamma-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry. Biochemistry 53(9):1511–1520. https://doi.org/10.1021/bi401536m

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Redhair M, Clouser AF, Atkins WM (2019) Hydrogen-deuterium exchange mass spectrometry of membrane proteins in lipid Nanodiscs. Chem Phys Lipids 220:14. https://doi.org/10.1016/j.chemphyslip.2019.02.007

    Article  CAS  PubMed  Google Scholar 

  42. Vahidi S, Bi Y, Dunn SD, Konermann L (2016) Load-dependent destabilization of the gamma-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry. Proc Natl Acad Sci U S A 113(9):2412–2417. https://doi.org/10.1073/pnas.1520464113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Donnarumma D, Maestri C, Giammarinaro PI, Capriotti L, Bartolini E, Veggi D, Petracca R, Scarselli M, Norais N (2018) Native State Organization of Outer Membrane Porins Unraveled by HDx-MS. J Proteome Res 17(5):1794–1800. https://doi.org/10.1021/acs.jproteome.7b00830

    Article  CAS  PubMed  Google Scholar 

  44. Cravello L, Lascoux D, Forest E (2003) Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteins. Rapid Commun Mass Spectrom 17(21):2387–2393. https://doi.org/10.1002/rcm.1207

    Article  CAS  PubMed  Google Scholar 

  45. Mysling S, Salbo R, Ploug M, Jorgensen TJ (2014) Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry. Anal Chem 86(1):340–345. https://doi.org/10.1021/ac403269a

    Article  CAS  PubMed  Google Scholar 

  46. Trabjerg E, Jakobsen RU, Mysling S, Christensen S, Jorgensen TJ, Rand KD (2015) Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow. Anal Chem 87(17):8880–8888. https://doi.org/10.1021/acs.analchem.5b01996

    Article  CAS  PubMed  Google Scholar 

  47. Forest E, Man P (2016) Conformational dynamics and interactions of membrane proteins by hydrogen/deuterium mass spectrometry. Methods Mol Biol 1432:269–279. https://doi.org/10.1007/978-1-4939-3637-3_17

    Article  CAS  PubMed  Google Scholar 

  48. Majumdar R, Manikwar P, Hickey JM, Arora J, Middaugh CR, Volkin DB, Weis DD (2012) Minimizing carry-over in an online pepsin digestion system used for the H/D exchange mass spectrometric analysis of an IgG1 monoclonal antibody. J Am Soc Mass Spectrom 23(12):2140–2148. https://doi.org/10.1007/s13361-012-0485-9

    Article  CAS  PubMed  Google Scholar 

  49. Claesen J, Burzykowski T (2017) Computational methods and challenges in hydrogen/deuterium exchange mass spectrometry. Mass Spectrom Rev 36(5):649–667. https://doi.org/10.1002/mas.21519

    Article  CAS  PubMed  Google Scholar 

  50. Brier S, Le Mignon M, Jain K, Lebrun C, Peurois F, Kellenberger C, Bordas-Le Floch V, Mascarell L, Nony E, Moingeon P (2018) Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen bet v 1. Allergy 73(5):1032–1040. https://doi.org/10.1111/all.13364

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The HDX-MS system was financed by the Equipex CACSICE (ANR-11-EQPX-0008). SB would like to thank Alexandre Chenal, Nadia Izadi-Prunyere, Mariette Matondo, Florence Cordier, Thierry Lang, Daniel Ladant, Iñaki Guijarro and Michael Nilges for their kindness and constant support over the last year.

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Authors and Affiliations

  1. Biochemistry of Macromolecular Interaction Unit, Department of Structural Biology and Chemistry, Institut Pasteur, CNRS UMR3528, Paris, France

    Darragh P. O’Brien & Alexandre Chenal

  2. Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK

    Darragh P. O’Brien

  3. Environment and Infectious Risks Unit, Department of Infection and Epidemiology, Institut Pasteur, Paris, France

    Véronique Hourdel

  4. Biological NMR Technological Platform, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, CNRS UMR3528, Paris, France

    Sébastien Brier

Authors
  1. Darragh P. O’Brien
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  2. Véronique Hourdel
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  3. Alexandre Chenal
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  4. Sébastien Brier
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Corresponding author

Correspondence to Sébastien Brier .

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Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Camilo Perez

  2. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Timm Maier

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O’Brien, D.P., Hourdel, V., Chenal, A., Brier, S. (2020). Hydrogen/Deuterium Exchange Mass Spectrometry for the Structural Analysis of Detergent-Solubilized Membrane Proteins. In: Perez, C., Maier, T. (eds) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0373-4_22

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  • DOI: https://doi.org/10.1007/978-1-0716-0373-4_22

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  • Publisher Name: Humana, New York, NY

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