Rhodopsin is the photoreceptor responsible for dim light vision, and the only member of the superfamily of G protein-coupled receptors (GPCRs) for which high-resolution crystal structures are available. Understanding rhodopsin activation has importance extending to the much larger superfamily of GPCRs which is targeted by roughly half of all current drugs. Rhodopsin activation begins with the absorption of a photon by the covalently bound chromophore retinal, which results in isomerization of 11-cis- to trans-retinylidene. Activation proceeds through a series of intermediates to the Meta-I (MI) state, which is in rapid exchange with the active form, Meta-II (MII). Hydrogen bonding of internal water is known to increase during the formation of MI, and the sensitivity of the MI/MII equilibrium to osmotic stress indicates a release of water from the protein upon formation of MII. However, despite a wide appreciation of the ubiquitous involvement of water in protein function, a specific role for water in rhodopsin activation has been a matter of conjecture.

I'll present our approach to the activation of Rhodopsin using large scale molecular dynamics and NMR. We combine NMR experiments with over 5 microseconds of all-atom molecular dynamics simulation of rhodopsin to propose that an increase in internal hydration has a direct role in defining the MI state. Two distint protonation states of Glu-181 were investigated. The simulation shows the formation of a rhodopsin structure, consistent with MI, where water channels connect the intradiscal space to the cytoplasm through the ligand binding domain and conserved D(E)RY and NPxxY motifs. In support of the hydration topography predicted by simulation, we present NMR evidence showing increased magnetization transfer from water via rhodopsin to the hydrophobic core of the lipid matrix upon MI formation. Our results provide a basis for a direct role of internal water in rhodopsin's activation mechanism.