Alpha-synuclein structures probed by 5-fluorotryptophan fluorescence and 19F NMR spectroscopy.
Alpha-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 approximately 0.2) indicate that all three indole side chains are exposed to the aqueous medium. Virtually identical single-exponential excited-state decays (tau approximately 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.
Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Gates R
GR
Winkler
Seth B
SB
Harkins
Jennifer C
JC
Lee
Harry B
HB
Gray
D005453:Fluorescence; D005461:Fluorine / Q000737:chemistry; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D051844:alpha-Synuclein / Q000737:chemistry*
D007554:Isotopes; D051844:alpha-Synuclein; D005461:Fluorine; C026927:5-fluorotryptophan; D014364:Tryptophan