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

Abstract

Background: 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. Results: The biarsenic fluorescent ligand F₂FlAsH binds to various wild-type G-protein α-subunits (αi1, αi2, αi3, αs_long, αs_short, 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-subunit and were from 5.7 to 8.2 for GTPγS, from 5.4 to 8.1 for GppNHp and from 4.8 to 8.2 for GDP and lastly up to 5.9 for GMP. While GDP and GMP increased the fluorescence anisotropy of F₂FlAsH complexes with αi-subunits, they had the opposite effect on the other αβγM complexes studied. Conclusions: Biarsenical ligands interact allosterically with endogenous G-protein α-subunits in a nucleotide-sensitive manner, so the presence or absence of guanine nucleotides has an effect on the fluorescence anisotropy, intensity and lifetime of F₂FlAsH-G-protein complexes.