TRYPTOPHAN-W207 IN TRANSDUCIN T-ALPHA IS THE FLUORESCENCE SENSOR OF THE G-PROTEIN ACTIVATION SWITCH AND IS INVOLVED IN THE EFFECTOR-BINDING

We have produced a recombinant transducin alpha subunit (rTalpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rTalpha. When reconstituted with excess Tbetagamma on retinal membrane, rTalpha displayed functional characteristics of wild-type Talpha vis a vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]Talpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTPgammaS binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]TalphaGDP-AlFx for PDEgamma, the effector subunit that binds most tightly to Talpha. [W207F]Talpha still bound in an activation-dependent way to PDEgamma, but with a 100-fold lower affinity than rTalpha. This suggests that W207 contributes to the G protein effector binding.