Characterization of oligomers during alpha-synuclein aggregation using intrinsic tryptophan fluorescence.
The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.
Dusa
Alexandra
A
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Kaylor
Joanna
J
Edridge
Shauna
S
Bodner
Nika
N
Hong
Dong-Pyo
DP
Fink
Anthony L
AL
D020106:Acrylamide / Q000378:metabolism; D019943:Amino Acid Substitution; D000682:Amyloid / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D005453:Fluorescence; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D017433:Protein Structure, Secondary / Q000235:genetics; D012997:Solvents; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D051844:alpha-Synuclein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000682:Amyloid; D000817:Anilino Naphthalenesulfonates; D012997:Solvents; D051844:alpha-Synuclein; D020106:Acrylamide; D014443:Tyrosine; D014364:Tryptophan; C007543:dityrosine