Temperature and pH dependence of the metarhodopsin I-Metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual waveleng th absorbance response of rod disk membrane (RDM) suspensions to a series o f small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a vari ety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been deter mined without a priori assumptions about these values, at temperatures betw een 0 and 15 degrees C and pHs from 6.2 to 8.2. Binding of G to MII in norm al RDM exhibits 1:1 stoichiometry (not cooperative), relatively weak, 2-4 x 10(4) M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6 , and a low thermal coefficient. The reported amount of active G remained c onstant even when its binding constant was reduced more than 10-fold at low pH. The method can readily be applied to the binding of MII to other prote ins or polypeptides that stabilize its conformation as MII. It appears capa ble of determining many of the essential physical constants of G protein co upled receptor interaction with immediate signaling partners and the effect of perturbation of environmental parameters on these constants.