Dual-emission fluorescent ESIPT probe for continuous monitoring of alpha-synuclein aggregation

DMYTRO A. YUSHCHENKO, JONATHAN A. FAUERBACH, SHYMALA THIRUNAVUKKUARASU, ELIZABETH JARES-ERIJMAN, THOMAS M. JOVIN.

San Francisco, California, Estados Unidos

Conferencia; Biophysical Society 54th Annual Meeting; 2010

Biophysical Society

The aggregation of presynaptic protein alpha-synuclein (AS) is associated with Parkinson´s disease and leads to fibrillar aggregates with a characteristic cross-beta-amyloid structure. The extrinsic dye thioflavin T (ThT) binds to these structures and is widely used for the identification and quantification of amyloid fibrils in vitro. However, it exhibits poor sensitivity and reproducibility, require sampling, and is insensitive to the early stages of aggregation. We have previously described the utility of pyrene [1]and aminonapthalene [2] probes for detecting early intermediates [1,2].Here we introduce a new sensor molecule for the continuous monitoring of AS aggregation denoted AS-140HC. Mutant AS (A140C, C-terminus) is labeled with the 3-hydroxychromone (3HC) dye MFC. MFC exhibits two bands in fluorescence (N* and T*) due to the Excited State Intramolecular Proton Transfer (ESIPT, [3]) . The ratio between the bands (IT*/IN*) is a reflects rigidity and the polarity and hyrogen bonding ability in the microenviroment of the probe. Addition of AS-140HC in range of 0.5-5% to wild type AS allows monitoring aggregation via the strong increase of the T* band of MFC (figure A). This change occurs at a much earlier stage of aggregation than ThioT (figure B) and permist the identification of numerous aggregation intermediates [4]. See also [5-7]Refs: [1] Shyamala JMB 2008; [2] Celej, BJ 2008; [3] ESIPT review, Alex BJ?; [4] poster of Fauerbach et al.; [5] Celej Biochemistry 2009; [6] Caarls J Fluor 2009; [7] poster of Shvadchak et al.