Advertisement

Two-Dimensional Crystallization of Membrane Proteins by Reconstitution Through Dialysis

  • Matthew C. Johnson
  • Tina M. Dreaden
  • Laura Y. Kim
  • Frederik Rudolph
  • Bridgette A. Barry
  • Ingeborg Schmidt-Krey
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 955)

Abstract

Studies of membrane proteins by two-dimensional (2D) crystallization and electron crystallography have provided crucial information on the structure and function of a rapidly growing number of these intricate proteins within a close-to-native lipid bilayer. Here we provide protocols for planning and executing 2D crystallization trials by detergent removal through dialysis, including the preparation of phospholipids and the dialysis setup. General factors to be considered, such as the protein preparation, solubilizing detergent, lipid for reconstitution, and buffer conditions are discussed. Several 2D crystallization conditions are highlighted that have shown great promise to grow 2D crystals within a surprisingly short amount of time. Finally, conditions for optimizing order and size of 2D crystals are outlined.

Key words

Two-dimensional crystallization 2D crystals Membrane protein Dialysis Lipid Detergent Electron crystallography 

Notes

Acknowledgments

We wish to thank our colleagues in the laboratory as well as in the cryo-EM community for helpful discussions over the years. We are particularly indebted to our collaborators for entrusting us with their valuable samples. Research in the Schmidt-Krey laboratory contributing to some of these observations was funded by NIH grant HL090630 and the Integrative Biosystems Institute.

References

  1. 1.
    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 109:219–234PubMedCrossRefGoogle Scholar
  2. 2.
    Jap BK, Zulauf M, Scheybani T, Hefti A, Baumeister W, Aebi U, Engel A (1992) 2D crystallization—from art to science. Ultra­microscopy 46:45–84PubMedCrossRefGoogle Scholar
  3. 3.
    Dolder M, Engel A, Zulauf M (1996) The micelle to vesicle transition of lipids and detergents in the presence of a membrane protein: towards a rationale for 2D crystallization. FEBS Lett 382:203–208PubMedCrossRefGoogle Scholar
  4. 4.
    Hasler L, Heymann JB, Engel A, Kistler J, Walz T (1998) 2D crystallization of membrane proteins: rationales and examples. J Struct Biol 121:162–171PubMedCrossRefGoogle Scholar
  5. 5.
    Kühlbrandt W (2003) Two-dimensional crystallization of membrane proteins: a practical guide. In: Schägger H, Hunte C (eds) Membrane protein purification and crystallization: a ­practical approach. Academic Press, San Diego, pp 253–284Google Scholar
  6. 6.
    Schmidt-Krey I (2007) Electron crystallography of membrane proteins: two-dimensional crystallization and screening by electron microscopy. Methods 41:417–426PubMedCrossRefGoogle Scholar
  7. 7.
    Abeyrathne PD, Chami M, Pantelic RS, Goldie KN, Stahlberg H (2010) Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection. Methods Enzymol 481:25–43PubMedCrossRefGoogle Scholar
  8. 8.
    Xu C, Rice WJ, He W, Stokes DL (2002) A structural model for the catalytic cycle of Ca(2+)-ATPase. J Mol Biol 8:201–211CrossRefGoogle Scholar
  9. 9.
    Ryan C, Stokes DL, Chen M, Zhang Z, Hardwicke PM (2004) Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum. J Biol Chem 13:5380–5386Google Scholar
  10. 10.
    Stokes DL, Delavoie F, Rice WJ, Champeil P, McIntosh DB, Lacapère JJ (2005) Structural studies of a stabilized phosphoenzyme intermediate of Ca2+-ATPase. J Biol Chem 280:18063–18072PubMedCrossRefGoogle Scholar
  11. 11.
    Chen Z, Akin BL, Stokes DL, Jones LR (2006) Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain. J Biol Chem 281:14163–14172PubMedCrossRefGoogle Scholar
  12. 12.
    Glaves JP, Trieber CA, Ceholski DK, Stokes DL, Young HS (2011) Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump. J Mol Biol 405:707–723PubMedCrossRefGoogle Scholar
  13. 13.
    Rigaud JL, Chami M, Lambert O, Levy D, Ranck JL (2000) Use of detergents in two-dimensional crystallization of membrane proteins. Biochim Biophys Acta Biomembr 1508:112–128CrossRefGoogle Scholar
  14. 14.
    Kühlbrandt W (1992) 2-dimensional crystallization of membrane proteins. Q Rev Biophys 25:1–49PubMedCrossRefGoogle Scholar
  15. 15.
    Zhao G, Johnson MC, Schnell JR, Kanaoka Y, Irikura D, Lam BK, Austen KF, Schmidt-Krey I (2010) Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring a particularly large combination of specific parameters. J Struct Biol 169:450–454PubMedCrossRefGoogle Scholar
  16. 16.
    Brisson A, Unwin PNT (1984) Tubular crystals of acetylcholine-receptor. J Cell Biol 99:1202–1211PubMedCrossRefGoogle Scholar
  17. 17.
    Iacovache I, Biasini M, Kowal J, Kukulski W, Chami M, van der Goot FG, Engel A, Rémigy HW (2009) The 2DX robot: a membrane protein 2D crystallization Swiss Army knife. J Struct Biol 169:370–378PubMedCrossRefGoogle Scholar
  18. 18.
    Kim C, Vink M, Hu M, Love J, Stokes DL, Ubarretxena-Belandia I (2010) An automated pipeline to screen membrane protein 2D crystallization. J Struct Funct Genomics 11:155–166PubMedCrossRefGoogle Scholar
  19. 19.
    Hu M, Vink M, Kim C, Derr K, Koss J, D’Amico K, Cheng A, Pulokas J, Ubarretxena-Belandia I, Stokes D (2010) Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins. J Struct Biol 171(1):102–110PubMedCrossRefGoogle Scholar
  20. 20.
    Rigaud JL, Mosser G, Lacapere JJ, Olofsson A, Levy D, Ranck JL (1997) Bio-beads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct Biol 118:226–235PubMedCrossRefGoogle Scholar
  21. 21.
    Schmidt-Krey I, Mutucumarana V, Haase W, Stafford DW, Kühlbrandt W (2006) Two-dimensional crystallization of human vitamin K-dependent γ-glutamyl carboxylase. J Struct Biol 157:437–442PubMedCrossRefGoogle Scholar
  22. 22.
    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 123:87–96PubMedCrossRefGoogle Scholar
  23. 23.
    Schmidt-Krey I, Kanaoka Y, Mills DJ, Irikura D, Haase W, Lam BK, Austen KF, Kühlbrandt W (2004) Human leukotriene C4 synthase at 4.5 Å resolution in projection. Structure 12:2009–2014PubMedCrossRefGoogle Scholar
  24. 24.
    Mitra AK, Miercke LJW, Turner GJ, Shand RF, Betlach MC, Stroud RM (1993) 2-dimensional crystallization of Escherichia-coli-expressed bacteriorhodopsin and its D96N variant—high-resolution structural studies in projection. Biophys J 65:1295–1306PubMedCrossRefGoogle Scholar
  25. 25.
    Tsai CJ, Ziegler C (2005) Structure determination of secondary transport proteins by electron crystallography: two-dimensional crystallization of the betaine uptake system BetP. J Mol Microbiol Biotechnol 10:197–207PubMedCrossRefGoogle Scholar
  26. 26.
    Nussberger S, Dorr K, Wang DN, Kühlbrandt W (1993) Lipid-protein interactions in crystals of plant light-harvesting complex. J Mol Biol 234:347–356PubMedCrossRefGoogle Scholar
  27. 27.
    Cevc G (1987) How membrane chain melting properties are regulated by the polar surface of the lipid bilayer. Biochemistry 26:6305–6310PubMedCrossRefGoogle Scholar
  28. 28.
    Mosser G (2001) Two-dimensional crystallogenesis of transmembrane proteins. Micron 32:517–540PubMedCrossRefGoogle Scholar
  29. 29.
    Kruse O, Hankamer B, Konczak C, Gerle C, Morris E, Radunz A, Schmid GH, Barber J (2000) Phosphatidylglycerol is involved in the dimerization of photosystem II. J Biol Chem 275:6509–6514PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Matthew C. Johnson
    • 1
  • Tina M. Dreaden
    • 2
  • Laura Y. Kim
    • 1
  • Frederik Rudolph
    • 1
  • Bridgette A. Barry
    • 2
  • Ingeborg Schmidt-Krey
    • 3
  1. 1.School of BiologyGeorgia Institute of TechnologyAtlantaUSA
  2. 2.School of Chemistry and BiochemistryGeorgia Institute of TechnologyAtlantaUSA
  3. 3.School of Biology, School of Chemistry and BiochemistryGeorgia Institute of TechnologyAtlantaUSA

Personalised recommendations