Recognition of Intermediate Species in Alpha-Synuclein Assembly Using ESIPT Dual Emission Probes

JONATHAN FAUERBACH, DMYTRO YUSCHENKO, DONNA ARNDT-JOVIN, ALEXANDER DEMCHENKO, THOMAS JOVIN Y ELIZABETH JARES-ERIJMAN

Kyiev, Ucrania.

Simposio; Vth International Symposium Supramolecular Systems in Chemistry and Biology; 2009

Alpha-synuclein (AS) is a protein whose aggregation in dopaminergic neurons is regarded as a main cause of Parkinson’s disease. AS is unfolded in solution, yet is able to misfold and self assemble leading to the formation of a number of different supramolecular structures that finally result in so called amyloid fibrils.There are presently few tools for monitoring the initial stages of amyloid protein self assembly. Thio-T assays only provide information regarding fibrillar structures, but there is a gap between monomer and fibrils which has not been filled.We have found that fluorescent dyes showing Excited State Proton Transfer (ESIPT) are a useful tool for monitoring the process of aggregation of amyloid proteins. These dyes exhibit a dual fluorescence in the form of a blue shifted band arising from the normal excited species (N*) and a red shifted band due to the excited state tautomer (T*), figure 1. The ratio between the bands is a measure of the difference in the microenvironment of the dye. It thus provides a good indicator of the folding processes occurring through the self assembly pathway (figure 2).Covalently labeled proteins (eg: AS A140C mutant) with MFC dyes have provided vital information about the initial stages of AS self organization. We have been able to correlate the spectral changes with the evolution of intermediate species through the use of Atomic Force Microscopy (AFM).It is the purpose of this work to better understand the process of aggregation and try to characterize intermediate species, particularly those related to cytotoxicity.