ABSTRACTWithin the complex aggregation process of Aβ-peptides into fibrils, oligomeric species, play a central role and reveal fundamental properties of the underlying mechanism of aggregation. In particular, low molecular weight aggregates have attracted increasing interest because of their role in cytotoxicity and neuronal apoptosis, typical of aggregation related diseases. One of the main techniques used to characterize such early stages of aggregation is fluorescence spectroscopy. To this end, Aβ-peptide chains are functionalized with fluorescent tags, often covalently bound to the disordered N-terminus region of the peptide, with the assumption that functionalization and presence of the fluorophore will not modify the process of self-assembly nor the final fibrillar structure. Up to date, experimental findings reveal size distributions of thermodynamically stable oligomers ranging from very narrow distributions of dimers to octamers, to very broad distributions up to 50-mers. In the present investigation we systematically study the effects of five of the most commonly used fluorophores on the aggregation of Aβ(1-40)-peptides. Time-resolved and single-molecule fluorescence spectroscopy have been chosen to monitor the oligomer populations at different fibrillation times, TEM, AFM and X-ray diffraction to investigate the structure of mature fibrils. While the structures of the mature fibrils were only slightly affected by the fluorescent tags, the sizes of the detected oligomeric species varied significantly depending on the chosen fluorophore. In particular, we relate the presence of high molecular weight oligomers (as found for the fluorophores HiLyte 647, Atto 647N and Atto 655) to net-attractive, hydrophobic fluorophore-peptide interactions, which are weak in the case of HiLyte 488, and Atto 488. The latter form low molecular weight oligomers only. Our findings reveal the potentially high impact of the properties of fluorophores on transient aggregates which needs to be included in the interpretation of experimental data of oligomers of fluorescently labeled peptides.
Deciphering Multiple Critical Parameters of Polymeric Self-Assembly by Fluorescence Spectroscopy of a Single Molecular Rotor BODIPY-C12
