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.