19F NMR studies of alpha-synuclein conformation and fibrillation.
Fibrils of the intrinsically disordered protein alpha-synuclein are hallmarks of Parkinson's disease. The fluorescent dye thioflavin T is often used to characterize fibrillation, but this assay may not provide quantitative information about structure and mechanism. To gain such information, we incorporated the 19F-labeled amino acid, 3-fluorotyrosine, into recombinant human alpha-synuclein at its endogenous tyrosine residues. Tyrosine 39 is in the positively charged N-terminal region of this 140-residue protein. The other three tyrosines, 125, 133, and 136, are near the C-terminus. 19F nuclear magnetic resonance spectroscopy was used to study several properties of labeled alpha-synuclein, including its conformation, conformational changes induced by urea, spermine, and sodium dodecyl sulfate (SDS), its interaction with SDS micelles, and the kinetics of fibril formation. The results show that the tyrosines are in disordered regions but that there is some structure near position 39 that is disrupted by urea. SDS binding alters the conformation near position 39, but the C-terminal tyrosines are disordered under all conditions. The NMR data also indicate that SDS-micelle-bound alpha-synuclein and the free protein exchange on the 10 ms time scale. Studies of fibrillation show the utility of 19F-labeled NMR. The data indicate that fibrillation is not accompanied by the formation of large quantities of low molecular weight intermediates. Although dye binding and 19F NMR data show that 1 mM SDS and 1 mM spermine accelerate aggregation compared to buffer alone, only the NMR data indicate that the species formed in SDS are smaller than those formed in buffer or buffer plus spermine. We conclude that 19F NMR spectroscopy is useful for obtaining residue-level, quantitative information about the structure, binding, and aggregation of alpha-synuclein.
Li
Conggang
C
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Lutz
Evan A
EA
Slade
Kristin M
KM
Ruf
Rebecca A S
RA
Wang
Gui-Fang
GF
Pielak
Gary J
GJ
D002021:Buffers; D004338:Drug Combinations; D005462:Fluorine Radioisotopes / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013096:Spermine / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002021:Buffers; D004338:Drug Combinations; D005462:Fluorine Radioisotopes; D051844:alpha-Synuclein; C001245:3-fluorotyrosine; D013096:Spermine; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine