Biarsenical ligands bind to endogenous G-protein α-subunits and enable allosteric sensing of nucleotide binding

BMC Biochemistry
Lauri TõntsonAgo Rinken

Abstract

Heterotrimeric G-proteins relay extracellular signals to intracellular effector proteins. Multiple methods have been developed to monitor their activity; including labeled nucleotides and biosensors based on genetically engineered G-proteins. Here we describe a method for monitoring unlabeled nucleotide binding to endogenous G-proteins α-subunits in a homogeneous assay based on the interaction of 4',5'-bis(1,2,3-dithioarsolan-2-yl)-2',7'-difluorofluorescein (F2FlAsH) with G-protein α-subunits. The biarsenic fluorescent ligand F2FlAsH binds to various wild-type G-protein α-subunits (αi1, αi2, αi3, αslong, αsshort, αolf, αq, α13) via high affinity As-cysteine interactions. This allosteric label enables real time monitoring of the nucleotide bound states of α-subunits via changes in fluorescence anisotropy and intensity of their F2FlAsH-complexes. We have found that different α-subunits displayed different signal amplitudes when interacting with F2FlAsH, being more sensitive to nucleotide binding to αi, αs, αolf and αq than to α13. Addition of nucleotides to F2FlAsH-labeled α-subunits caused concentration-dependent effects on their fluorescence anisotropy. pEC50 values of studied nucleotides depended on the subtype of the α-subuni...Continue Reading

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Citations

Jun 9, 2016·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Ago RinkenSergei Kopanchuk
Dec 8, 2020·The FEBS Journal·Alexey Bondar, Josef Lazar

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Methods Mentioned

BETA
GTPase
Fluorescence
tandem affinity chromatography

Software Mentioned

GraphPad
Li
FLIM
GraphPad Prism

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