galactose-H+ transport protein, GaIP, of Escherichia coli

The binding of the transport inhibitor, forskolin, to the galactose-HI symporter, GalP, of Escherichia coli was evaluated by equilibrium and time-resolved fluorescence measurements. A quench in protein fluorescence of 8-12% was observed upon the binding of forskolin. The overall dissociation constant (Kd) for forskolin determined by fluorescence titration ranged between 1.2 and 2.2 suM, which is similar to that reported from equilibrium dialysis measurements of the binding of [3H]forskolin (Kd = 0.91.4 uM). The kinetics of forskolin binding were measured by stopped-flow fluorescence methods. The protein fluorescence was quenched in a biphasic manner; the faster of these two rates was dependent on the concentration of forskolin and was interpreted as the initial binding step from which both the association (k.n) and dissociation (kor,) rate constants were determined. The association and dissociation rate constants were 5.4-6.2 ,tM-' s-' and 5.1-11.5 s-' respectively, and the Kd was calculated to be 1.5 ,#M. The binding of forskolin was inhibited by D-galactose, but not by L-galactose, and displacement by