Abstact

We describe a straightforward method for purifying and optimizing human green cone opsin (GCO), which we then used for biophysical and structural studies of a GCO mutant, GCO(E129Q). Our results show that in dark-state GCO, residue E129 enables long-wavelength light absorption, presumably by acting as the counterion for the protonated retinal Schiff base. Notably, the Schiff base pKa in dark-state GCO(E129Q) appears to be markedly lower (pKa approximately 4) than in the rhodopsin equivalent, Rho(E113Q) (pKa approximately 7), indicating distinct electrostatic environments at the retinal attachment site. Functional studies show that light-activated GCO(E129Q) decays more slowly and activates more G-protein than wild-type GCO (GCO(WT)). To identify the basis for these differences, we determined the structure of active GCO(E129Q) bound to a G-protein. We first developed a streamlined workflow to identify conditions that enhance GCO(E129Q) binding to G-proteins. This approach involved screening GCO(E129) binding to Galpha-CT resin (beads bearing tethered Galpha C-terminal peptides), followed by small-scale pull-down assays using 1D4 antibody beads to detect co-purification of GCO(E129) with a Venus-tagged mini-G-protein. Using the optimized conditions, we determined a 3.0-A cryo-EM structure of the GCO(E129)-G-protein complex. Comparison with rhodopsin and our recent 3.0-A structure of GCO(WT) reveals that the active-state architectures are largely similar, with several intriguing differences. Together, these results establish a generalizable, streamlined approach for biophysical and structural analysis of cone opsins and provide new mechanistic insight into the activation and signaling properties of GCO.