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

-Synuclein Structures Probed by 5-Fluorotryptophan Fluorescence and 19F NMR Spectroscopy
Received: January 3, 2006
In Final Form: February 9, 2006
Web Release Date: March 16, 2006
Gates R. Winkler, Seth B. Harkins, Jennifer C. Lee,* and Harry B. Gray*
J. Phys. Chem. B,
ACS Publications
Copyright? 2006 American Chemical Society
Beckman Institute, California Institute of Technology, Pasadena, California 91125
-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 ( 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.
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