Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology

Abstract

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all alpha-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

Improvement of heat-induced fibril assembly of soy β-conglycinin (7S Globulins) at pH 2.0 through electrostatic screening

“…The formation of highly-ordered amyloid-like fibrils can be generally monitored by using Th T fluorescence spectroscopy, since Th T has a high selectivity for amyloid fibrils (Foderà et al, 2008;Nilsson, 2004;Žerovnik et al, 2007). More importantly, Th T does not affect or only slightly affects the early formation of fibrils, making this technique suitable for in situ fibril detection (Foderà et al, 2008).…”

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

“…Trifluoroethanol (TFE), 1 the simplest alcohol with a CF3 group, and its derivatives are most commonly used as anaesthetics. It is toxic to blood, male reproductive system, brain, upper respiratory tract and eyes [9]. There are two main effects of TFE on peptides and proteins: first the stabilization of helical, b-turn and b-hairpin structures and second the disruption of the native tertiary structure of intact proteins leading to aggregates [10].…”

The in Vivo and in Vitro Aggregation Properties of Globular Proteins Correlate With Their Conformational Stability: The SH3 Case

“…7,8 This effect has been characterized extensively in vitro by protein engineering using both disease-related and non-related polypeptide models. [9][10][11][12][13][14] Fewer studies have addressed the influence of polypeptide properties on their aggregation within the cell, [15][16][17][18][19] and only a few have compared systematically the in vitro and in vivo aggregation behaviour of proteins. 17,[20][21][22] The in vivo aggregation of polypeptides does not necessarily have to correlate with their in vitro properties, because the protein quality machinery modulates the accumulation of aggregation-prone polypeptidic chains by facilitating their folding, masking hydrophobic regions and targeting improperly folded proteins towards degradation pathways.…”