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

摘要

Photon absorption by rhodopsin is proposed to lead to an activation pathway that is describednby the extended reaction scheme Meta I h Meta IIa h Meta IIb h Meta IIbH+, where Meta IIbH+ is thoughtnto be the conformational substate that activates the G protein transducin. Here we test this extended schemenfor rhodopsin in a membrane bilayer environment by investigating lipid perturbation of the activationnmechanism. We found that symmetric membrane lipids having two unsaturated acyl chains, such as 1,2-ndioleoyl-sn-glycero-3-phosphocholine (DOPC), selectively stabilize the Meta IIa substate in the abovenmechanism. By combining FTIR and UV-visible difference spectroscopy, we characterized the structuralnand functional changes involved in the transition to the Meta IIa intermediate, which links the inactive MetanI intermediate with the Meta IIb states formed by helix rearrangement. Besides the opening of the Schiffnbase ionic lock, the Meta IIa substate is characterized by an activation switch in a conserved water-mediatednhydrogen-bonded network involving transmembrane helices H1/H2/H7, which is sensed by its key residuenAsp83. On the other hand, movement of retinal toward H5 and its interaction with another interhelical H3H5 network mediated by His211 and Glu122 is absent in Meta IIa. The latter rearrangement takes placenonly in the subsequent transition to Meta IIb, which has been previously associated with movement of H6.nOur results imply that activating structural changes in the H1/H2/H7 network are triggered by disruption ofnthe Schiff base salt bridge and occur prior to other chromophore-induced changes in the H3/H5 networknand the outward tilt of H6 in the activation process.