Lipid-Dependent Ensemble of Conformational Substates in Rhodopsin Activation Studied by FTIR and UV-Visible Spectroscopy

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G protein-coupled receptors (GPCRs) transmit signals across lipid membranes where rhodopsin is an important prototype. Here we address the role of soft matter in GPCR function for this class of integral membrane proteins [1,2]. Our hypothesis is the process involves an ensemble of activated states affected by membrane protein-lipid bilayer interactions [1]. Rhodopsin was investigated in lipid membranes (native disk membranes, POPC, DOPC) by Fourier transform infrared (FTIR) and UV-visible spectroscopy. Application of different spectroscopic methods together with pH titration at various temperatures allowed us to identify distributions of states in the MI-to-MII activating equilibrium and determine equilibrium constants and thermodynamic parameters of the transitions. Analysis of pH titration curves indicated that more than one activated state exists [3] supporting the concept of rhodopsin as a dynamically activated receptor. Furthermore, temperature changes had a crucial effect on rhodopsin activation enabling coexistence and simultaneous observation of multiple inactive and active states. Variation of the lipid composition indicated the POPC bilayer noticeably shifted the equilibrium to the inactive MI state [1] while the DOPC behavior was similar to native membranes. To improve data reduction, we analyzed the MI-to-MII equilibrium for the whole range of FTIR spectra where changes were observed between the MI and MII states. The results show the distribution of pKa and alkaline end-point values for all temperatures and different lipid compositions, indicating that activation of rhodopsin yields a conformational ensemble (collectively designated as MII state). We hypothesize that collective large-scale helical fluctuations in the metarhodopsin equilibrium are coupled to activation of transducin in visual signaling. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] U. Chawla et al. (2020) Angew. Chem. Int. doi.org/10.1002/anie.202003342. [3] M. Mahalingam et al. (2008) PNAS 105, 17795-17800.