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 H 2 O using two-dimensional infrared (2D-IR) spectroscopy. Conventional IR studies have been performed in D 2 O to avoid spectral congestion despite distinct H-D isotope effects. We observed a slowdown of the INS fibrillation process in D 2 O compared to that in H 2 O. 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 H 2 O and D 2 O. 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 D 2 O is due to the kinetic isotope effect at an early stage of fibrillation of INS in D 2 O, i.e. , enhanced dimer formation of INS in D 2 O. 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 H 2 O, which will open the way for observing protein dynamics under biological conditions without IR spectroscopic interference by water vibrations.