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Atomic force microscopy images and dynamic light scattering measurements are used to understand the mechanism underlying the solubilization of insulin fibrils by the aqueous solutions of dimethyl sulfoxide (DMSO) without perturbing their [beta]-pleated structure. The findings revealed that the DMSO-induced ensembles of insulin molecules are closely related to elementary building blocks of amyloid fibrils.

The resonance Raman spectra for various complexes are studied to analyze the formation of an intensely violet inclusion complex of insulin amyloid fibrils and molecular iodine, which acts as a misfolded protein and a nanoscale scaffold. The analysis provides a new tool for studying the amyloids and also presents a new model for designing the protein-based drug delivery media by explaining the molecular basis of their varying strain-dependent biological activity.

A model cosolvent, ethanol, has profound and diversified effects on the amyloidogenic self-assembly of insulin, yielding spectroscopically and morphologically distinguishable forms of beta-aggregates. The alcohol reduces hydrodynamic radii of insulin molecules, decreases enthalpic costs associated with aggregation-prone intermediate states, and accelerates the aggregation itself.

Research reveals that insulin aggregation occurs in two phases. The tertiary contacts are loosened and endothermic unfolding leads to a bulky intermediate, followed by slow, irreversible, and exothermic aggregation as determined by infrared spectroscopy and ultra-sensitive scanning calorimetry.

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 ± 2% of amide protons are protected. Of those ‘trapped’ protons, a further 25 ± 2 or 35 ± 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 °C or 0.1 MPa and 100 °C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 °C. A complete deuteration of H2O-grown fibrils occurs when the β-sheet structure is reassembled in a 75 wt % DMSO/D2O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular fl-sheet motive. In disagreement with the concept of ‘amyloid fibers as water-filled nanotubes’ [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591–5595], elution of D2O-grown fibrils with H2O is complete, which is reflected by the vanishing of D2O bending vibrations at 1214 cm-1. This implies the absence of ‘trapped water’ within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation. [ABSTRACT FROM AUTHOR]