REAL-TIME CONFORMATION CHANGES IN THE RETINAL PHOSPHODIESTERASE GAMMA-SUBUNIT MONITORED BY RESONANCE ENERGY-TRANSFER

The gamma subunits of the retinal cGMP phosphodiesterase (gamma(PDE)) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding pr otein transducin (alpha(T)). In order to characterize conformational c hanges in the 87-amino acid gamma(PDE) subunit that may accompany the activation of the holoenzyme, gamma(PDE) was labeled with the fluoresc ent probes 5-iodoacetamideofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements. 5-Iodoacetamidofluoresc ein specifically labeled a cystein residue at position 68 and served a s a resonance energy transfer donor. The site of modification of eosin -5-isothiocyanate, which served as the resonance energy transfer accep tor, was determined to be within the first seven residues of the amino terminus of gamma(PDE). Energy transfer between the labeled sites on free, unbound gamma(PDE) indicated that they were separated by a dista nce of 63 Angstrom, consistent with a random conformation. Upon bindin g that catalytic alpha beta subunits of the PDE, the distance between the two probes on gamma(PDE) increased to 77 Angstrom. Binding of the labeled gamma(PDE) by alpha(T) . guanosine 5'-3'-O-(thio)triphosphate did not affect the distance between the probes under conditions where the PDE was activated. These activated alpha(T) to gamma(PDE), which i s essential for the stimulation of PDE activity, does not impart signi ficant alterations in the tertiary structure of the gamma(PDE) molecul e. They also support a model for PDE activation that places active alp ha(T) in a complex with the holoenzyme.