Rhodopsin is a dynamically active receptor in lipid membranes

G-protein-coupled receptors transmit signals across cellular membranes and regulate multiple biological functions with rhodopsin as an important prototype. Here we investigated the distribution of substates in rhodopsin activation unavailable by X-ray crystallography due to use of cryogenic temperatures and absence of the membrane environment [1]. Our hypothesis was that activation involves dynamic allostery whereby an ensemble of states is affected by the membrane soft matter (lipids and water) as well as pH and temperature [1-3]. We investigated rhodopsin in native retinal disk membranes and POPC or DOPC lipids using FTIR and UV-visible spectroscopy. By observing the pH dependence of the activating transition for various spectroscopic bands, we determined the distributions of states upon photoactivation. Data reduction and analysis of pH titration curves revealed equilibrium constants and thermodynamic parameters for the transition from inactive Meta-I to the active Meta-II state. Furthermore, we calculated the distribution of pKa and alkaline end-point values at all wave numbers of FTIR spectra where intensities change upon activation. We discovered that temperature not only shifts the rhodopsin equilibrium to the active state, but also enables coexistence of multiple inactive and active states. Our data support the concept of a dynamically active receptor, where the equilibrium of substates facilitates the high reaction rate for the next vision step (transducin activation). The calculated enthalpy and entropy values suggest partial unfolding of the receptor when forming of the active Meta-II state. Our results are consistent with the idea of activated rhodopsin as a conformational ensemble of states representing different tiers of the energy landscape, which are partially determined by an enthalpy-entropy folding funnel.