Journal of Bioenergetics and Biomembranes

, Volume 28, Issue 1, pp 13–27 | Cite as

Strategies for crystallizing membrane proteins

  • R. Michael Garavito
  • Daniel Picot
  • Patrick J. Loll


Crystallizing membrane proteins remains a challenging endeavor despite the increasing number of membrane protein structures solved by X-ray crystallography. The critical factors in determining the success of the crystallization experiments are the purification and preparation of membrane protein samples. Moreover, there is the added complication that the crystallization conditions must be optimized for use in the presence of detergents although the methods used to crystallize most membrane proteins are, in essence, straightforward applications of standard methodologies for soluble protein crystallization. The roles that detergents play in the stability and aggregation of membrane proteins as well as the colloidal properties of the protein-detergent complexes need to be appreciated and controlledbefore and during the crystallization trials. All X-ray quality crystals of membrane proteins were grown from preparations of detergent-solubilized protein, where the heterogeneous natural lipids from the membrane have been replaced by ahomogeneous detergent environment. It is the preparation of such monodisperse, isotropic solutions of membrane proteins that has allowed the successful application of the standard crystallization methods routinely used on soluble proteins. In this review, the issues of protein purification and sample preparation are addressed as well as the new refinements in crystallization methodologies for membrane proteins. How the physical behavior of the detergent, in the form of micelles or protein-detergent aggregates, affects crystallization and the adaptation of published protocols to new membrane protein systems are also addressed. The general conclusion is that many integral membrane proteins could be crystallized if pure and monodisperse preparations in a suitable detergent system can be prepared.

Key words

Membrane proteins crystallization nonionic detergents protein-detergent interactions monodispersity 


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  1. Allen, J. P., Feher, G., Yeates, T. O., Komiya, K., and Rees, D. C. (1987).Proc. Natl. Acad. Sci. USA 84, 5730–5734.PubMedGoogle Scholar
  2. Andreu, J. M. (1985).Methods Enzymol. 117, 346–354.PubMedGoogle Scholar
  3. Bordier, C. (1981).J. Biol. Chem. 256, 1604–1609.PubMedGoogle Scholar
  4. Bott, R. R., Navia, M. A., and Smith, J. L. (1982).J. Biol. Chem. 257, 9883–9886.PubMedGoogle Scholar
  5. Buchanan, S. K., Fritzsch, G., Ermler, U., and Michel, H. (1993).J. Mol. Biol. 230, 1311–1314.CrossRefPubMedGoogle Scholar
  6. Carter, C. W., Jr. (1990).Methods: A Companion to Methods in Enzymology 1, 12–24.CrossRefGoogle Scholar
  7. Chang, C.-H., Schiffer, M., Tiede, D., Smith, U., and Norris, J. R. (1985).J. Mol. Biol. 186, 201–203.CrossRefPubMedGoogle Scholar
  8. Chang, C.-H., Tiede, D., Tang, J., Smith, U., Norris, J. R., and Schiffer, M. (1986).FEBS Lett. 205, 82–86.CrossRefPubMedGoogle Scholar
  9. Cowan, S. W., Schirmer, T., Rummel, R., Steiert, M., Ghosh, R., Pauptit, R., Jansonius, J. N., and Rosenbusch, J. P. (1992).Nature (London) 358, 727–733.CrossRefGoogle Scholar
  10. Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y., and McPherson, A. (1994).Acta Cryst. D50, 414–423.Google Scholar
  11. D'Arcy, A. (1994).Acta Cryst. D50, 469–471.Google Scholar
  12. De Grip, W. J. (1982).Methods Enzymol. 81, 256–265.PubMedGoogle Scholar
  13. Deisenhofer, J., Epp, O., Miki, K., Huber, R., and Michel, H. (1985).Nature (London) 318, 618–624.CrossRefGoogle Scholar
  14. Eisele, J.-L., and Rosenbusch, J. P. (1989).J. Mol. Biol. 206, 209–212.CrossRefPubMedGoogle Scholar
  15. Garavito, R. M., and Picot, D. (1990).Methods: A Companion to Methods in Enzymology 1, 57–69.CrossRefGoogle Scholar
  16. Garavito, R. M., Hinz, U., and Neuhaus, J.-M. (1984).J. Biol. Chem. 259, 4254–4257.PubMedGoogle Scholar
  17. Garavito, R. M., and Rosenbusch, J. P. (1986).Methods in Enzymol. 125, 309–328.Google Scholar
  18. Garavito, R. M., Jenkins, J. A., Jansonius, J. N., Karlsson, R., and Rosenbusch, J. P. (1983).J. Mol. Biol. 164, 313–327.CrossRefPubMedGoogle Scholar
  19. Garavito, R. M., Markovic-Housley, Z., and Jenkins, J. A. (1986).J. Cryst. Growth 76, 701–709.CrossRefGoogle Scholar
  20. Giege, R., Dock, A. C., Kern, D., Lorber, B., Thierry, J. C., and Moras, D. (1986).J. Crys. Growth 76, 554–561.CrossRefGoogle Scholar
  21. Gouaux, J. E., Braha, O., Hobaugh, M. R., Song, L., Cheley, S., Shustak, C., and Bayley, H. (1994).Proc. Natl. Acad. Sci. USA 91, 12828–12831.PubMedGoogle Scholar
  22. Harlan, J. E. (1993).Solution Structure and Function of Ligand Interactions with Prostaglandin H2 Synthase, Ph.D. Thesis, University of Chicago.Google Scholar
  23. Helenius, A., and Simons, K. (1975).Biochim. Biophys. Acta 415, 69–79.Google Scholar
  24. Helenius, A., McCaslin, D. R., Fries, E., and Tanford, C. (1979).Methods Enzymol. 56, 734–749.PubMedGoogle Scholar
  25. Hermann, K. W. (1966).J. Colloid Interface Sci. 22, 352–358.CrossRefGoogle Scholar
  26. Hermann, K. W., Brushmiller, J. G., and Courchene W. L. (1966).J. Phys. Chem. 70, 2909–2918.Google Scholar
  27. Hubbard, S. R., Wei, L., Ellis, L., and Hendrickson, W. A. (1994).Nature (London) 372, 746–754.CrossRefGoogle Scholar
  28. Jancarik, J., and Kim, S.-H. (1991).J. Appl. Cryst. 24, 409–411.CrossRefGoogle Scholar
  29. Kameyama, K., and Takagi, T. (1990).J. Colloid Interface Sci. 137, 1–10.CrossRefGoogle Scholar
  30. Kates, M. (1986).Techniques in Lipidology, Elsevier, New York.Google Scholar
  31. Kingston, R. L., Baker, H. M., and Baker, E. N. (1994).Acta Cryst. D50, 429–440.Google Scholar
  32. Kleymann, G., Ostermeier, C., Ludwig, B., Skerra, A., and Michel, H. (1995).Bio/Technology 13, 155–160.CrossRefPubMedGoogle Scholar
  33. Krauss, N., Hinrichs, W., Witt, I., Fromme, P., Pritzkow, W., Dauter, Z., Betzel, C., Wilson, K. S., Witt, H. T., and Saenger, W. (1993).Nature (London) 361, 326–331.CrossRefGoogle Scholar
  34. Kresheck, G. C. (1981).Chem. Phys. Lipids 29, 69–74.CrossRefGoogle Scholar
  35. Kühlbrandt, W. (1988).Q. Rev. Biophys. 21, 429–477.PubMedGoogle Scholar
  36. Külbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994).Nature (London) 367, 614–621.CrossRefGoogle Scholar
  37. Le Maire, M., Kwee, S., Andersen, J., and Møller, J. (1983).Eur J. Biochem. 129, 525–532.PubMedGoogle Scholar
  38. Lorber, B., Bishop, J. B., and DeLucas, L. J. (1990).Biophys. Biochim. Acta 1023, 254–265.Google Scholar
  39. McDermott, G., Prince, S. M., Freer, A. A., Hawthornthwaite-Lawless, Papiz, M. Z., Cogdell, R. J., and Isaacs, N. W. (1995).Nature (London) 374, 517–521.Google Scholar
  40. McPherson, A. (1982).Preparation and Analysis of Protein Crystals, Wiley, New York.Google Scholar
  41. Michel, H. (1982a).J. Mol. Biol. 158, 567–572.CrossRefPubMedGoogle Scholar
  42. Michel, H. (1982b).EMBO J. 1, 1267–1271.Google Scholar
  43. Michel, H. (1983).Trends Biochem. Sci. 8, 56–59.CrossRefGoogle Scholar
  44. Michel, H. (1991).Crystallization of Membrane Proteins, CRC Press, Boca Raton, pp. 73–88.Google Scholar
  45. Mitchell, D. J., Tiddy, G. J. T., Waring, L., Waring, T., Waring, B., and McDonald, M. P. (1983).J. Chem. Soc. Faraday Trans. 79, 975–1000.CrossRefGoogle Scholar
  46. Møller, J., and Le Maire, M. (1993).J. Biol. Chem. 268, 18659–18672.PubMedGoogle Scholar
  47. Papiz, M. Z., Hawthornwaite, A. M., Cogdell, R. J., Woolley, K. J., Wightman, P. A., Ferguson, L. A., and Lindsay, J. G. (1989).J. Mol. Biol. 209, 833–835.CrossRefPubMedGoogle Scholar
  48. Pebay-Peyroula, E., Garavito, R. M., Rosenbusch, J. P., Zulauf, M., and Timmins, P. A. (1995).Structure,3, 1051–1059.CrossRefPubMedGoogle Scholar
  49. Picot, D., Loll, P. J., and Garavito, R. M. (1994).Nature (London) 367, 243–249.CrossRefGoogle Scholar
  50. Privé, G., Kaback, R. H., and Eisenberg, D. (1994).Acta Cryst. D50, 375–379.Google Scholar
  51. Reiss-Husson, F. (1992).Crystallization of Nucleic Acids and Proteins: A Practical Approach (Ducruix, A., and Giege, R., eds.), IRL Press, New York, pp. 175–193.Google Scholar
  52. Roepe, P. D., and Kaback, H. R. (1989).Proc. Nat. Acad. Sci (USA) 86, 6087–6091.Google Scholar
  53. Rosen, M. J. (1978).Surfactants and Interfacial Phenomena, Wiley, New York.Google Scholar
  54. Roth, M., Lewitt-Bentley, A., Michel, H., Deisenhofer, J., Huber, R., and Oesterhelt, D. (1989).Nature London 340, 659–662.CrossRefGoogle Scholar
  55. Roth, M., Arnoux, B., Ducruix, A., and Reiss-Husson, F. (1991).Biochemistry 30, 9403–9413.CrossRefPubMedGoogle Scholar
  56. Roxby, R. W., and Mills, B. P. (1990).J. Phys. Chem. 94, 456–59.CrossRefGoogle Scholar
  57. Scarborough, G. A. (1994).Acta Cryst. D50, 643–649.Google Scholar
  58. Schertler, G. F. X., Bartunik, H. D., Michel, H., and Oesterhelt, D. (1993).J. Mol. Biol. 234, 156–164.CrossRefPubMedGoogle Scholar
  59. Schirmer, T., Keller, T. A., Wang, Y. F., and Rosenbusch, J. P. (1995).Science 267, 512–514.PubMedGoogle Scholar
  60. Song, L., and Gouaux, J. E. (1995).Methods Enzymol., in press.Google Scholar
  61. Sowadski, J. M. (1994).Curr. Top. Struct. Biol. 4, 761–764.CrossRefGoogle Scholar
  62. Stauffer, K. A., Page, M. G. P., Hardmeyer, A., Keller, T. A., and Pauptit, R. (1990).J. Mol. Biol. 211, 297–299.CrossRefPubMedGoogle Scholar
  63. Steck, T. L., and Fox, C. F. (1972).Membrane Molecular Biology (Fox, C. F., and Keith, A. D., eds.), Sinauer Associates Inc., Stamford, pp. 27–75.Google Scholar
  64. Stura, E. A., Satterthwait, A. C., Calvo, J. C., Kaslow, D. C., and Wilson, I. A. (1994).Acta Cryst. D50, 448–455.Google Scholar
  65. Tanford, C. (1980).The Hydrophobic Effect, Wiley, New York.Google Scholar
  66. Teeter, M. (1984).Proc. Natl. Acad. Sci. USA 81, 6014–6017.Google Scholar
  67. Thiyagarajan, P., and Tiede, D. M. (1994).J. Phys. Chem. 98, 10343–10351.CrossRefGoogle Scholar
  68. Timmins, P. A., Leonhard, M., Weltzien, H. U., Wacker, T., and Welte, W. (1988).FEBS Letters 238, 361–368.CrossRefGoogle Scholar
  69. Timmins, P. A., Hauk, J., Wacker, T., and Welte, W. (1991).FEBS Lett. 280, 115–120.CrossRefPubMedGoogle Scholar
  70. Weber, P. C. (1990).Methods: A Companion to Methods in Enzymology 1, 31–37.CrossRefGoogle Scholar
  71. Weckstrom, K. (1985).FEBS Letters 192, 220–224.CrossRefPubMedGoogle Scholar
  72. Weiss, M. S., Wacker, T., Nestel, U., Woitzik, X., Weckesser, J., Kreutz, W., Welte, W., and Schulz, G. (1990).FEBS Lett. 267, 268–272.CrossRefPubMedGoogle Scholar
  73. Weiss, M. S., Abele, U., Weckesser, J., Welte, W., Schiltz, E., and Schulz, G. E. (1991).Science 254, 1627–1630.PubMedGoogle Scholar
  74. Weltzien, H. U., Richter, G., and Ferber, E. (1979).J. Biol. Chem. 254, 3652–3657.PubMedGoogle Scholar
  75. Wennerström, H., and Lindman, B. (1979).Phys. Rep. 52, 1–86.CrossRefGoogle Scholar
  76. Wilson, I. A., Skehel, J. J., and Wiley, D. C. (1981).Nature (London) 289, 366–373.CrossRefGoogle Scholar
  77. Zulauf, M. (1991).Crystallization of Membrane Proteins (Michel, H., ed.), CRC Press, Boca Raton, pp. 54–71.Google Scholar
  78. Zulauf, M., and Hayter, J. B. (1982).Colloid Polym. Sci. 260, 1023–1028.CrossRefGoogle Scholar
  79. Zulauf, M., and Rosenbusch, J. P. (1983).J. Phys. Chem.,87, 856–862.CrossRefGoogle Scholar
  80. Zulauf, M., Fuerstenberger, U., Grabo, M., Jaeggi, P., Regenass, M., and Rosenbusch, J. P. (1989).Methods in Enzymol. 172, 528–538.Google Scholar

Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • R. Michael Garavito
    • 1
  • Daniel Picot
    • 2
  • Patrick J. Loll
    • 3
  1. 1.Department of Biochemistry and Molecular BiologyThe University ChicagoChicago
  2. 2.Institut de Biologie Physico-ChimiqueParisFrance
  3. 3.Department of PharmacologyUniversity of Pennsylvania School of MedicinePhiladelphia

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