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)
Erickson
J W
JW
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401, USA.
Mittal
R
R
Cerione
R A
RA
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D004735:Energy Transfer; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes; D006150:Guanine Nucleotides / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D013439:Sulfhydryl Reagents / Q000737:chemistry; D015293:Transducin / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006150:Guanine Nucleotides; D013439:Sulfhydryl Reagents; C012957:eosine-5-isothiocyanate; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin; D008239:Lysine; D003545:Cysteine; D004801:Eosine Yellowish-(YS)