Stabilization of an intermediate activation state for transducin by a fluorescent GTP analogue

Biochemistry
Sekar Ramachandran, R A Cerione

Abstract

The GTP-binding protein (G protein), transducin, serves as a key molecular switch in vertebrate vision through the tight regulation of its GTP-binding (activation)/GTP hydrolytic (deactivation) cycle by the photoreceptor rhodopsin. To better understand the structure-function characteristics of transducin activation, we have set out to identify spectroscopic probes that bind to the guanine nucleotide-binding site of this G protein and maintain its ability to interact with its specific cellular target/effector, the cyclic GMP phosphodiesterase (PDE). In this study, we describe the characterization of a fluorescently labeled GTP analogue, BODIPY-FL GTPgammaS (BOD-GTPgammaS), that binds to the alpha subunit of transducin (alpha(T)) in a rhodopsin- and Gbetagamma-dependent manner, similar to the binding of GTP or GTPgammaS, with an apparent dissociation constant of 100 nM. The rhodopsin-dependent binding of BOD-GTPgammaS to alpha(T) is slow, relative to the rate of binding of GTPgammaS, particularly under conditions where rhodopsin must act catalytically to stimulate the exchange of BOD-GTPgammaS for GDP on multiple alpha(T) subunits. This reflects a slower rate of dissociation of rhodopsin and Gbetagamma from alpha(T)-BOD-GTPgammaS...Continue Reading

References

Jan 1, 1994·Methods in Enzymology·R A Cerione
May 2, 1997·The Journal of Biological Chemistry·A E GranovskyN O Artemyev
Jul 17, 1998·Nature·T IiriH R Bourne
Feb 5, 2002·Annual Review of Physiology·Vadim Y ArshavskyEdward N Pugh

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Citations

Sep 18, 2009·Protein Science : a Publication of the Protein Society·Heidi E HammAnita M Preininger
Dec 13, 2005·Biochemical and Biophysical Research Communications·Francis S Willard, David P Siderovski

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