The amyloid fibril of bovine insulin and its renaturing intermediates were studied by using Raman optical activity (ROA). In the spectrum of the amyloid, the sharp +/- ROA couplet of amide I band characteristic of the β-sheet-rich proteins was observed, together with a sharp peak at 1271 cm(-1) characteristic of a turn structure. The shoulder ROA peak of the native insulin at ~ 1340 cm(-1), which was assigned to the hydrated α-helix, was not observed in the amyloid, suggesting that the hydrated α-helix was converted to the parallel β-sheet structure in the amyloid. Recovery of the amyloid to the native state was also monitored by ROA. The intermediate states showed distinct features from the amyloid or native ones. The intermediates did not show a characteristic ROA peak of the poly(L-proline) II helix at ~ 1318 cm(-1). The hydrated α-helix ROA peak was not recovered in the intermediate states. In a process of the amyloid formation, at first the hydrated α-helix of the native insulin is converted to a specific partially unfolded structure, and then, it was converted to the parallel β-sheet structure with many turns.

Institute of Organic Chemistry and Biochemistry Academy of Sciences Prague Czech Republic

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