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 s f9 cells, using a baculovirus system. Deletion of the myristoylation s ite near the N-terminal increased the solubility and allowed the purif ication of rTalpha. When reconstituted with excess Tbetagamma on retin al membrane, rTalpha displayed functional characteristics of wild-type Talpha vis a vis its coupled receptor, rhodopsin and its effector, cG MP phosphodiesterase (PDE). We further mutated a tryptophan, W207, whi ch is conserved in all G proteins and is suspected to elicit the fluor escence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]Talpha mutant displayed high a ffinity receptor binding and underwent a conformational switch upon re ceptor-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 a ctivate the PDE. To characterize better its effector-activating intera ction 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.