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-sulf (MIANS) results in an enhancement in the MIANS fluorescence. The alpha(T)GDP-induced fluorescence enhancement is dosedependent and yields an apparent K-d value of similar to 3 mu M. Activation of alpha(T)GDP 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 CIT ''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) The reactive lysine residue on the alpha(T) subunit also appears to be located in a conformationally-sensitive region, since previous studies have shown that the modification of this residue within an alpha(T)GTP gamma S complex prevents the stimulation (by alpha(T)) of the cyclic GMP phosphodiesterase. However, the lysine-modified alpha(T)GTP gamma S complex still binds to Y gamma DE With high affinity, suggesting that an activated alpha(T) subunit must make at least two contacts with the effector enzyme, with one contact being responsible for high-affinity binding and the other (which is sensitive to lysine modification) being responsible for stimulation of enzyme activity. Here we present evidence that the reactive lysine on the alpha(T) subunit is lysine 267 located within the guanine ring binding domain of alpha(T). The distance between cysteine 68 on YPDE and lysine 267 on alpha(T) was measured through resonance energy transfer, using (iodoacetamido)fluorescein (IAF) attached to cysteine 68 as the energy donor and eosin isothiocyanate (EITC) attached to lysine 267 as the energy acceptor. On the basis of the observed efficiency of energy transfer at saturation, the distance between these residues is estimated to be 45 Angstrom. This measured distance is larger than that separating a previously proposed stimulatory contact site for the gamma(PDE) and the guanine ring binding domain, based on the crystal structure of alpha(T)GTP gamma S, and will be considered within the context of different models for the high-affinity binding of the alpha(T) subunit to its effector.