title: Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis.
abstract: The aggregation of α-synuclein (α-Syn) is linked to Parkinson's disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways.
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
authors.forename: Zhengjian
authors.initials: Z
authors.lastname: Lv
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Krasnoslobodtsev
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ysselstein
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Rochet
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York; Tri-Institutional Training Program in Chemical Biology, Weill Cornell Medical College, and Rockefeller University, and Memorial Sloan-Kettering Cancer Center, New York, New York.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
authors.affiliation: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska. Electronic address: ylyubchenko@unmc.edu.
authors.forename: Yuri L
authors.initials: YL
authors.lastname: Lyubchenko
mesh_terms: D017354:Point Mutation; D055503:Protein Multimerization; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics
chemical_list: D051844:alpha-Synuclein