How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

Abstract

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.

Soluble pre-fibrillar tau and β-amyloid species emerge in early human Alzheimer’s disease and track disease progression and cognitive decline

“…However, the utility of such SDS-stable oligomeric species must be questioned when considering that the presence of SDS, even at low concentrations (0.2 %) can greatly enhanced the abundance of dimeric [80] trimeric [10] and higher, multimeric Aβ species [5, 31, 32, 66]. Furthermore, and in line with the impact of heat and reducing agents such DTT to modulate amyloidogenic β-sheet aggregation [13, 45, 69], we demonstrate that the relative abundance of *56 was strongly reduced and any correlation with disease progression lost in the absence of such treatments. This suggests that some of the previous data may be due to technical artefacts.…”

Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

“…Pairs of cysteines can oxidize to form disulfide bonds, which contribute to the correct fold and functionality of proteins (40). Importantly, inter-or intramolecular disulfide bonding has been shown to promote or at least stabilize amyloid fibers (41,42). Furthermore, reducing environments can inhibit or delay amyloidogenesis by blocking intermolecular bonding and thereby preventing fiber growth (34,43).…”

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

“…The exposure of hydrophobic regions able to bind ANS is also associated with the formation of soluble early aggregates of lysozyme at pH 12.2. 59 ANS has similarly been reported to interact with oligomeric aggregates of other proteins; 34 for example, well before the formation of amyloid fibrils able to bind ThT, insulin begins to undergo a series of structural changes, detected by binding with ANS. 41 Crucially, regions of exposed hydrophobicity have been strongly implicated in the cytotoxicity of protein aggregates, as it has been proposed that the pathogenic nature of prefibrillar aggregates could lie in the exposure of groups that are normally buried in a folded protein.…”