Biophysical and structural analysis of human green cone opsin

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 ≈4) than in the rhodopsin equivalent, Rho_E113Q (pKa ≈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 Gα-CT resin (beads bearing tethered Gα 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-Å cryo-EM structure of the GCO_E129-G-protein complex. Comparison with rhodopsin and our recent 3.0-Å 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.