On the binding of Thioflavin-T to HET-s amyloid fibrils assembled at pH 2.
Amyloid fibrils are ordered beta-sheet protein or peptide polymers. The benzothiazole dye Thioflavin-T (ThT) shows a strong increase in fluorescence upon binding to amyloid fibrils and has hence become the most commonly used amyloid-specific dye. In spite of this widespread use, the mechanism underlying specific binding and fluorescence enhancement upon interaction with amyloid fibrils remains largely unknown. Recent contradictory reports have proposed radically different modes of binding. We have studied the interaction of ThT with fibrils of the prion forming domain of the fungal HET-s prion protein assembled at pH 2 in order to try to gain some insight into the general mechanism of ThT-binding and fluorescence. We found that ThT does not bind to HET-s(218-289) fibrils as a micelle as previously proposed in the case of insulin fibrils. We have measured binding kinetics, affinity and stoichiometry at pH values above and below the pI of the HET-s(218-289) fibrils and found that binding is dramatically affected by pH and ionic strength. Binding is poor at acidic pH, presumably as a result of repulsive electrostatic interaction between the positively charged ThT molecule and the fibril surface. Finally, we found that ThT acquires chiral properties when it is fibril-bound. These results are discussed in relation to the different ThT-binding modes that have been proposed.
Sabaté
Raimon
R
Laboratoire de Génétique Moléculaire des Champignons, Institut de Biochimie et de Génétique Cellulaires, UMR 5095 CNRS/Université de Bordeaux 2, 1 rue Camille St Saëns, 33077 Bordeaux cedex, France. sabate@ibgc.cnrs.fr
Lascu
Ioan
I
Saupe
Sven J
SJ
D000682:Amyloid / Q000737:chemistry*; D052160:Benzothiazoles; D005456:Fluorescent Dyes / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry; D007700:Kinetics; D048468:Podospora / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011328:Prions; D011485:Protein Binding; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013844:Thiazoles / Q000737:chemistry*
D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D005656:Fungal Proteins; C083891:HET-S protein, Podospora anserina; D007328:Insulin; D011108:Polymers; D011328:Prions; D013844:Thiazoles; C009462:thioflavin T