Pyrene labeled α-synuclein: tracking the early stages of amyloid protein aggregation

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

Salzburgo, Austria

Conferencia; 10th Conference on Methods & Applications of Fluorescence; 2007

The aggregation of α-synuclein (AS), a presynaptic protein, plays an important role in the etiology of Parkinson’s disease. The low molecular weight oligomers or protofibrils adopting the β-sheet structure characteristic of amyloid proteins are presumed to be the cytotoxic species [1].  Unfortunately, currently employed techniques for following the kinetics of AS aggregation e.g. the fluorescence enhancement of thioflavin-T detect only the fibrillar species formed at the later stages of the reaction. Moreover, these assays are not continuous and lack reproducibility. We have devised a new fluorescence aggregation assay that is continuous and can detect the formation of oligomeric intermediates [2]. The approach is based on fluorescent tagging of functionally neutral ala-to-cysteine mutants of AS. Pyrene conjugates of AS at three positions in the AS sequence were used: N-terminal (residue 18), the core NAC region (residue 90), and the C-terminal region (residue 140). Pyrene was selected as fluorescence probe because of its long fluorescence lifetime, environmental sensitivity, and high fluorescence anisotropy in the immobilized state. Different spectral properties of pyrene were monitored during AS aggregation: fluorescence intensity, spectral distribution of the monomer emission, excimer formation, steady state and time resolved anisotropy and fluorescence lifetime. All of these parameters changed in a systematic manner right from the onset of aggregation. The formation of lower molecular weight oligomers were evident in both the wild type and genetic (A53T, A30P familial mutations) variants of AS, but the responses differed according to the position of the pyrene tag and the mutation. The pyrene labeled AS assay represents the first continuous method to follow the early kinetics of amyloid protein aggregation. We believe that this new assay will provide a convenient platform for high throughput screening of potential therapeutic drugs for Parkinsons’s disease (aggregation inhibitors, antagonists) as well as basic science investigation.