Use of resonance energy transfer to determine the proximity of the guanine nucleotide binding site of transducin relative to a conformationally-sensitive site on the gamma subunit of the cyclic GMP phosphodiesterase.

In this work, we have used resonance energy transfer to determine the relative positions of a reactive cysteine residue on the gamma subunit of the retinal cyclic GMP phosphodiesterase (gamma PDE) and a reactive lysine residue on the alpha subunit of transducin (alpha T). The single cysteine residue on gamma PDE (residue 68) is located at a site that is sensitive to the binding of both the inactive and active forms of alpha T. This is demonstrated by the finding that the addition of an alpha T-GDP complex to a gamma PDE subunit labeled with the environmentally-sensitive probe 2-(4-maleimidoanilino)naphthalene-6-sulfonate (MIANS) results in an enhancement in the MIANS fluorescence. The alpha TGDP-induced fluorescence enhancement is dose-dependent and yields an apparent Kd value of approximately 3 microM. Activation of alpha TGDP by aluminum fluoride, when bound to the MIANS-labeled gamma PDE (M-gamma PDE), then results in a quenching of the MIANS fluorescence. The aluminum fluoride-induced change in M-gamma PDE fluorescence occurs on a time scale identical to that observed for changes in the intrinsic alpha T fluorescence that correspond to activating conformational changes in the alpha T "switch II" region. These results suggest that the induction of the activated state of the alpha T subunit results in a change in conformation close to cysteine 68 in gamma PDE.(ABSTRACT TRUNCATED AT 250 WORDS)