Structural Transitions Associated with the Assembly Dynamics of Transthyretin Amyloid Protofibrils

Abstract

Transthyretin (TTR) is a serum protein involved in the transport of thyroxine and co-transport of retinol. Several neurodegenerative diseases are associated with the aggregation of TTR into amyloid fibrils, but the molecular mechanisms of the assembly remain unknown. Here we used AFM to identify intermediates along the assembly-disassembly of TTR amyloid protofibrils. By using dynamic force spectroscopy, we also characterized the mechanical response of protofibrils at different growth stages before and after their partial disassembly.During TTR assembly, annular oligomers with 15.8 ± 2.3 nm in diameter appeared after 3-5 days of incubation. These annular oligomers displayed a tendency to associate laterally, forming linear structures that preceded the appearance of amyloid protofibrils. In other proteins, similar annular oligomeric structures have been implicated in amyloid cytotoxicity, but it remains unclear to which extent they represent an on pathway intermediate. Upon solvent exchange, dramatic morphological rearrangements occurred leading to the partial disassembly of protofibrils. During disassembly, annular oligomers with 7.0 ± 0.6 nm appeared, suggesting that the annular arrangement is a common motif in the TTR assembly and disassembly pathways. Force spectroscopy of both native TTR and early protofibrils contained a sawtooth pattern with ∼4 nm periodicity. Conceivably, this pattern corresponds to successive structural transitions related to the sequential unfolding of the s-strands within the TTR monomer. Force spectra of TTR protofibrils also revealed a time-dependent increase in the length of the manipulated structure, indicating that the axial association between monomers stabilizes with time. Thus, stabilization of intermonomeric contacts appears to be a much slower process than that of mere assembly, possibly involving additional structural rearrangements of the monomer within the protofibrils.