Heterogeneity of aggregates in the fibrillation mechanisms of proteins probed by time resolved fluorescence

Abstract

Under appropriate conditions almost all proteins are able to aggregate to form long, well-ordered and beta-sheet rich arrays known as amyloid fibrils. The formation of such structures involves complex intra and intermolecular interactions modulated by the structure and dynamics of the native protein, and by the physico-chemical properties of the solvent. Multiple interactions and cross-feedback during the aggregation pathway cause different ultimate aggregates’ morphologies and the possible simultaneous occurrence of multiple species. The structural definition of such assemblies is complicated by the polymorphism of the amyloid fibrils. Aim of this study is to inquire on the different nature of the intermediates formed through the aggregation pathways and on the final different fibrillar morphologies. We report an experimental study on 1-Anilino-8-Naphtalenesulphonate and Thioflavin T fluorescence decays to characterize the different fibrillar morphologies rising from different model proteins. Simple straight beta-aggregated proto-fibrillar structures are compared with more heterogeneous morphologies resulting from kinetics involving multiple interconnected steps. Fluorescence lifetime distributions of the different probes can be related with the conformational heterogeneity of proteins during the aggregation pathways, so representing a fingerprint of morphological differences which are perpetuated at all structural levels. In particular, changes in the position of the center of the distribution can be related to differences in the average environment of the excited fluorophore. The width of the lifetime distributions characterizes the strength and the variety of the mechanisms influencing the micro-environmental properties of the fluorophore, giving information on the number of accessible conformational substates in the fibrillar morphology and on the interconversion among substates during the lifetime of the excited state.