Normal and Reversed Supramolecular Chirality of Insulin Fibrils Probed by Vibrational Circular Dichroism at the Protofilament Level of Fibril Structure

Abstract

Fibrils are β-sheet-rich aggregates that are generally composed of several protofibrils and may adopt variable morphologies, such as twisted ribbons or flat-like sheets. This polymorphism is observed for many different amyloid associated proteins and polypeptides. In a previous study we proposed the existence of another level of amyloid polymorphism, namely, that associated with fibril supramolecular chirality. Two chiral polymorphs of insulin, which can be controllably grown by means of small pH variations, exhibit opposite signs of vibrational circular dichroism (VCD) spectra. Herein, using atomic force microscopy (AFM) and scanning electron microscopy (SEM), we demonstrate that indeed VCD supramolecular chirality is correlated not only by the apparent fibril handedness but also by the sense of supramolecular chirality from a deeper level of chiral organization at the protofilament level of fibril structure. Our microscopic examination indicates that normal VCD fibrils have a left-handed twist, whereas reversed VCD fibrils are flat-like aggregates with no obvious helical twist as imaged by atomic force microscopy or scanning electron microscopy. A scheme is proposed consistent with observed data that features a dynamic equilibrium controlled by pH at the protofilament level between left- and right-twist fibril structures with distinctly different aggregation pathways for left- and right-twisted protofilaments.

Dehydration-induced Initial Conformational Change of Hydrated Proteins Detected by the Changes in Vibrational Circular Dichroism Activity

“…Actually, the aggregation of proteins induces highly regular structures with right-handed optical activity such as insulin and lysozyme fibrils. 26,29 In contrast, the initial increase in left-handed optical activity reflects the increase in the structural regularity of Mb and casein that leads to their aggregation. Here, the chirality of individual amino acid molecules in both the proteins should not be changed because their compositions were not changed due to dehydration.…”

“…However, the increase has not been observed in several representative types of secondary structures. 14,[23][24][25][26][27][28][29] Here we propose two possible origins of the increase in the left-handed optical activity in both Mb and casein. One is that the increase may sensitively reflect the increase in the partial structural regularity of individual Mb or casein before their aggregation.…”

“…It is worth mentioning that such an increase in right-handed optical activity in the amide I band was also reported in the aggregation or fibrillogenesis of proteins and peptides such as polyalanine octamer, insulin, and lysozyme. [26][27][28][29] We examined the influence of the orientation of the aggregated Mb on IR and VCD spectra. The apparently aggregated Mb film was prepared by the conservation inside the TOM-BOX for 10 h after 40 min of N2 drying of the Mb stock solution.…”

“…This is because circular dichroism activity sensitively reflects the slight changes in conformations of proteins. 14,[23][24][25][26][27][28][29] To avoid strong IR interference from bulk water, we prepared hydrated protein films.…”

An optically transparent thin-layer electrochemical cell for the study of vibrational circular dichroism of chiral redox-active molecules

“…7,8 Consequently, VCD has become an essential tool employed in biophysical, biochemical, and pharmaceutical research laboratories. [9][10][11][12][13][14][15][16] To the best of our knowledge, the only VCD spectra of chiral organic radicals have been reported by Mori et al 17 However, these radical cations were obtained on addition of a chemical oxidant, so that many aspects of the chiroptical activity (e.g., the reversibility of the oxidation, and optically transparent thin-layer electrochemical (OTTLE) 18 cell applicable for VCD measurements on electrochemically generated radical ions, using a commercially available VCD spectrometer.…”