Raman optical activity study on insulin amyloid‐ and prefibril intermediate

Abstract

The amyloid fibril of bovine insulin and its renaturing intermediates were studied by using Raman optical activity (ROA). In the spectrum of the amyloid, the sharp +/- ROA couplet of amide I band characteristic of the β-sheet-rich proteins was observed, together with a sharp peak at 1271 cm(-1) characteristic of a turn structure. The shoulder ROA peak of the native insulin at ~ 1340 cm(-1), which was assigned to the hydrated α-helix, was not observed in the amyloid, suggesting that the hydrated α-helix was converted to the parallel β-sheet structure in the amyloid. Recovery of the amyloid to the native state was also monitored by ROA. The intermediate states showed distinct features from the amyloid or native ones. The intermediates did not show a characteristic ROA peak of the poly(L-proline) II helix at ~ 1318 cm(-1). The hydrated α-helix ROA peak was not recovered in the intermediate states. In a process of the amyloid formation, at first the hydrated α-helix of the native insulin is converted to a specific partially unfolded structure, and then, it was converted to the parallel β-sheet structure with many turns.

Use of Raman and Raman optical activity for the structural characterization of a therapeutic monoclonal antibody formulation subjected to heat stress

“…ROA spectra have been reported for human serum albumin, bovine insulin, Concanavalin A, lysozyme, and immunoglobulin G among others. The technique has also been successfully applied to detection of amyloid formation in insulin solutions . On the ROA spectrum, different conformations within proteins have well‐defined, specific band assignments in the amide I region .…”

Conformational analyses of peptides and proteins by vibrational Raman optical activity

“…Very recently, the first ROA observations of the amyloid fibril and its renaturing process were reported for insulin [70] by using an incident circularly polarized ROA instrument [71]. Although extensive studies were done on this system by using UV circular dichroism (UVCD) [72][73][74][75], electron microscopy [72,73,[75][76][77], fluorescence spectroscopy using dyes [72][73][74], IR [75,78], Raman [79][80][81], mass spectrometry [82], and so on (see [74] and references therein), adequate molecular structures have not yet been obtained, especially for the intermediate states, because of a lack of analytical tools with a sufficient sensitivity to the protein structure and applicability to time-dependent colloidal samples.…”

“…4 Schematic presentation of the possible secondary structural changes during the renaturation of the amyloid fibril of insulin based on ref. [70]. The position of the hydrated α helix in native insulin is still not clear.…”

“…a Modulation method used in ref. [70] by fixed quarterwave plate (static Q) and inserted half-wave plates (H1 and H2). b Conventional modulation method by the rotated quarter-wave plate (Q) very fast equilibria.…”

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

“…[13][14][15][16][17] However, initial conformational changes of proteins under denaturation have not been detected spectroscopically. The study on the initial conformational changes has been limited to the usage of simulations.…”