Detection and characterization of (early) intermediates in the aggregation of alpha-synuclein

Rolf Vermeij (BPE group)

Objective

The project as a whole aims at gaining more insight into the structure and function of a-synuclein in monomeric or fibrillar states. Structure of the protein is being investigated by single-pair-FRET methods (population distributions and folding: not this project) and by the use of environmentally sensitive dyes (bulk spectroscopy mainly). Labels will be introduced at strategic positions in the protein via introduction of cysteine-residues at strategic positions by point-mutations. Focus of these methods will be on early stages of the aggregation, since both the “normal” (unfolded, native, monomeric) and the “toxic” (protofibrillar) forms of synuclein occur in these early stages. New dyes or protein-dye constructs will have to be designed to gain information on early folding in the protein. Initially, pyrene was chosen as environmentally sensitive fluorescent label for these studies. This label shows sensitivity to hydrophilicity of the environment (intensity ratio of vibronic bands) and due to its longer lifetime it can also show proximity of a second dye moiety by excimer formation.