Tryptophan Probes at the a-Synuclein and Membrane Interface

Understanding how environmental factors affect the conformational dynamics of alpha-synuclein (alpha-syn) is of great importance because the accumulation and deposit of aggregated alpha-syn in the brain are intimately connected to Parkinson's disease etiology. Measurements of steady-state and time-resolved fluorescence of single tryptophan-containing alpha-syn variants have revealed distinct phospholipid vesicle and micelle interactions at residues 4, 39, 94, and 125. Our circular dichroism data confirm that Trp mutations do not affect alpha-syn membrane binding properties (apparent association constant K-a(app) similar to 1 x 10(7) M-1 for all synucleins) saturating at an estimated lipid-to-protein molar ratio of 380 or approximately 120 proteins covering similar to 7% of the surface area of an 80 nm diameter vesicle. Fluorophores at positions 4 and 94 are the most sensitive to the lipid bilayer with pronounced spectral blue-shifts (W4: Delta lambda(max) similar to 23 nm; W94: Delta lambda(max) similar to 10 nm) and quantum yield increases (W4, W94: similar to 3 fold), while W39 and W125 remain primarily water-exposed. Time-resolved fluorescence data show that all sites (except W125) have subpopulations that interact with the membrane.