Intrinsic biophysical monitors of transducin activation: fluorescence, UV-visible spectroscopy, light scattering, and evanescent field techniques

Information on the temporal order, the kinetics, and the energetics of each of the reaction intermediates is required in order to fully elucidate the mechanism of catalytic interaction of G proteins with their receptors. Intrinsic monitors, such as light scattering, absorption, and fluorescence, exploit endogenous properties of proteins, and therefore offer the advantage of leaving the system under investigation undisturbed. Such assays currently available for G proteins include the well-known intrinsic fluorescence changes of a tryptophan residue located near the G protein's active center. In addn., the visual system offers UV-visible spectrophotometric assays in which the retinal chromophore itself serves as an intrinsic reporter group. These assays include monitoring the transducin sensitive formation of the active metarhodopsin-II (MII) conformation, as well as the photoregeneration of dark state rhodopsin from MII. A third technique is kinetic light scattering, which makes use of the unique activation-dependent soly. of the rod G protein. While these monitors are highly specialized, more generally applicable monitors have been developed in recent years and successfully tested with the visual system. This family of techniques is based on the evanescent field in the vicinity of a reflecting optical surface, and includes surface plasmon resonance and resonant mirror spectroscopy. Applications and limitations of these techniques are described. (c) 2000 Academic Press. [on SciFinder (R)]

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Methods in Enzymology, 315, Vertebrate Phototransduction and the Visual Cycle, Pt. A, 471-489

 Record created 2006-02-27, last modified 2018-01-27

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