Multiparametric fluorescence detection of early stages in the amyloid protein alpha-synuclein

SHYAMALA THIRUNAVUKKUARASU, ELIZABETH A JARES-ERIJMAN AND THOMAS M JOVIN

JOURNAL OF MOLECULAR BIOLOGY

Año: 2008 vol. 378 p. 1064 - 1073

0022-2836

    The aggregationof  alpa-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°C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio(II/IIII), 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(0–160ns) 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.