Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature‐induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native‐like α‐helical structure. On heating to 70°C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT‐IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from α‐helical to β‐sheet structure. At later stages, EM shows the development of fibrils with well‐defined repetitive morphologies including structures with a periodic helical twist of ∼450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.

Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimer's disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in β-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric β-sheets. Sequences predicted to be highly favorable on this basis were found experimentally to self-associate efficiently into β-sheets, whereas point mutations predicted to be unfavorable for this structure inhibited polymerization. However, the property to form polymeric β-sheets is not a sufficient requirement for fibril formation because, under the conditions used here, preformed β-sheets from these peptides with charged residues form well defined fibrils only if the total net charge of the molecule is ±1. This finding illustrates the delicate balance of interactions involved in the formation of fibrils relative to more disordered aggregates. The present results, in conjunction with x-ray fiber diffraction, electron microscopy, and Fourier transform infrared measurements, have allowed us to propose a detailed structural model of the fibrils.