α-Synuclein Structures Probed by 5-Fluorotryptophan Fluorescence and 19F NMR Spectroscopy

α-Synuclein, the main protein component of fibrillar deposits found in Parkinson's disease, is intrinsically disordered in vitro. Site-specific information on the protein conformation has been obtained by biosynthetic incorporation of an unnatural amino acid, 5-fluorotryptophan (5FW), into the recombinant protein. Using fluorescence and 19F NMR spectroscopy, we have characterized three proteins with 5FW at positions 4, 39, and 94. Steady-state emission spectra (maxima at 353 nm; quantum yields ≈ 0.2) indicate that all three indole side chains are exposed to the aqueous medium. Virtually identical single-exponential excited-state decays (τ ≈ 3.4 ns) were observed in all three cases. Single 19F NMR resonances were measured for W4, W39, and W94 at −49.0 ± 0.1 ppm. Our analysis of the spectroscopic data suggests that the protein conformations are very similar in the regions near the three sites.