Sequential Rearrangement of Interhelical Networks Upon Rhodopsin Activation in Membranes: The Meta ll_a Conformational Substate

摘要

Photon absorption by rhodopsin is proposed to lead to an activation pathway that is described by the extended reaction scheme Meta I ? Meta II_a ? Meta II_b ? Meta II_bH~+, where Meta II_bH~+ is thought to be the conformational substate that activates the G protein transducin. Here we test this extended scheme for rhodopsin in a membrane bilayer environment by investigating lipid perturbation of the activation mechanism. We found that symmetric membrane lipids having two unsaturated acyl chains, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), selectively stabilize the Meta II_a substate in the above mechanism. By combining FTIR and UV-visible difference spectroscopy, we characterized the structural and functional changes involved in the transition to the Meta II_a intermediate, which links the inactive Meta I intermediate with the Meta II_b states formed by helix rearrangement. Besides the opening of the Schiff base ionic lock, the Meta II_a substate is characterized by an activation switch in a conserved water-mediated hydrogen-bonded network involving transmembrane helices H1/H2/H7, which is sensed by its key residue Asp83. On the other hand, movement of retinal toward H5 and its interaction with another interhelical H3/ H5 network mediated by His211 and Glu122 is absent in Meta II_a. The latter rearrangement takes place only in the subsequent transition to Meta II_b, which has been previously associated with movement of H6. Our results imply that activating structural changes in the H1/H2/H7 network are triggered by disruption of the Schiff base salt bridge and occur prior to other chromophore-induced changes in the H3/H5 network and the outward tilt of H6 in the activation process.