The origin of secondary structure transitions and peptide self-assembly propensity in trifluoroethanol-water mixtures

Abstract

AbstractThe formation of transient helical intermediates, implicated in the early-stages of amyloid formation in amyloidogenic peptides, is thought to be enhanced by membrane-peptide interactions. Uperin 3.5 is a seventeen-residue antimicrobial, amyloidogenic peptide that forms amyloid in phosphate buffered saline (PBS). The role of 2,2,2-trifluoroethanol (TFE) concentration, a known α-helical stabiliser, in modulating aggregation of Uperin 3.5 peptide in membrane-mimetic TFE:water mixtures was investigated. Thioflavin T (ThT) fluorescence assays showed complete inhibition of aggregation at higher concentrations of TFE (≥ 20% TFE:water v/v). However, a five-to-seven-fold increase in fibrillation kinetics was observed at 10% TFE:water mixtures in comparison to aggregation in a buffer. Further, aggregation in TFE:water mixtures was only observed upon addition of buffer. Interestingly, circular dichroism (CD) spectra showed the appearance of partial helical structures in 10% TFE:water, which transitioned to β-sheet rich structures only after addition of buffer. Microsecond time-scale molecular dynamics (MD) simulations of multiple U3.5 peptides in both salt-free and salt-containing TFE:water mixtures showed that changes in the local environment of peptide residues determined the structural transition and aggregation trajectories for U3.5. Consistent with experiments, the greatest extent of aggregation was observed for low TFE concentration (10% TFE:water simulations), characterised by faster formation of helical intermediates (oligomers). While the presence of 10% TFE efficiently induced partial helical structure in individual U3.5 peptides, it did not impede peptide-peptide interactions, thus enabling peptide aggregation. Addition of salt, screened like-charge repulsion between positively charged residues of different peptides, leading to stronger inter-peptide interactions. Significantly, the presence of salt determined subsequent structural transitions in the helical intermediates; either forming a predominantly α-helical oligomer in salt-free solutions or a β-sheet-rich oligomer in salt-containing solutions.