Real Time Conformational Changes in the Retinal Phosphodiesterase γ Subunit Monitored by Resonance Energy Transfer

The γ subunit of the retinal cGMP phosphodiesterase (γPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the α subunit of the GTP-binding protein transducin (αT). In order to characterize conformational changes in the 87-amino acid γPDE subunit that may accompany the activation of the holoenzyme, γPDE was labeled with the fluorescent probes 5-iodoacetamidofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements. 5-Iodoacetamidofluorescein specifically labeled a cysteine 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 γPDE. Energy transfer between the labeled sites on free, unbound γPDE indicated that they were separated by a distance of 63 Å, consistent with a random conformation. Upon binding the catalytic αβ subunits of the PDE, the distance between the two probes on γPDE increased to 77 Å. Binding of the labeled γPDE by αT·;guanosine 5′-3-O-(thio)triphosphate did not affect the distance between the probes under conditions where the PDE was activated. These data are consistent with the view that the binding of activated αT to γPDE, which is essential for the stimulation of PDE activity, does not impart significant alterations in the tertiary structure of the γPDE molecule. They also support a model for PDE activation that places active αT in a complex with the holoenzyme.