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Reconstitution of Detergent-Solubilized Membrane Proteins into Proteoliposomes and Nanodiscs for Functional and Structural Studies

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Structure and Function of Membrane Proteins

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

Abstract

Reconstitution of detergent-solubilized membrane proteins into phospholipid bilayers allows for functional and structural studies under close-to-native conditions that greatly support protein stability and function. Here we outline the detailed steps for membrane protein reconstitution to result in proteoliposomes and nanodiscs. Reconstitution can be achieved via a number of different strategies. The protocols for preparation of proteoliposomes use detergent removal via dialysis or via nonpolar polystyrene beads, or a mixture of the two methods. In this chapter, the protocols for nanodiscs apply polystyrene beads only. Proteoliposome preparation methods allow for substantial control of the lipid-to-protein ratio, from minimal amounts of phospholipid to high concentrations, type of phospholipid, and mixtures of phospholipids. In addition, dialysis affords a fairly large degree of control and variation of parameters such as rate of reconstitution, temperature, buffer conditions, and proteoliposome size. For the nanodisc approach, which is highly advantageous for ensuring equal access to both membrane sides of the protein as well as fast reconstitution of only a single membrane protein into a well-defined bilayer environment in each nanodisc, the protocols outline how a number of these parameters are more restricted in comparison to the proteoliposome protocols.

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References

  1. Gao Y, Cao E, Julius D, Cheng Y (2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 16:347–351

    Article  Google Scholar 

  2. Schmidt-Krey I (2007) Electron crystallography of membrane proteins: two-dimensional crystallization and screening by electron microscopy. Methods 41:417–426

    Article  CAS  Google Scholar 

  3. Kühlbrandt W (1992) Two-dimensional crystallization of membrane proteins. Q Rev Biophys 25:1–49

    Article  Google Scholar 

  4. Jap BK, Zulauf M, Scheybani T, Hefti A, Baumeister W, Aebi U, Engel A (1992) 2D crystallization: from art to science. Ultramicroscopy 46:45–84

    Article  CAS  Google Scholar 

  5. Engel A, Hoenger A, Hefti A, Henn C, Ford RC, Kistler J, Zulauf M (1992) Assembly of 2-D membrane protein crystals: dynamics, crystal order, and fidelity of structure analysis by electron microscopy. J Struct Biol 109:219–234

    Article  CAS  Google Scholar 

  6. Vink M, Derr K, Love J, Stokes DL, Ubarretxena-Belandia I (2007) A high-throughput strategy to screen 2D crystallization trials of membrane proteins. J Struct Biol 160:295–304

    Article  CAS  Google Scholar 

  7. Stahlberg H, Biyani N, Engel A (2015) 3D reconstruction of two-dimensional crystals. Arch Biochem Biophys 581:68–77

    Article  CAS  Google Scholar 

  8. Rigaud J-L, Mosser G, Lacapère J-J, Olofsson A, Lévy D, Ranck J-L (1997) Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct Biol 118:226–235

    Article  CAS  Google Scholar 

  9. Rigaud J-L, Lévy D, Mosser G, Lambert O (1998) Detergent removal by non-polar polystyrene beads. Eur Biophys J 27:305–319

    Article  CAS  Google Scholar 

  10. Bayburt TH, Carlson JW, Sligar SG (1998) Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer. J Struct Biol 123:37–44

    Article  CAS  Google Scholar 

  11. Bayburt TH, Grinkova YV, Sligar SG (2002) Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett 2:853–856

    Article  CAS  Google Scholar 

  12. Denisov IG, Grinkova YV, Lazarides AA, Sligar SG (2004) Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J Am Chem Soc 126:3477–3487

    Article  CAS  Google Scholar 

  13. Denisov IG, Sligar SG (2017) Nanodiscs in membrane biochemistry and biophysics. Chem Rev 117:4669–4713

    Article  CAS  Google Scholar 

  14. Yeh V, Lee T-Y, Chen C-W, Kuo P-C, Shiue J, Chu L-K, Yu T-Y (2018) Highly efficient transfer of 7TM membrane protein from native membrane to covalently circularized nanodisc. Sci Rep 8:13501

    Article  Google Scholar 

  15. Knowles TJ, Finka R, Smith C, Lin YP, Dafforn T, Overduin M (2009) Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J Am Chem Soc 131:7484–7485

    Article  CAS  Google Scholar 

  16. Esmaili M, Acevedo-Morantes C, Wille H, Overduin M (2020) The effect of hydrophobic alkyl sidechains on size and solution behaviors of nanodiscs formed by alternating styrene maleamic copolymer. Biochim Biophys Acta Biomembr 1862:183360

    Article  CAS  Google Scholar 

  17. Schmidt-Krey I, Lundqvist G, Morgenstern R, Hebert H (1998) Parameters for the two-dimensional crystallization of the membrane protein microsomal glutathione transferase. J Struct Biol 123:87–96

    Article  CAS  Google Scholar 

  18. Schmidt-Krey I, Mutucumarana V, Haase W, Stafford DW, Kühlbrandt W (2007) Two-dimensional crystallization of human vitamin K-dependent γ-glutamyl carboxylase. J Struct Biol 157:437–442

    Article  CAS  Google Scholar 

  19. Zhao G, Johnson MC, Schnell JR, Kanaoka Y, Haase W, Irikura D, Lam BK, Schmidt-Krey I (2010) Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring adjustment of a particularly large combination of specific parameters. J Struct Biol 169:450–454

    Article  CAS  Google Scholar 

  20. Schmidt-Krey I, Rubinstein JL (2011) Electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles. Micron 42:107–116

    Article  CAS  Google Scholar 

  21. Johnson MC, Schmidt-Krey I (2014) Towards a general protocol to form single-layered 2D crystal sheets of membrane proteins for electron crystallography. Microsc Microanal 20:1240–1241

    Article  Google Scholar 

  22. Uddin YM, Schmidt-Krey I (2015) Inducing two-dimensional crystallization of membrane proteins by dialysis for electron crystallography. Methods Enzymol 557:351–562

    Article  CAS  Google Scholar 

  23. Grinkova YV, Denisov IG, Sligar SG (2010) Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Eng Des Sel 23:843–848

    Article  CAS  Google Scholar 

  24. Ritchie TK, Grinkova YV, Bayburt TH, Denisov IG, Zolnerciks JK, Atkins WM, Sligar SG (2009) Chapter 11—reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464:211–231

    Article  CAS  Google Scholar 

  25. Rouck JE, Krapf JE, Roy J, Huff HC, Das A (2017) Recent advances in nanodisc technology for membrane protein studies (2012-2017). FEBS Lett 591:2057–2088

    Article  CAS  Google Scholar 

  26. Hagn F, Etzkorn M, Raschle T, Wagner G (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135:1919–1925

    Article  CAS  Google Scholar 

  27. Nasr ML, Baptista D, Strauss M, Sun Z-YJ, Grigoriu S, Huser S, Plückthun A, Hagn F, Walz T, Hogle JM, Wagner G (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14:49–52

    Article  CAS  Google Scholar 

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Author notes

  1. Kerry Strickland and Kasahun Neselu contributed equally.

Authors and Affiliations

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

    Kerry M. Strickland, Carolann L. Espy & Ingeborg Schmidt-Krey

  2. Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA, USA

    Kerry M. Strickland & Nael A. McCarty

  3. School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA

    Kasahun Neselu, Arshay J. Grant, Carolann L. Espy & Ingeborg Schmidt-Krey

Authors
  1. Kerry M. Strickland
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  2. Kasahun Neselu
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  3. Arshay J. Grant
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  4. Carolann L. Espy
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  5. Nael A. McCarty
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  6. Ingeborg Schmidt-Krey
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Corresponding author

Correspondence to Ingeborg Schmidt-Krey .

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

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Strickland, K.M., Neselu, K., Grant, A.J., Espy, C.L., McCarty, N.A., Schmidt-Krey, I. (2021). Reconstitution of Detergent-Solubilized Membrane Proteins into Proteoliposomes and Nanodiscs for Functional and Structural Studies. 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_2

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  • DOI: https://doi.org/10.1007/978-1-0716-1394-8_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1393-1

  • Online ISBN: 978-1-0716-1394-8

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