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

Abstract

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 b H + ; where Meta II b H + 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 re-arrangement. 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.

Solid-state 2 H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

“…According to our molecular dynamics simulations (45), helices H1-H4 make up the TM core of the receptor (4,17). The rhodopsin core is stabilized by two ionic locks involving the PSB and the E(D)RY motif with their counterions, plus three hydrogen-bonded networks and several conserved microdomains (27). Light-induced retinal isomerization destabilizes the inactive receptor conformation (17,18).…”

“…Activation entails large-scale movements of helices H5 and H6 together with H7 (19,23), exposing transducin (G t ) recognition sites on the C2 and C3 cytoplasmic loops (22). A current picture (17,27,44) is that photon absorption leads to the major reactions (17,44):…”

“…In Fig. 5 we combine the 2 H NMR relaxation data with our FTIR studies (17,27) and other measurements (18,19,23,40,45) of μs-ms functional protein movements. In the Meta I state, the C9-Me acts as a hinge point that prevents roll of the entire ligand.…”

“…Spectroscopic approaches including spin-label EPR (23), 13 C NMR (18,(24)(25)(26), and Fourier transform infrared (FTIR) (17,27) studies are thus needed to establish the activation mechanism of the photoreceptor as it underlies the visual process. Solid-state NMR spectroscopy (28) is particularly important, as it gives knowledge of both protein structure and dynamics in a membrane lipid environment (29).…”

Intramolecular Interactions That Induce Helical Rearrangement upon Rhodopsin Activation

“…Although the helical arrangement of Meta-II a is comparable with those of the dark state or its precursors, the intramolecular interactions in Meta-II a should be altered, and these alterations should readily induce significant helical rearrangements. Thus, identification of the intramolecular interactions in Meta-II a is essential for understanding the mechanism by which photoactivated rhodopsin adopts the active conformation (18).…”