This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The visual photoreceptor rhodopsin is a prototypical seven transmembrane helical G-protein coupled receptor (GPCR) that is responsible for light detection under dim light conditions in the scotopic visual system. The detailed structure of the binding site of the covalently attached 11-cis retinylidene chromophore has been determined, confirming data from functional characterization. However, the entry pathway in the opsin apoprotein for the hydrophobic ligand 11-cis retinal is unknown. Here we show that the primary ligand entry site is located in the transmembrane region between helices 5 and 6, suggesting a hydrophobic binding and unbinding pathway for the ligand through the bilayer interior. In addition to the primary entry site, a particular residue located in the second extracellular loop has a significant effect on chromophore regeneration rate, probably through an indirect effect on the helix 5-helix 6 pathway. Multi-nanosecond free energy calculations of ligand binding in a membrane model of rhodopsin were performed based on reversible MD simulations using umbrella potentials. The weighted histogram analysis method (WHAM) was employed to eliminate the effects of the biasing umbrella potentials and to recover potential of mean force (PMF) free energy profiles. The results are compared with irreversible steering molecular dynamics (SMD) simulations applying the Jarzynski nonequilibrium equality, and with multi-configurational thermodynamic integration (MCTI). The free energy profiles along the proposed entry pathway support the ligand gating mechanism suggested by complementary tryptophan fluorescence resonance energy transfer (FRET) experiments on site-directed mutant and wild type receptors. These show four orders of magnitude changes of all-trans retinal release and 11-cis retinal binding kinetics depending on side chains in the entry pathway. The results suggest high energy barriers for ligand entry and exit. Hydrogen bond donor side chains along the pathway lead to kinetic trapping of retinal. Models of members of the family of visual pigments based on sequence homology suggest that these findings could explain the rapid recovery of the receptors following bleaching in the photopic system for bright-light and color vision.
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