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, 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-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.

Figure 1

The effect of 10 μM GTPγS on the formation of complexes between F₂FlAsH and G-proteins in the presence (A) or absence of βγM subunits (B). Data are presented as the time-dependent attenuation of fluorescence anisotropy in comparison to GTPγS-untreated F₂FlAsH-G-protein complexes. The preparations used were αolf (1), αq (2), αs_short (3), αs_long (4), α13 (5), none (6) and βγM (7). Data is from two or three independent experiments carried out in duplicate and presented as mean ± SD as error bars.

Figure 2

The effect of 10 μM GTPγS (solid lines) and 10 μM GDP (dotted lines) on the formation of complexes between F₂FlAsH and Gi-protein heterotrimers. Data are presented as the time-dependent change in fluorescence anisotropy in comparison to nucleotide-untreated F₂FlAsH-Gi-protein complexes. Data is from two or three independent experiments carried out in duplicate and presented as mean ± SD as error bars.

Figure 3

Effect of GTPγS on the fluorescence anisotropy of F₂FlAsH-G-protein preparations. The fluorescence anisotropy of α-subunits with (A, B) or without (C, D) βγM subunits, in the absence (solid bars) or presence of 10 μM GTPγS (striped bars), was measured after 6 h of incubation at 28°C (A and C) and again after subsequent heat treatment for 1 h at 70°C, (B and D). Data is from two independent experiments carried out in duplicate, presented as mean ± SD as error bars.

Figure 4

Concentration-dependent effects of nucleotides on the fluorescence anisotropy of F₂FlAsH-αβγM complexes. The data was collected after incubation of the complexes with different concentrations of GTPγS (red circles), GppNHp (green squares), GDP (blue pyramids) or GMP (magenta triangles). Measurements were taken at different timepoints for the various α-subunits: A - αs_long at 6 h; B - αs_short at 6 h; C - α13 at 14 h; D - αq at 6 h; E - αolf at 6 h; F - αi1 at 2 h; G - αi2 at 2 h; and H - αi3 at 2 h. Data is from two or three independent experiments in duplicate, presented as mean ± SD as error bars.