Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Aug;79(2):1053–1065. doi: 10.1016/S0006-3495(00)76359-4

Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry.

E J Nettleton 1, P Tito 1, M Sunde 1, M Bouchard 1, C M Dobson 1, C V Robinson 1
PMCID: PMC1301001  PMID: 10920035

Abstract

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.

Full Text

The Full Text of this article is available as a PDF (362.4 KB).

Selected References

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

  1. Bai Y., Milne J. S., Mayne L., Englander S. W. Primary structure effects on peptide group hydrogen exchange. Proteins. 1993 Sep;17(1):75–86. doi: 10.1002/prot.340170110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benson M. D. Familial amyloidotic polyneuropathy. Trends Neurosci. 1989 Mar;12(3):88–92. doi: 10.1016/0166-2236(89)90162-8. [DOI] [PubMed] [Google Scholar]
  3. Booth D. R., Sunde M., Bellotti V., Robinson C. V., Hutchinson W. L., Fraser P. E., Hawkins P. N., Dobson C. M., Radford S. E., Blake C. C. Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature. 1997 Feb 27;385(6619):787–793. doi: 10.1038/385787a0. [DOI] [PubMed] [Google Scholar]
  4. Brange J., Andersen L., Laursen E. D., Meyn G., Rasmussen E. Toward understanding insulin fibrillation. J Pharm Sci. 1997 May;86(5):517–525. doi: 10.1021/js960297s. [DOI] [PubMed] [Google Scholar]
  5. Brange J., Dodson G. G., Edwards D. J., Holden P. H., Whittingham J. L. A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin). Proteins. 1997 Apr;27(4):507–516. [PubMed] [Google Scholar]
  6. Brange J, Vølund A. Insulin analogs with improved pharmacokinetic profiles. Adv Drug Deliv Rev. 1999 Feb 1;35(2-3):307–335. doi: 10.1016/s0169-409x(98)00079-9. [DOI] [PubMed] [Google Scholar]
  7. Burke M. J., Rougvie M. A. Cross- protein structures. I. Insulin fibrils. Biochemistry. 1972 Jun 20;11(13):2435–2439. doi: 10.1021/bi00763a008. [DOI] [PubMed] [Google Scholar]
  8. Canet D., Sunde M., Last A. M., Miranker A., Spencer A., Robinson C. V., Dobson C. M. Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants. Biochemistry. 1999 May 18;38(20):6419–6427. doi: 10.1021/bi983037t. [DOI] [PubMed] [Google Scholar]
  9. Fabris D., Fenselau C. Characterization of allosteric insulin hexamers by electrospray ionization mass spectrometry. Anal Chem. 1999 Jan 15;71(2):384–387. doi: 10.1021/ac980753s. [DOI] [PubMed] [Google Scholar]
  10. Gejyo F., Yamada T., Odani S., Nakagawa Y., Arakawa M., Kunitomo T., Kataoka H., Suzuki M., Hirasawa Y., Shirahama T. A new form of amyloid protein associated with chronic hemodialysis was identified as beta 2-microglobulin. Biochem Biophys Res Commun. 1985 Jun 28;129(3):701–706. doi: 10.1016/0006-291x(85)91948-5. [DOI] [PubMed] [Google Scholar]
  11. Glenner G. G., Eanes E. D., Bladen H. A., Linke R. P., Termine J. D. Beta-pleated sheet fibrils. A comparison of native amyloid with synthetic protein fibrils. J Histochem Cytochem. 1974 Dec;22(12):1141–1158. doi: 10.1177/22.12.1141. [DOI] [PubMed] [Google Scholar]
  12. Goldman J., Carpenter F. H. Zinc binding, circular dichroism, and equilibrium sedimentation studies on insulin (bovine) and several of its derivatives. Biochemistry. 1974 Oct 22;13(22):4566–4574. doi: 10.1021/bi00719a015. [DOI] [PubMed] [Google Scholar]
  13. Harper J. D., Lansbury P. T., Jr Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu Rev Biochem. 1997;66:385–407. doi: 10.1146/annurev.biochem.66.1.385. [DOI] [PubMed] [Google Scholar]
  14. Hua Q. X., Weiss M. A. Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition. Biochemistry. 1991 Jun 4;30(22):5505–5515. doi: 10.1021/bi00236a025. [DOI] [PubMed] [Google Scholar]
  15. Jeffrey P. D., Coates J. H. An equilibrium ultracentrifuge study of the self-association of bovine insulin. Biochemistry. 1966 Feb;5(2):489–498. doi: 10.1021/bi00866a014. [DOI] [PubMed] [Google Scholar]
  16. Kelly J. W. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr Opin Struct Biol. 1998 Feb;8(1):101–106. doi: 10.1016/s0959-440x(98)80016-x. [DOI] [PubMed] [Google Scholar]
  17. Krimm S., Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv Protein Chem. 1986;38:181–364. doi: 10.1016/s0065-3233(08)60528-8. [DOI] [PubMed] [Google Scholar]
  18. Küster B., Mann M. Identifying proteins and post-translational modifications by mass spectrometry. Curr Opin Struct Biol. 1998 Jun;8(3):393–400. doi: 10.1016/s0959-440x(98)80075-4. [DOI] [PubMed] [Google Scholar]
  19. Lashuel H. A., Lai Z., Kelly J. W. Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation. Biochemistry. 1998 Dec 22;37(51):17851–17864. doi: 10.1021/bi981876+. [DOI] [PubMed] [Google Scholar]
  20. Lord R. S., Gubensek F., Rupley J. A. Insulin self-association. Spectrum changes and thermodynamics. Biochemistry. 1973 Oct 23;12(22):4385–4391. doi: 10.1021/bi00746a014. [DOI] [PubMed] [Google Scholar]
  21. Mirza U. A., Chait B. T. Effects of anions on the positive ion electrospray ionization mass spectra of peptides and proteins. Anal Chem. 1994 Sep 15;66(18):2898–2904. doi: 10.1021/ac00090a017. [DOI] [PubMed] [Google Scholar]
  22. Nettleton E. J., Sunde M., Lai Z., Kelly J. W., Dobson C. M., Robinson C. V. Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry. J Mol Biol. 1998 Aug 21;281(3):553–564. doi: 10.1006/jmbi.1998.1937. [DOI] [PubMed] [Google Scholar]
  23. Pocker Y., Biswas S. B. Self-association of insulin and the role of hydrophobic bonding: a thermodynamic model of insulin dimerization. Biochemistry. 1981 Jul 21;20(15):4354–4361. doi: 10.1021/bi00518a019. [DOI] [PubMed] [Google Scholar]
  24. Rostom A. A., Robinson C. V. Disassembly of intact multiprotein complexes in the gas phase. Curr Opin Struct Biol. 1999 Feb;9(1):135–141. doi: 10.1016/s0959-440x(99)80018-9. [DOI] [PubMed] [Google Scholar]
  25. Sluzky V., Tamada J. A., Klibanov A. M., Langer R. Kinetics of insulin aggregation in aqueous solutions upon agitation in the presence of hydrophobic surfaces. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9377–9381. doi: 10.1073/pnas.88.21.9377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith G. D., Swenson D. C., Dodson E. J., Dodson G. G., Reynolds C. D. Structural stability in the 4-zinc human insulin hexamer. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7093–7097. doi: 10.1073/pnas.81.22.7093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sunde M., Serpell L. C., Bartlam M., Fraser P. E., Pepys M. B., Blake C. C. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J Mol Biol. 1997 Oct 31;273(3):729–739. doi: 10.1006/jmbi.1997.1348. [DOI] [PubMed] [Google Scholar]
  28. Vecchio G., Bossi A., Pasta P., Carrea G. Fourier-transform infrared conformational study of bovine insulin in surfactant solutions. Int J Pept Protein Res. 1996 Aug;48(2):113–117. doi: 10.1111/j.1399-3011.1996.tb00820.x. [DOI] [PubMed] [Google Scholar]
  29. WAUGH D. F. A mechanism for the formation of fibrils from protein molecules. J Cell Physiol Suppl. 1957 May;49(Suppl 1):145–164. doi: 10.1002/jcp.1030490415. [DOI] [PubMed] [Google Scholar]
  30. Wei J. A., Lin Y. Z., Zhou J. M., Tsou C. L. FTIR studies of secondary structures of bovine insulin and its derivatives. Biochim Biophys Acta. 1991 Oct 11;1080(1):29–33. doi: 10.1016/0167-4838(91)90107-b. [DOI] [PubMed] [Google Scholar]

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

RESOURCES