RESONANCE ENERGY-TRANSFER AS A DIRECT MONITOR OF GTP-BINDING PROTEIN EFFECTOR INTERACTIONS - ACTIVATED ALPHA-TRANSDUCIN BINDING TO THE CGMP PHOSPHODIESTERASE IN THE BOVINE PHOTOTRANSDUCTION CASCADE

Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP-gamma-S-bound alpha-subunit of transducin (alpha(T)GTP-gamma-S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido)fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while the alpha(T)GTP-gamma-S was labeled with eosin-5-isothiocyanate (EITC-alpha(T)GTP-gamma-S and served as the energy acceptor. The EITC-alpha(T)GTP-gamma-S species was able to quench a significant percentage of the IAF-PDE fluorescence (typically greater-than-or-equal-to 30%) due to resonance energy transfer between the IAF and EITC moieties. The quenching by the EITC-alpha(T)GTP-gamma-S species was dose-dependent, saturable (K(d) = 21 nM), and specific for the GTP-gamma-S-bound form of the alpha-T subunit. Limited trypsin treatment of the IAF-PDE, which selectively removes a fluorescein-labeled gamma-subunit (gamma-PDE), Completely eliminates the quenching of the IAF fluorescence by the EITC-alpha(T)GTP-gamma-S complex. Although the EITC-alpha(T)GTP-gamma-S complex competes with the unlabeled alpha(T)GTP-gamma-S for a binding site on the IAF-PDE, as well as for a site on the native PDE, it is not able to stimulate PDE activity. Thus, the modification of a single EITC-reactive residue on the alpha(T)GTP-gamma-S complex prevents this subunit from eliciting a key activation event within the retinal effector enzyme.