Dual Lifetimes for Complexes between Glutathione-S-transferase (hGSTA1-1) and Product-like Ligands Detected by Single-Molecule Fluorescence Imaging.

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione S-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate.