Assays for activation of opsin by all-trans-retinal

The data collected with the techniques discussed in this chapter suggest significant differences between the active conformation(s) of the opsin/atr complex, which are reversibly formed in the dark, and the active conformation (R*) of the meta-II photoproduct. First, there is good evidence for noncovalent opsin/atr complexes with considerable activity (although covalent binding of atr is found in mutant opsins. Even more intriguing, all-trans-retinal in an amount that saturates the activity of the opsin/atr complex toward Gt does not measurably inhibit the access of 11-cis-retinal to the light-sensitive binding site during regeneration (Fig. 2C). On the other hand, forced protonation at or near Glu-134 appears to be an integral mechanism for both the meta-II and the opsin-like activities (Fig. 4). Thus, it is not inconceivable that these two activities of the receptor arise from two fundamentally different conformations, one meta-II-like and one opsin-like. They would be similar with respect to the Gt (or RK) protein-protein interaction but different in their mode of retinal-protein interaction.