Monitoring Conformational Changes During Agonist Release from a GPCR in Real Time: Transmembrane Helix 6 Resets as All-Trans Retinal is Released from Rhodopsin

Abstract

The active metarhodopsin II (MII) conformation of rhodopsin loses activity when it releases its agonist, all-trans retinal, and decays to the inactive apoprotein, opsin. Here, we studied how the conformational change and retinal release are linked. In our experiments, we used tryptophan induced quenching of fluorescence (TrIQ) to observe movement of transmembrane helix 6 (TM6) through an attached fluorescent probe, while simultaneously measuring intrinsic tryptophan fluorescence to monitor retinal release. The data show TM6 moves from its position in the active structure back into the helical bundle in opsin (indicative of the inactive conformation) with a rate matching that of retinal exit; both processes exhibit a t1/2 of ∼15 minutes at 20°C in 0.05% dodecyl maltoside at pH 6.0. We further tested the correlation of these events in two ways. First, we carried out an Arrhenius analysis and determined near identical activation energies for TM6 resetting and retinal release (∼23 kcal/mol). Additionally, we tested the effect of altering the rate of retinal release on the rate of the conformational change. The release process was slowed by introducing a mutation to the Schiff base counterion, E113Q. When retinal exit was slowed, the rate of TM6 resetting decelerated to the same degree. Likewise, accelerating retinal release by using hydroxylamine to hydrolyze the Schiff base linkage caused a corresponding acceleration of the rate of TM6 movements. With these tools in hand, we have begun investigating the dynamics and energetic of other movements in the protein, with the goal of constructing a more comprehensive model for receptor deactivation following light-activation, as well as testing how mutations (such as retinitis pigmentosa), small molecules, and other proteins can effect these events.