Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein.
The aggregation of alpha-synuclein is believed to be a critical step in the etiology of Parkinson's disease. A variety of biophysical techniques were used to investigate the aggregation and fibrillation of alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by Trp, and two others by Phe, in order to permit fluorescence resonance energy transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic light-scattering and other probes, indicate the existence of a transient oligomer, whose population reaches a maximum at the end of the lag time. This oligomer, in which the alpha-synuclein is in a partially folded conformation, is subsequently converted into fibrils, and has physical properties that are distinct from those of the monomer and fibrils. In addition, another population of soluble oligomers was observed to coexist with fibrils at completion of the reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer. Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating that the C-terminal domain is not part of the fibril core. No FRET was observed in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core. Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates and competing pathways.
Kaylor
Joanna
J
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
Bodner
Nika
N
Edridge
Shauna
S
Yamin
Ghiam
G
Hong
Dong-Pyo
DP
Fink
Anthony L
AL
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D052160:Benzothiazoles; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D051843:Synucleins; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D051844:alpha-Synuclein
D000817:Anilino Naphthalenesulfonates; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; C497604:SNCA protein, human; D051843:Synucleins; D013844:Thiazoles; D051844:alpha-Synuclein; C009462:thioflavin T; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan