Influence of denaturants on amyloid β42 aggregation kinetics

Abstract

Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide associated with Alzheimer’s disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aβ42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.

In Situ Observation of Chemically Induced Protein Denaturation at Solvated Interfaces

“…Although the spatial resolution of the liquid-based AFM is limited to relatively large changes in single protein shape or dissociation into subunits, the benefits of this approach could start to address an important unmet need to investigate conformational changes and hydrophobic hydration effects that can occur during the denaturation. For example, it has been recently shown that urea can modulate growth and aggregation kinetics of amyloid beta peptides implicated in the pathology of Alzheimer’s disease, , which we anticipate can now be visualized, and morphological changes quantified one protein at a time.…”

The Action of Chemical Denaturants: From Globular to Intrinsically Disordered Proteins

“…A comprehensive analysis of the impact that urea and guanidinium have on the aggregation process of the peptide Aβ42, a natively unfolded peptide whose aggregation is believed to be heavily implicated in the insurgence and progression of Alzheimer's disease, has been reported by Linse, Knowles, and colleagues [125]. These authors demonstrated that urea, as a non-ionic denaturant, reduces the overall aggregation rate with a stronger effect on nucleation compared to the elongation steps.…”

“…Chemical denaturants may operate at different stages of the amyloid formation process and may either favor or disfavor it [125]. In some cases, it has been observed a reduction of the lag phase.…”

Molecular Mechanisms of Inhibition of Protein Amyloid Fibril Formation: Evidence and Perspectives Based on Kinetic Models

“…Consequently, denaturants are more likely to inhibit than accelerate fibril formation from disordered proteins. Aβ42 fibrillation is inhibited by urea [ 134 , 135 ] and guanidinium chloride at concentrations above 1 M [ 135 , 136 ] due to a decrease in the rate of both primary and secondary nucleation. Interestingly, at low concentrations, guanidinium chloride act as an osmolyte, increasing the rate of beta-amyloid fibrillation, likely due to the electrostatic interactions with the ions formed upon its dissociation.…”