Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry.
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.
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, Oxford, OX1 3QT, United Kingdom.
E J
EJ
Nettleton
P
P
Tito
M
M
Sunde
M
M
Bouchard
C M
CM
Dobson
C V
CV
Robinson
D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D019281:Dimerization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D011485:Protein Binding; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D015032:Zinc / Q000737:chemistry
D000682:Amyloid; D007328:Insulin; D046911:Macromolecular Substances; D015032:Zinc