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 protein transducin (alpha(T)). In order to characterize conformational changes in the 87-amino acid gamma(PDE) subunit that may accompany the activation of the holoenzyme, gamma(PDE) was labeled with the fluorescent probes 5-iodoacetamideofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements. 5-Iodoacetamidofluorescein specifically labeled a cystein residue at position 68 and served as a resonance energy transfer donor. The site of modification of eosin-5-isothiocyanate, which served as the resonance energy transfer acceptor, 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 distance of 63 Angstrom, consistent with a random conformation. Upon binding 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 is essential for the stimulation of PDE activity, does not impart significant alterations in the tertiary structure of the gamma(PDE) molecule. They also support a model for PDE activation that places active alpha(T) in a complex with the holoenzyme.