Reversible Assembly of a Drug Peptide into Amyloid Fibrils: A Dynamic Circular Dichroism Study.

The common view on the amyloid fibril formation is that it is a multistep process that involves many oligomeric intermediate species, which leads to a high degree of polymorphism. This view derives from numerous kinetic studies whose vast majority was carried out with amyloid β fragments or other pathological amyloidogenic sequences. Yet, it is not clear whether the mechanisms inferred from these studies are universal and also apply to functional amyloids, in particular to peptide hormones which form reversible amyloid structures. In the present work, we study the self-assembly properties of atosiban, a nonapeptide drug, whose sequence is very close to those of the oxytocin and vasopressin hormones. We show that this very soluble peptide consistently self-assembles into 7 nm wide amyloid fibrils above a critical aggregation concentration (2-10 w/w % depending on the buffer conditions). The peptide system is characterized in details, from the monomeric to the assembled form, with osmotic concentration measurements, transmission electron microscopy, small-angle X-ray scattering, infrared and fluorescence spectroscopy, and circular dichroism (CD). We have followed in situ the fibril assembly with fluorescence and synchrotron radiation CD and noticed that the peptide undergoes conformational changes during the process. However, several lines of evidence point toward the association of monomers and dimers into fibrils without passing through oligomeric intermediate species contrary to what is usually reported for pathogenic amyloids. The native β-hairpin conformation of the monomer could explain the straightforward assembly. The tyrosine stacking is also shown to play an important role.