New Insights into the Self-Assembly of Insulin Amyloid Fibrils:  An H−D Exchange FT-IR Study

Abstract

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.1 MPa and 100 degrees C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 degrees C. A complete deuteration of H(2)O-grown fibrils occurs when the beta-sheet structure is reassembled in a 75 wt % DMSO/D(2)O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular beta-sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D(2)O-grown fibrils with H(2)O is complete, which is reflected by the vanishing of D(2)O bending vibrations at 1214 cm(-)(1). This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.

Lability Landscape and Protease Resistance of Human Insulin Amyloid: A New Insight into Its Molecular Properties

“…[31][32][33][34][35][36][37][38][39][40][41][42][43][44] Here, we demonstrate that the stability and lability of insulin amyloid are strongly pH dependent and, therefore, can be prevented and tightly controlled during the insulin production and application processes. A focus in the amyloid field concerns how amyloid depositions can be triggered and propagated in the body.…”

“…[32][33][34] Under a variety of destabilizing conditions, insulin also forms amyloid fibrils, which are characterized by different arrangements of constituting protofilaments and, therefore, vary in morphology. [31][32][33][34][35][36][37][38][39] The formation of insoluble insulin aggregates can lead to a significant reduction of the functionally active substance in formulations, posing serious problems in biopharmaceutical applications. [40][41][42][43][44] Regular injections of insulin can also cause long-term effects, such as depositions of insulin amyloid, which have been observed in diabetic patients after repeated injections.…”

Conformational analyses of peptides and proteins by vibrational Raman optical activity

“…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. As far as we know, NMR spectroscopy has not been applied to the amyloid system, perhaps due to the difficulty of the measurement and analysis for this aggregated system.…”

Proteomic analysis optimization: Selective protein sample on-column retention in reverse-phase liquid chromatography☆

“…DMSO has been also identified as a denaturing reagent for membrane proteins and was found to assist in protein unfolding [21,22]. It has been also reported to compete for amide hydrogen atoms with carbonyl groups thus collapsing protein secondary structure supported by hydrogen bonding in native proteins [21].…”

“…DMSO has been also identified as a denaturing reagent for membrane proteins and was found to assist in protein unfolding [21,22]. It has been also reported to compete for amide hydrogen atoms with carbonyl groups thus collapsing protein secondary structure supported by hydrogen bonding in native proteins [21]. In a study of beta-amyloid peptide components of Alzheimer's plaques, DMSO protein solutions exhibited unprotected monomeric protein entities comparable to these obtained from 8 M urea solutions [22].…”