Direct observation of protein structural transitions through entire amyloid aggregation processes in water using 2D-IR spectroscopy.

Amyloid proteins that undergo self-assembly to form insoluble fibrillar aggregates have attracted much attention due to their role in biological and pathological significance in amyloidosis. This study aims to understand the amyloid aggregation dynamics of insulin (INS) in HO using two-dimensional infrared (2D-IR) spectroscopy. Conventional IR studies have been performed in DO to avoid spectral congestion despite distinct H-D isotope effects. We observed a slowdown of the INS fibrillation process in DO compared to that in HO. The 2D-IR results reveal that different quaternary structures of INS at the onset of the nucleation phase caused the distinct fibrillation pathways of INS in HO and DO. A few different biophysical analysis, including solution-phase small-angle X-ray scattering combined with molecular dynamics simulations and other spectroscopic techniques, support our 2D-IR investigation results, providing insight into mechanistic details of distinct structural transition dynamics of INS in water. We found the delayed structural transition in DO is due to the kinetic isotope effect at an early stage of fibrillation of INS in DO, , enhanced dimer formation of INS in DO. Our 2D-IR and biophysical analysis provide insight into mechanistic details of structural transition dynamics of INS in water. This study demonstrates an innovative 2D-IR approach for studying protein dynamics in HO, which will open the way for observing protein dynamics under biological conditions without IR spectroscopic interference by water vibrations.