Hydrophobic Mismatch Modulates the Kinetics of G Protein Binding and Receptor Conformation Change

A recent study demonstrated that rhodopsin in 14:0,14:1 PC (hydrophobic thickness = 21.4 angstroms) has 8% less helical content than rhodopsin in 18:0,18:1 PC (hydrophobic thickness = 29.2 angstroms). We investigated the effects of hydrophobic thickness on rates of MII and transducin (Gt) binding and phospholipid dynamics and packing order. Purified rhodopsin was reconstituted in liposomes consisting of 14:0,14:1 PC and 18:0,18:1 PC at a lipid:protein ratio of 200. Kinetics of MII formation and Gt binding where measured with flash photolysis, and membrane properties were assessed via time-resolved fluorescence anisotropy decay measurements of diphenylhexatriene (DPH). MII formation was analyzed in terms of the square model. Analysis of the DPH anisotropy decay data in terms of the P2-P4 model showed that lipid dynamics and fractional free volume (fv) were higher in the 14:0,14:1 PC membrane. Previous studies demonstrate that an increase in these two bilayer properties is associated with enhanced MII formation, but in this case equilibrium concentration of MII and the rate of MII formation was higher in 18:0,18:1 PC at all temperatures. Analysis of the temperature dependence of the kinetics in terms of reaction rate theory showed this was chiefly due to increased activation enthalpy for two of the forward rates; Lumi to MI-380 and MI-480 to MII. At 30 oC in 18:0,18:1 PC, MII formed with a time constant of 0.69 ms and the MII-Gt complex formed in 0.79 ms. This near-immediate formation of MII-Gt following MII is similar to what is observed for rhodopsin in the native membrane. In 14:0,14:1 PC MII formed in 5.43 ms and MII-G complex formed in 37.6 ms. This long lag between appearance of MII and Gt binding demonstrates that hydrophobic mismatch has deleterious consequences for G protein-coupled signaling.