A homogeneous method to measure nucleotide exchange by α-subunits of heterotrimeric G-proteins using fluorescence polarization.
The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [³⁵S]guanosine 5'-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg(2+) concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.
Muller
Robin E
RE
Department of Pharmacology, UNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365, USA.
Klein
Klara R
KR
Hutsell
Stephanie Q
SQ
Siderovski
David P
DP
Kimple
Adam J
AJ
D005454:Fluorescence Polarization; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction
D044385:GTP-Binding Protein alpha Subunits; D016244:Guanosine 5'-O-(3-Thiotriphosphate)