Crowding Alone Cannot Account for Cosolute Effects on Amyloid Aggregation

Abstract

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. This effect has previously been attributed to the large volume excluded by inert cellular solutes, sometimes termed “macromolecular crowding”. A different family of cosolutes that are commonly used as osmoprotectants by cells (termed osmolytes) exhibited in several instances an inhibitory effect on amyloid formation. To learn more about the difference in action between these two groups of cosolutes, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates to form amyloid fibrils. We found that, in the presence of polyols (acting as osmolytes) and polyethylene glycols (crowders), the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with solute concentration, and grew with crowder molecular volume as would also be predicted by molecular crowding models. Once aggregation was initiated and followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy, the polyols glycerol and sorbitol increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. This difference in effect between the two cosolute groups cannot be accounted for by the purely entropic excluded volume interactions that are responsible for crowding. We therefore suggest that other forces, enthalpic by nature, may contribute to the cosolute effects acting on amyloid formation.