Time-Resolved Small-Angle X-Ray Scattering Study of the Early Formation of Amyloid Protofibrils on a Apomyoglobin Mutant

Abstract

Early prefibrillar intermediates are a key issue in amyloid researches, as they show the highest cytotoxicity with respect to mature fibrils, which are less toxic or even harmless. However, the early aggregation process is largely unknown, since nucleation events are rare, and elongation and reorganization processes are very fast. Time-resolved synchrotron Small Angle X-ray Scattering (SAXS), coupled to extended model fitting analysis, has been used to provide in a millisecond temporal range the structural and aggregational properties of the early and soluble protofibrils formed by a model protein after a pH jump. The amyloidogenic apomyoglobin mutant W7FW14F has been investigated as fibril former: this mutant, which is in a monomeric, partly folded state at acidic pH, undergoes at neutral pH a nucleation-dependent polymerization reaction, resulting in the formation of cytotoxic amyloid fibrils similar to those detected for proteins involved in amyloid diseases. SAXS evidenced that oligomerization of W7FW14F in solution happens in less than 100 ms after the pH jump from 4.0 to 7.0., while the resulting pattern of protein prefibrillation reveals the simultaneous presence of worm-like species and of cylindrically-shaped aggregates, whose structural features mainly change after 20 ms from the solution pH jump. Model fitting analysis gives the composition of the different oligomers and their relative concentration as a function of time, suggesting that protofibril formation occurs through the presence of aggregation and rearrangement competing processes and through the contribution of multiple coexisting elongated and worm-like protein species. The possibility to use SAXS in monitoring the effects of cosolvents and/or pharmaceutical agents in modifying or preventing the early amyloid aggregation patterns is then demonstrated.