Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheralsite that reveals conformational interactions between the peripheral and acylation sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entranc e. Recent studies have shown that the peripheral site contributes to cataly tic efficiency by transiently binding substrates on their way to the acylat ion site, but the question of whether the peripheral site makes other contr ibutions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiat ion of a conformational change that is transmitted allosterically to the ac ylation site to alter catalysis, However, evidence for conformational inter actions between these sites has been difficult to obtain, Here we report th at thioflavin T, a fluorophore widely used to detect amyloid structure in p roteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 mum. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the g reatest enhancement of fluorescence for the binding of a fluorophore to ACh E yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes wi th AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4. 2. The observation of this partial quenching of thioflavin T fluorescence i s a major advance in the study of AChE, for two reasons. First, it allows t hioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initia tes a change in the local AChE conformation at the peripheral site that que nches the fluorescence of bound thioflavin T, The data provide strong evide nce in support of a conformational interaction between the two AChE sites.