title: Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties.
abstract: Substantial evidence suggests that the fibrillation of alpha-synuclein is a critical step in the development of Parkinson's disease. In vitro, alpha-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric alpha-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in alpha-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of alpha-synuclein are likely to be crucial to the fibrillation process.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95964, USA. natasa.poklar@bf.uni-lj.si
authors.forename: Natasa Poklar
authors.initials: NP
authors.lastname: Ulrih
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Barry
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Fink
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000682:Amyloid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D020125:Mutation, Missense; D010300:Parkinson Disease / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014443:Tyrosine / Q000235:genetics; D051844:alpha-Synuclein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000682:Amyloid; D011994:Recombinant Proteins; D051844:alpha-Synuclein; D014443:Tyrosine; D000409:Alanine