Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Feb;76(2):1048–1062. doi: 10.1016/S0006-3495(99)77270-X

Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions.

H Wille 1, S B Prusiner 1
PMCID: PMC1300055  PMID: 9916037

Abstract

The structural transition from the cellular prion protein (PrPC) that is rich in alpha-helices to the pathological form (PrPSc) that has a high beta-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation.

Full Text

The Full Text of this article is available as a PDF (769.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akowitz A., Sklaviadis T., Manuelidis E. E., Manuelidis L. Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations. Microb Pathog. 1990 Jul;9(1):33–45. doi: 10.1016/0882-4010(90)90038-r. [DOI] [PubMed] [Google Scholar]
  2. Bessen R. A., Marsh R. F. Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol. 1994 Dec;68(12):7859–7868. doi: 10.1128/jvi.68.12.7859-7868.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolton D. C., McKinley M. P., Prusiner S. B. Identification of a protein that purifies with the scrapie prion. Science. 1982 Dec 24;218(4579):1309–1311. doi: 10.1126/science.6815801. [DOI] [PubMed] [Google Scholar]
  4. Bomford R., Stapleton M., Winsor S., Beesley J. E., Jessup E. A., Price K. R., Fenwick G. R. Adjuvanticity and ISCOM formation by structurally diverse saponins. Vaccine. 1992;10(9):572–577. doi: 10.1016/0264-410x(92)90435-m. [DOI] [PubMed] [Google Scholar]
  5. Byler D. M., Susi H. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers. 1986 Mar;25(3):469–487. doi: 10.1002/bip.360250307. [DOI] [PubMed] [Google Scholar]
  6. Caughey B. W., Dong A., Bhat K. S., Ernst D., Hayes S. F., Caughey W. S. Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry. 1991 Aug 6;30(31):7672–7680. doi: 10.1021/bi00245a003. [DOI] [PubMed] [Google Scholar]
  7. Caughey B., Chesebro B. Prion protein and the transmissible spongiform encephalopathies. Trends Cell Biol. 1997 Feb;7(2):56–62. doi: 10.1016/S0962-8924(96)10054-4. [DOI] [PubMed] [Google Scholar]
  8. Caughey B., Raymond G. J., Kocisko D. A., Lansbury P. T., Jr Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies. J Virol. 1997 May;71(5):4107–4110. doi: 10.1128/jvi.71.5.4107-4110.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang Q., Liu H., Chen J. Fourier transform infrared spectra studies of protein in reverse micelles: effect of AOT/isooctane on the secondary structure of alpha-chymotrypsin. Biochim Biophys Acta. 1994 Jun 12;1206(2):247–252. [PubMed] [Google Scholar]
  10. Cohen F. E., Prusiner S. B. Pathologic conformations of prion proteins. Annu Rev Biochem. 1998;67:793–819. doi: 10.1146/annurev.biochem.67.1.793. [DOI] [PubMed] [Google Scholar]
  11. Dolder M., Engel A., Zulauf M. The micelle to vesicle transition of lipids and detergents in the presence of a membrane protein: towards a rationale for 2D crystallization. FEBS Lett. 1996 Mar 11;382(1-2):203–208. doi: 10.1016/0014-5793(96)00180-9. [DOI] [PubMed] [Google Scholar]
  12. Endo T., Groth D., Prusiner S. B., Kobata A. Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein. Biochemistry. 1989 Oct 17;28(21):8380–8388. doi: 10.1021/bi00447a017. [DOI] [PubMed] [Google Scholar]
  13. Gabizon R., McKinley M. P., Prusiner S. B. Properties of scrapie prion proteins in liposomes and amyloid rods. Ciba Found Symp. 1988;135:182–196. doi: 10.1002/9780470513613.ch12. [DOI] [PubMed] [Google Scholar]
  14. Gabizon R., McKinley M. P., Prusiner S. B. Purified prion proteins and scrapie infectivity copartition into liposomes. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4017–4021. doi: 10.1073/pnas.84.12.4017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gajdusek D. C. Transmissible and non-transmissible amyloidoses: autocatalytic post-translational conversion of host precursor proteins to beta-pleated sheet configurations. J Neuroimmunol. 1988 Dec;20(2-3):95–110. doi: 10.1016/0165-5728(88)90140-3. [DOI] [PubMed] [Google Scholar]
  16. Gasset M., Baldwin M. A., Fletterick R. J., Prusiner S. B. Perturbation of the secondary structure of the scrapie prion protein under conditions that alter infectivity. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):1–5. doi: 10.1073/pnas.90.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glaeser R. M., Downing K. H. High-resolution electron crystallography of protein molecules. Ultramicroscopy. 1993 Dec;52(3-4):478–486. doi: 10.1016/0304-3991(93)90064-5. [DOI] [PubMed] [Google Scholar]
  18. Harrison P. M., Bamborough P., Daggett V., Prusiner S. B., Cohen F. E. The prion folding problem. Curr Opin Struct Biol. 1997 Feb;7(1):53–59. doi: 10.1016/s0959-440x(97)80007-3. [DOI] [PubMed] [Google Scholar]
  19. Hsiao K. K., Groth D., Scott M., Yang S. L., Serban H., Rapp D., Foster D., Torchia M., Dearmond S. J., Prusiner S. B. Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9126–9130. doi: 10.1073/pnas.91.19.9126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hunter G. D., Kimberlin R. H., Gibbons R. A. Scrapie: a modified membrane hypothesis. J Theor Biol. 1968 Sep;20(3):355–357. doi: 10.1016/0022-5193(68)90135-5. [DOI] [PubMed] [Google Scholar]
  21. Hunter G. D., Millson G. C. Attempts to release the scrapie agent from tissue debris. J Comp Pathol. 1967 Jul;77(3):301–307. doi: 10.1016/0021-9975(67)90039-4. [DOI] [PubMed] [Google Scholar]
  22. Jarrett J. T., Lansbury P. T., Jr Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell. 1993 Jun 18;73(6):1055–1058. doi: 10.1016/0092-8674(93)90635-4. [DOI] [PubMed] [Google Scholar]
  23. Kersten G. F., Spiekstra A., Beuvery E. C., Crommelin D. J. On the structure of immune-stimulating saponin-lipid complexes (iscoms). Biochim Biophys Acta. 1991 Feb 25;1062(2):165–171. doi: 10.1016/0005-2736(91)90388-o. [DOI] [PubMed] [Google Scholar]
  24. Kimberlin R. H., Millson G. C., Hunter G. D. An experimental examination of the scrapie agent in cell membrane mixtures. 3. Studies of the operational size. J Comp Pathol. 1971 Jul;81(3):383–391. doi: 10.1016/0021-9975(71)90026-0. [DOI] [PubMed] [Google Scholar]
  25. Landau E. M., Rosenbusch J. P. Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14532–14535. doi: 10.1073/pnas.93.25.14532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Malone T. G., Marsh R. F., Hanson R. P., Semancik J. S. Membrane-free scrapie activity. J Virol. 1978 Mar;25(3):933–935. doi: 10.1128/jvi.25.3.933-935.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marsh R. F., Dees C., Castle B. E., Wade W. F., German T. L. Purification of the scrapie agent by density gradient centrifugation. J Gen Virol. 1984 Feb;65(Pt 2):415–421. doi: 10.1099/0022-1317-65-2-415. [DOI] [PubMed] [Google Scholar]
  28. McKinley M. P., Meyer R. K., Kenaga L., Rahbar F., Cotter R., Serban A., Prusiner S. B. Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis. J Virol. 1991 Mar;65(3):1340–1351. doi: 10.1128/jvi.65.3.1340-1351.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Millson G. C., Hunter G. D., Kimberlin R. H. An experimental examination of the scrapie agent in cell membrane mixtures. II. The association of scrapie activity with membrane fractions. J Comp Pathol. 1971 Apr;81(2):255–265. doi: 10.1016/0021-9975(71)90100-9. [DOI] [PubMed] [Google Scholar]
  30. Millson G. C., Hunter G. D., Kimberlin R. H. The physico-chemical nature of the scrapie agent. Front Biol. 1976;44:243–266. [PubMed] [Google Scholar]
  31. Pan K. M., Baldwin M., Nguyen J., Gasset M., Serban A., Groth D., Mehlhorn I., Huang Z., Fletterick R. J., Cohen F. E. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10962–10966. doi: 10.1073/pnas.90.23.10962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pebay-Peyroula E., Rummel G., Rosenbusch J. P., Landau E. M. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science. 1997 Sep 12;277(5332):1676–1681. doi: 10.1126/science.277.5332.1676. [DOI] [PubMed] [Google Scholar]
  33. Prusiner S. B., Bolton D. C., Groth D. F., Bowman K. A., Cochran S. P., McKinley M. P. Further purification and characterization of scrapie prions. Biochemistry. 1982 Dec 21;21(26):6942–6950. doi: 10.1021/bi00269a050. [DOI] [PubMed] [Google Scholar]
  34. Prusiner S. B., Groth D. F., Cochran S. P., Masiarz F. R., McKinley M. P., Martinez H. M. Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent. Biochemistry. 1980 Oct 14;19(21):4883–4891. doi: 10.1021/bi00562a028. [DOI] [PubMed] [Google Scholar]
  35. Prusiner S. B., Hadlow W. J., Garfin D. E., Cochran S. P., Baringer J. R., Race R. E., Eklund C. M. Partial purification and evidence for multiple molecular forms of the scrapie agent. Biochemistry. 1978 Nov 14;17(23):4993–4999. doi: 10.1021/bi00616a021. [DOI] [PubMed] [Google Scholar]
  36. Prusiner S. B., McKinley M. P., Bowman K. A., Bolton D. C., Bendheim P. E., Groth D. F., Glenner G. G. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 1983 Dec;35(2 Pt 1):349–358. doi: 10.1016/0092-8674(83)90168-x. [DOI] [PubMed] [Google Scholar]
  37. Prusiner S. B. Prion diseases and the BSE crisis. Science. 1997 Oct 10;278(5336):245–251. doi: 10.1126/science.278.5336.245. [DOI] [PubMed] [Google Scholar]
  38. Prusiner S. B., Scott M. R., DeArmond S. J., Cohen F. E. Prion protein biology. Cell. 1998 May 1;93(3):337–348. doi: 10.1016/s0092-8674(00)81163-0. [DOI] [PubMed] [Google Scholar]
  39. Prusiner S. B., Scott M., Foster D., Pan K. M., Groth D., Mirenda C., Torchia M., Yang S. L., Serban D., Carlson G. A. Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell. 1990 Nov 16;63(4):673–686. doi: 10.1016/0092-8674(90)90134-z. [DOI] [PubMed] [Google Scholar]
  40. Riesner D., Kellings K., Post K., Wille H., Serban H., Groth D., Baldwin M. A., Prusiner S. B. Disruption of prion rods generates 10-nm spherical particles having high alpha-helical content and lacking scrapie infectivity. J Virol. 1996 Mar;70(3):1714–1722. doi: 10.1128/jvi.70.3.1714-1722.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Safar J., Ceroni M., Gajdusek D. C., Gibbs C. J., Jr Differences in the membrane interaction of scrapie amyloid precursor proteins in normal and scrapie- or Creutzfeldt-Jakob disease-infected brains. J Infect Dis. 1991 Mar;163(3):488–494. doi: 10.1093/infdis/163.3.488. [DOI] [PubMed] [Google Scholar]
  42. Safar J., Roller P. P., Gajdusek D. C., Gibbs C. J., Jr Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J Biol Chem. 1993 Sep 25;268(27):20276–20284. [PubMed] [Google Scholar]
  43. Safar J. The folding intermediate concept of prion protein formation and conformational links to infectivity. Curr Top Microbiol Immunol. 1996;207:69–76. doi: 10.1007/978-3-642-60983-1_6. [DOI] [PubMed] [Google Scholar]
  44. Safar J., Wille H., Itri V., Groth D., Serban H., Torchia M., Cohen F. E., Prusiner S. B. Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med. 1998 Oct;4(10):1157–1165. doi: 10.1038/2654. [DOI] [PubMed] [Google Scholar]
  45. Semancik J. S., Marsh R. F., Geelen J. L., Hanson R. P. Properties of the scrapie agent-endomembrane complex from hamster brain. J Virol. 1976 May;18(2):693–700. doi: 10.1128/jvi.18.2.693-700.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sklaviadis T., Dreyer R., Manuelidis L. Analysis of Creutzfeldt-Jakob disease infectious fractions by gel permeation chromatography and sedimentation field flow fractionation. Virus Res. 1992 Dec;26(3):241–254. doi: 10.1016/0168-1702(92)90016-3. [DOI] [PubMed] [Google Scholar]
  47. Stahl N., Baldwin M. A., Hecker R., Pan K. M., Burlingame A. L., Prusiner S. B. Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid. Biochemistry. 1992 Jun 2;31(21):5043–5053. doi: 10.1021/bi00136a600. [DOI] [PubMed] [Google Scholar]
  48. Telling G. C., Haga T., Torchia M., Tremblay P., DeArmond S. J., Prusiner S. B. Interactions between wild-type and mutant prion proteins modulate neurodegeneration in transgenic mice. Genes Dev. 1996 Jul 15;10(14):1736–1750. doi: 10.1101/gad.10.14.1736. [DOI] [PubMed] [Google Scholar]
  49. Telling G. C., Parchi P., DeArmond S. J., Cortelli P., Montagna P., Gabizon R., Mastrianni J., Lugaresi E., Gambetti P., Prusiner S. B. Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science. 1996 Dec 20;274(5295):2079–2082. doi: 10.1126/science.274.5295.2079. [DOI] [PubMed] [Google Scholar]
  50. Wille H., Zhang G. F., Baldwin M. A., Cohen F. E., Prusiner S. B. Separation of scrapie prion infectivity from PrP amyloid polymers. J Mol Biol. 1996 Jun 21;259(4):608–621. doi: 10.1006/jmbi.1996.0343. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES