Mechanisms of early amyloid formation and the confining influence of macromolecular crowding

Molecular insight into the time dependence of structure and composition of prefibrillar aggregates di-rectly reveal the basic mechanisms and kinetics of structure formation. This aspect is not only of broad interest for biomedical questions, as soluble amyloid oligomers are evidenced to be the primary cause for many wide-spread diseases, but also for the engineering of novel materials. The project aims to get a detailed understanding on the early prefibrillar growth process of amyloids and the influence of outer constraints on size and stability of the aggregates as well as on available aggregation pathways. We want to access this field from a polymer-physics perspective and expect fundamental insights into the mechanisms of protein self-assembly and oligomer growth processes. In particular we want to comparatively study the early oligomeric states of the A-peptides, A(1-40) and A(1-42), which are known to exhibit different fibrillation kinetics, using a combined approach of two-color single-molecule fluorescence spectroscopy and small and wide angle X-ray scattering (SAXS,WAXS). Single-molecule spectroscopy is capable to reveal oligomeric states and state populations even of unstable and rare intermediates, SAXS and WAXS experiments enable to follow the time course of the total amounts of monomers, intermediates and fibrils and provide valuable structural information. The obtained infor-mation will be used to develop rate-kinetic models describing the on- and off pathways of aggregation, which will be further used to study the effects of spatial confinement on the derived aggregation mechanisms by using macromolecular crowding as an outer constraint. Macromolecular crowding allows mimicking cell-like constraints, thus the project will help to understand the influences of con-finement on amyloid aggregation and fibrillation kinetics at the molecular level and we plan to extend our investigations to other proteins with opposing aggregation tendencies in confinement.