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.
Winkler
Gates R
GR
Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Harkins
Seth B
SB
Lee
Jennifer C
JC
Gray
Harry B
HB
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