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A direct fluorescence-based assay for RGS domain GTPase accelerating activity by Francis S. Willard; Adam J. Kimple; Christopher A. Johnston; David P. Siderovski is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is A direct fluorescence-based assay for RGS domain GTPase accelerating activity about?

Diverse extracellular signals regulate seven transmembrane-spanning receptors to modulate cellular physiology. These receptors signal primarily through activation of heterotrimeric guanine nucleotide binding proteins (G proteins). A major determinant of heterotrimeric G protein signaling in vivo and in vitro is the intrinsic GTPase activity of the Ga subunit. RGS (regulator of G protein signaling) domain-containing proteins are GTPase accelerating proteins specific for Ga subunits. In this article, we describe the use of the ribose-conjugated fluorescent guanine nucleotide analog BODIPYFL-GTP as a spectroscopic probe to measure intrinsic and RGS protein-catalyzed nucleotide hydrolysis by Ga o . BODIPYFL-GTP bound to Ga o exhibits a 200% increase in fluorescence quantum yield. Hydrolysis of BODIPYFL-GTP to BODIPYFL-GDP reduces the quantum yield to 27% above its unbound value. We demonstrate that BODIPYFL-GTP can be used as a rapid real-time probe for measuring RGS domain-catalyzed GTP hydrolysis by Ga o . We demonstrate the effectiveness of this assay in the analysis of loss-of-function point mutants of both Ga o and RGS12. This assay should be useful in screening for and analyzing

Who reads A direct fluorescence-based assay for RGS domain GTPase accelerating activity?

Author

Francis S. Willard; Adam J. Kimple; Christopher A. Johnston; David P. Siderovski

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2005

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)