Exploration of insulin amyloid polymorphism using Raman spectroscopy and imaging

Abstract

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature, and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90 . The intensity ratio of two Raman bands at 1672 and 1657 cm-1 due to {beta}-sheet and -helix structures was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the {beta}-sheet structure was higher in the case with salts, especially at a higher temperature and with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond ([~]514 cm-1) and the ratio of the tyrosine doublet R(I850/I826) were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis (PCA). Especially, amyloid fibrils with Na2SO4 media formed the g-g-g conformation of disulfide bond at a higher rate and without any salts; on the contrary, the g-g-g conformation was partially transformed into the g-g-t conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three-dimensional structural differences of amyloid polymorphisms. The present results indicate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.\n\nStatement of SignificanceOur results revealed three Raman markers distinguishing amyloid fibril polymorphisms caused by salt and temperature effects; the relative proportion of protein secondary structures (-helix and {beta}-sheet), the ratio of tyrosine doublet, and the conformational differences of disulfide bonds. The lower values of tyrosine doublet in the case with salts were interpreted as the anions rob the hydration water from proteins which induced protein misfolding. Using these parameters, Raman images captured their higher order structural differences in situ without labeling. The images of hydrogen bonds strength variations due to tyrosine doublet is believed to include significant novelty. The present results imply the potential of Raman imaging for use as a diagnostic imaging tool for tissues with amyloid-induced diseases.