The mechanism of amyloid‐fibril formation by stefin B: Temperature and protein concentration dependence of the rates

Abstract

Cystatins, a family of structurally related cysteine proteinase inhibitors, have proved to be useful model system to study amyloidogenesis. We have extended previous studies of the kinetics of amyloid-fibril formation by human stefin B (cystatin B) and some of its mutants, and proposed an improved model for the reaction. Overall, the observed kinetics follow the nucleation and growth behavior observed for many other amyloidogenic proteins. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N(I) irreversible transitions with equivalent rates (k(I)), which fitted with N(I) = 64), fibril growth and nonproductive oligomerization, best explained by an off-pathway state with a rate-limiting escape rate. Three energies of activation were derived from global fitting to the minimal kinetic scheme, and independently through the fitting of the individual component rates. Nucleation was found to be a first-order process within an oligomeric species with an enthalpy of activation of 55 +/- 4 kcal mol(-1). Fibril growth was a second-order process with an enthalpy of activation (27 +/- 5 kcal mol(-1)), which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The highest enthalpy of activation (95 +/- 5 kcal mol(-1) at 35 degrees C), characteristic of a substantial degree of unfolding as observed prior to domain-swapping reactions, equated with the escape rate of the off-pathway oligomeric state.

Conformational behaviour and aggregation of chickpea cystatin in trifluoroethanol: Effects of epicatechin and tannic acid

“…Members of http://dx.doi.org/10.1016/j.abb.2014.08.015 0003-9861/Ó 2014 Elsevier Inc. All rights reserved. this family show predisposition for fibrillation and have recently been proven to be useful model system to study amyloidogenesis [12][13][14]. The aim this study was to investigate how a plant protein CPC (chick pea cystatin) isolated from chick pea behaves in the process of aggregation and fibrillation as only few reports are available on fibrillation of cystatins in general and no report is found on plant cystatins in particular.…”

Evaluation of polyphenols as possible therapeutics for amyloidoses: Comparative analysis of Kaempferol and Catechin

“…Members of this family show predisposition for fibrillation and has recently been proven to be useful model system to study amyloidogenesis [15]. GBC thus served as an attractive model for studying its likelihood of foraying into aggregated amyloid state during acid denaturation.…”

Interaction between Oligomers of Stefin B and Amyloid-β in Vitro and in Cells

“…Stefin B has a much higher tendency than stefin A to form dimers, higher oligomers, and amyloid fibrils, nevertheless, the stefin A domain-swapped dimer could be prepared under more extreme solution conditions and its structure was determined by NMR (22,23). The model for the mechanism of stefin B fibrillization involves off-pathway lower oligomer formation (probably involving domain-swapping, due to a high energetic barrier) and a larger nucleus in the order of 30 dimers, explaining both an unusual behavior at higher protein concentrations and a relatively long lag phase (21).…”