Multiparametric fluorescence detection of early stages in the amyloid protein aggregation of pyrene-labeled alpha-synuclein.
The aggregation of alpha-synuclein, a presynaptic protein, has an important role in the etiology of Parkinson's disease. Oligomers or protofibrils adopting the cross-beta-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of alpha-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of alpha-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 degrees C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (I(I)/I(III)), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molecular mass and close intermolecular association in a manner dependent on position in the sequence and the presence of point mutations. The time-resolved decays (0-160 ns) of intensity and anisotropy exhibited complex, characteristic features. The new assay constitutes a convenient platform for the high-throughput screening of agents useful in the diagnosis and therapy of Parkinson's disease as well as in basic investigations.
Thirunavukkuarasu
Shyamala
S
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Jares-Erijman
Elizabeth A
EA
Jovin
Thomas M
TM
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism; D016880:Anisotropy; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010300:Parkinson Disease / Q000175:diagnosis / Q000209:etiology / Q000473:pathology; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011721:Pyrenes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000682:Amyloid; D005456:Fluorescent Dyes; D011721:Pyrenes; D051844:alpha-Synuclein; C030984:pyrene