On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Abstract

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10−5 s−1). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.

Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods

“…From the fitted kinetic data to a sigmoidal function [ 45 ], the obtained values of the half-time for aggregation (t 1/2 ) and the apparent rate constant (k app = 1/τ) are 138.0 h and 0.028 h -1 , respectively. The lag time (t lag [ThT] = t 1/2 −2τ), deduced from these parameters, has a value of 66.6 h. Generally, these parameters are dependent of several factors such as the protein concentration, temperature, pH and cosolvents [ 27 – 41 ].…”

Gold nanocolloid–protein interactions and their impact on β-sheet amyloid fibril formation

“…Those parameters are dependent on the conditions used for the brillation as protein concentration, solvents, temperature and pH. 38,40,41 Eqn (1) tted all curves adequately, providing the values of t lag and apparent rate of bril formation (k) summarised in Table S1 (ESI †). At t SA ¼ 0 h, no uorescence was observed in any sample, indicating that no b-sheet aggregates or brils were formed at this stage.…”

Quinopeptide formation associated with the disruptive effect of epigallocatechin-gallate on lysozyme fibrils

“…Fibrils prepared in an acidic medium can be disassembled by SDS into lysozyme monomers and hydrolysis-originated fragments (discussed subsequently). In contrast, at a high pH, lysozyme transforms into aggregates by intermolecular disulfide cross-linking [29,30]. Although the molecular mechanisms of lysozyme fibrillation were different under acidic and alkaline conditions, EGCG exhibited an inhibitory effect on amyloid formation under both conditions.…”