Skip to main content
SpringerLink
Log in

Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering

  • Protocol
  • First Online:
Structure and Function of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2302))

Abstract

This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370–377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5(12):993–996. https://doi.org/10.1038/nrd2199

    Article  CAS  PubMed  Google Scholar 

  2. Carpenter EP, Beis K, Cameron AD, Iwata S (2008) Overcoming the challenges of membrane protein crystallography. Curr Opin Struct Biol 18(5):581–586. https://doi.org/10.1016/j.sbi.2008.07.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lin S-H, Guidotti G (2009) Purification of membrane proteins. In: Burgess RR, Deutscher MP (eds) Methods in enzymology, vol 463. Academic, New York, NY, pp 619–629. https://doi.org/10.1016/S0076-6879(09)63035-4

    Chapter  Google Scholar 

  4. Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666(1–2):105–117. https://doi.org/10.1016/j.bbamem.2004.04.011

    Article  CAS  PubMed  Google Scholar 

  5. Wiener MC (2004) A pedestrian guide to membrane protein crystallization. Methods 34(3):364–372. https://doi.org/10.1016/j.ymeth.2004.03.025

    Article  CAS  PubMed  Google Scholar 

  6. Hong X, Weng YX, Li M (2004) Determination of the topological shape of integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria in the detergent solution by small-angle X-ray scattering. Biophys J 86(2):1082–1088. https://doi.org/10.1016/s0006-3495(04)74183-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gimpl K, Klement J, Keller S (2016) Characterising protein/detergent complexes by triple-detection size-exclusion chromatography. Biol Proced Online 18:4. https://doi.org/10.1186/s12575-015-0031-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Breyton C, Gabel F, Lethier M, Flayhan A, Durand G, Jault JM, Juillan-Binard C, Imbert L, Moulin M, Ravaud S, Hartlein M, Ebel C (2013) Small angle neutron scattering for the study of solubilised membrane proteins. Eur Phys J E Soft Matter 36(7):71. https://doi.org/10.1140/epje/i2013-13071-6

    Article  CAS  PubMed  Google Scholar 

  9. Garg S, Porcar L, Woodka AC, Butler PD, Perez-Salas U (2011) Noninvasive neutron scattering measurements reveal slower cholesterol transport in model lipid membranes. Biophys J 101(2):370–377. https://doi.org/10.1016/j.bpj.2011.06.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shull CG, Wollan EO (1948) X-ray, electron, and neutron diffraction. Science 108(2795):69–75. https://doi.org/10.1126/science.108.2795.69

    Article  CAS  PubMed  Google Scholar 

  11. Oliver RC, Naing S-H, Weiss KL, Pingali SV, Lieberman RL, Urban VS (2018) Contrast-matching detergent in small-angle neutron scattering experiments for membrane protein structural analysis and ab Initio modeling. JoVE 140:e57901

    Google Scholar 

  12. Trewhella J (2006) Neutrons reveal how nature uses structural themes and variation in biological regulation. Physica B 385:825–830

    Article  Google Scholar 

  13. Oliver RC, Pingali SV, Urban VS (2017) Designing mixed detergent micelles for uniform neutron contrast. J Phys Chem Lett 8(20):5041–5046. https://doi.org/10.1021/acs.jpclett.7b02149

    Article  CAS  PubMed  Google Scholar 

  14. Naing SH, Oliver RC, Weiss KL, Urban VS, Lieberman RL (2018) Solution structure of an intramembrane aspartyl protease via small angle neutron scattering. Biophys J 114(3):602–608. https://doi.org/10.1016/j.bpj.2017.12.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Midtgaard SR, Darwish TA, Pedersen MC, Huda P, Larsen AH, Jensen GV, Kynde SAR, Skar-Gislinge N, Nielsen AJZ, Olesen C, Blaise M, Dorosz JJ, Thorsen TS, Venskutonyte R, Krintel C, Moller JV, Frielinghaus H, Gilbert EP, Martel A, Kastrup JS, Jensen PE, Nissen P, Arleth L (2018) Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering. FEBS J 285(2):357–371. https://doi.org/10.1111/febs.14345

    Article  CAS  PubMed  Google Scholar 

  16. Ashkar R, Bilheux HZ, Bordallo H, Briber R, Callaway DJE, Cheng X, Chu XQ, Curtis JE, Dadmun M, Fenimore P, Fushman D, Gabel F, Gupta K, Herberle F, Heinrich F, Hong L, Katsaras J, Kelman Z, Kharlampieva E, Kneller GR, Kovalevsky A, Krueger S, Langan P, Lieberman R, Liu Y, Losche M, Lyman E, Mao Y, Marino J, Mattos C, Meilleur F, Moody P, Nickels JD, O’Dell WB, O’Neill H, Perez-Salas U, Peters J, Petridis L, Sokolov AP, Stanley C, Wagner N, Weinrich M, Weiss K, Wymore T, Zhang Y, Smith JC (2018) Neutron scattering in the biological sciences: progress and prospects. Acta Crystallogr D Struct Biol 74(Pt 12):1129–1168. https://doi.org/10.1107/S2059798318017503

    Article  CAS  PubMed  Google Scholar 

  17. Meilleur F, Weiss KL, Myles DA (2009) Deuterium labeling for neutron structure-function-dynamics analysis. Methods Mol Biol 544:281–292. https://doi.org/10.1007/978-1-59745-483-4_18

    Article  CAS  PubMed  Google Scholar 

  18. Erez E, Fass D, Bibi E (2009) How intramembrane proteases bury hydrolytic reactions in the membrane. Nature 459(7245):371

    Article  CAS  Google Scholar 

  19. Lemberg MK, Bland FA, Weihofen A, Braud VM, Martoglio B (2001) Intramembrane proteolysis of signal peptides: an essential step in the generation of HLA-E epitopes. J Immunol 167(11):6441–6446. https://doi.org/10.4049/jimmunol.167.11.6441

    Article  CAS  PubMed  Google Scholar 

  20. McLauchlan J, Lemberg MK, Hope G, Martoglio B (2002) Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J 21(15):3980–3988. https://doi.org/10.1093/emboj/cdf414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wolfe MS (2019) Structure and function of the gamma-secretase complex. Biochemistry 58(27):2953–2966. https://doi.org/10.1021/acs.biochem.9b00401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wolfe MS, Xia W, Ostaszewski BL, Diehl TS, Kimberly WT, Selkoe DJ (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398(6727):513–517. https://doi.org/10.1038/19077

    Article  CAS  PubMed  Google Scholar 

  23. Chow VW, Mattson MP, Wong PC, Gleichmann M (2010) An overview of APP processing enzymes and products. NeuroMolecular Med 12(1):1–12. https://doi.org/10.1007/s12017-009-8104-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Katz JJ, Crespi HL (1966) Deuterated organisms: cultivation and uses. Science 151(3715):1187–1194. https://doi.org/10.1126/science.151.3715.1187

    Article  CAS  PubMed  Google Scholar 

  25. Dang S, Wu S, Wang J, Li H, Huang M, He W, Li YM, Wong CC, Shi Y (2015) Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH. Proc Natl Acad Sci U S A 112(11):3344–3349. https://doi.org/10.1073/pnas.1502150112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bai XC, Yan C, Yang G, Lu P, Ma D, Sun L, Zhou R, Scheres SHW, Shi Y (2015) An atomic structure of human gamma-secretase. Nature 525(7568):212–217. https://doi.org/10.1038/nature14892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Li X, Dang S, Yan C, Gong X, Wang J, Shi Y (2013) Structure of a presenilin family intramembrane aspartate protease. Nature 493(7430):56–61. https://doi.org/10.1038/nature11801

    Article  CAS  PubMed  Google Scholar 

  28. Naing SH, Kalyoncu S, Smalley DM, Kim H, Tao X, George JB, Jonke AP, Oliver RC, Urban VS, Torres MP, Lieberman RL (2018) Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog. J Biol Chem 293(13):4653–4663. https://doi.org/10.1074/jbc.RA117.001436

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Naing SH, Vukoti KM, Drury JE, Johnson JL, Kalyoncu S, Hill SE, Torres MP, Lieberman RL (2015) Catalytic properties of intramembrane aspartyl protease substrate hydrolysis evaluated using a FRET peptide cleavage assay. ACS Chem Biol 10(9):2166–2174. https://doi.org/10.1021/acschembio.5b00305

    Article  CAS  PubMed  Google Scholar 

  30. Johnson JL, Kalyoncu S, Lieberman RL (2016) Lessons from an alpha-helical membrane enzyme: expression, purification, and detergent optimization for biophysical and structural characterization. Methods Mol Biol 1432:281–301. https://doi.org/10.1007/978-1-4939-3637-3_18

    Article  CAS  PubMed  Google Scholar 

  31. Holme T (1970) Enzymes-laboratory scale production. Process Biochem 5:62–66

    CAS  Google Scholar 

  32. Törnkvist M, Larsson G, Enfors S-O (1996) Protein release and foaming in Escherichia coli cultures grown in minimal medium. Bioprocess Eng 15(5):231–237. https://doi.org/10.1007/bf02391583

    Article  Google Scholar 

  33. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552. https://doi.org/10.1385/1-59259-584-7:531

    Article  CAS  PubMed  Google Scholar 

  34. Norholm MH, Light S, Virkki MT, Elofsson A, von Heijne G, Daley DO (2012) Manipulating the genetic code for membrane protein production: what have we learnt so far? Biochim Biophys Acta 1818(4):1091–1096. https://doi.org/10.1016/j.bbamem.2011.08.018

    Article  CAS  PubMed  Google Scholar 

  35. Koch AL (1970) Turbidity measurements of bacterial cultures in some available commercial instruments. Anal Biochem 38(1):252–259. https://doi.org/10.1016/0003-2697(70)90174-0

    Article  CAS  PubMed  Google Scholar 

  36. VanAken T, Foxall-VanAken S, Castleman S, Ferguson-Miller S (1986) Alkyl glycoside detergents: synthesis and applications to the study of membrane proteins. Methods Enzymol 125:27–35. https://doi.org/10.1016/s0076-6879(86)25005-3

    Article  CAS  PubMed  Google Scholar 

  37. Feroz H, Vandervelden C, Ikwuagwu B, Ferlez B, Baker CS, Lugar DJ, Grzelakowski M, Golbeck JH, Zydney AL, Kumar M (2016) Concentrating membrane proteins using ultrafiltration without concentrating detergents. Biotechnol Bioeng 113(10):2122–2130. https://doi.org/10.1002/bit.25973

    Article  CAS  PubMed  Google Scholar 

  38. Zhou R, Shi Y, Yang G (2017) Expression, purification, and enzymatic characterization of intramembrane proteases. Methods Enzymol 584:127–155. https://doi.org/10.1016/bs.mie.2016.09.046

    Article  CAS  PubMed  Google Scholar 

  39. Torres-Arancivia C, Ross CM, Chavez J, Assur Z, Dolios G, Mancia F, Ubarretxena-Belandia I (2010) Identification of an archaeal presenilin-like intramembrane protease. PLoS One 5(9). https://doi.org/10.1371/journal.pone.0013072

Download references

Acknowledgments

SANS studies on IAPs in the Lieberman lab are supported by NSF grant 1817796. The Office of Biological and Environmental Research supported research at Oak Ridge National Laboratory’s Center for Structural Molecular Biology (CSMB), using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy.

Author information

Authors and Affiliations

  1. School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Yuqi Wu & Raquel L. Lieberman

  2. Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

    Kevin L. Weiss

Authors
  1. Yuqi Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kevin L. Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Raquel L. Lieberman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Raquel L. Lieberman .

Editor information

Editors and Affiliations

  1. School of Biological Sciences and School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Ingeborg Schmidt-Krey

  2. School of Physics, Georgia Institute of Technology, Atlanta, GA, USA

    James C. Gumbart

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wu, Y., Weiss, K.L., Lieberman, R.L. (2021). Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering. In: Schmidt-Krey, I., Gumbart, J.C. (eds) Structure and Function of Membrane Proteins. Methods in Molecular Biology, vol 2302. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1394-8_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1394-8_12

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1393-1

  • Online ISBN: 978-1-0716-1394-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation