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  • Title: Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated alpha-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade.
    Author: Erickson JW, Cerione RA.
    Journal: Biochemistry; 1991 Jul 23; 30(29):7112-8. PubMed ID: 1713060.
    Abstract:
    Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP gamma S-bound alpha subunit of transducin (alpha T GTP gamma S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido) fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while the alpha T GTP gamma S was labeled with eosin-5-isothiocyanate (EITC-alpha T GTP gamma S) and served as the energy acceptor. The EITC-alpha T GTP gamma S species was able to quench a significant percentage of the IAF-PDE fluorescence (typically greater than or equal to 30%) due to resonance energy transfer between the IAF and EITC moieties. The quenching by the EITC-alpha T GTP gamma S species was dose-dependent, saturable (Kd = 21 nM), and specific for the GTP gamma S-bound form of the alpha T subunit. Limited trypsin treatment of the IAF-PDE, which selectively removes a fluorescein-labeled gamma subunit (gamma PDE), completely eliminates the quenching of the IAF fluorescence by the EITC-alpha T GTP gamma S complex. Although the EITC-alpha T GTP gamma S complex competes with the unlabeled alpha T GTP gamma S for a binding site on the IAF-PDE, as well as for a site on the native PDE, it is not able to stimulate PDE activity. Thus, the modification of a single EITC-reactive residue on the alpha T GTP gamma S complex prevents this subunit from eliciting a key activation event within the retinal effector enzyme.
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