Figure 1. Optimization workflow for the exemplar Gαq biosensor.

(a-c) RLuc8 donor positioning. (a) The inactive Gαq/Gβ1/γ2 crystal structure (PDB 3AH8) defined regions within the alpha-helical domain (red box) in close proximity to the N-terminus of the Gγ-subunit (green box). Twenty Gαq-RLuc8 chimeric proteins were generated between αA-αB and αB-αC helices. (b) Gαq-RLuc8 chimeras were evaluated in duplicate using the prototypic Gαq-coupled NT₁R neurotensin receptor. Performance was evaluated as fold-increase in dynamic range (Net BRET2) relative to the reference construct (insertion of RLuc8 after Lys97 in Gαq was named position 98). (c) The top five RLuc8 positions (119, 122, 123, 125, 126; boxed) were confirmed (N=3) and Gαq(125)-RLuc8 was chosen as the optimal chimeric donor (panel c, boxed). (d) Gγ-GFP2 optimization: the Gαq(125)-RLuc8 chimera was tested alongside each of 12 N-terminally fused Gγ-GFP2 constructs and a co-precipitated mixture of Gβ1–4 subunits. The Gγ9-GFP2 chimera provided the largest signal (N=3). (e) Gβ optimization: Gαq(125)-RLuc8 and Gγ9-GFP2 were used to screen each of four Gβ subunits (N=3). Stepwise optimization determined that Gαq(125)-RLuc8/Gβ3γ9-GFP2 was the optimal biosensor composition.