Rhodopsin is a prototypical member of G protein-coupled receptors (GPCRs) whose activation and conformational changes can be studied using UV-visible and FTIR spectroscopy. Our hypothesis is that the photoreceptor activation is influenced by the soft matter (water and lipids) and proteins in a membrane environment. Hydration primarily influences metarhodopsin-II (MII) formation rather than deformation of the lipid bilayer. Our goal was to develop an accurate and simple experiment using UV-visible spectroscopy to investigate the influence of the soft matter and G-protein-derived peptide on the activation equilibrium of metarhodopsin-I (MI) and MII. Understanding the influence of  thermodynamic parameters upon the rhodopsin activation is an important objective. Surprisingly, we observed that the light-dark difference UV-visible spectra were significantly time-dependent close to ambient temperature, which required monitoring of the spectral changes due to deprotonation of the retinal Schiff base in the active MII state. The time-dependent changes were fitted to an exponential decay to obtain the initial value and as well as the change in rate constant. The MII  protonated Schiff base (PSB) fraction was calculated and plotted as a function of pH at various time points. Titration curves of initial values and partial decay of the MII fraction displayed similar tendencies consistent with our previous observations. Furthermore, acid denaturation assays were conducted to address the question of whether the changes are due to the hydrolysis of the Schiff base linkage. Alternatively, the observed time-dependent changes may be due to generation of the storage form metarhodopsin-III (MIII), which is yet to be investigated. The effects of the lipid to protein ratio and the G-protein derived peptides upon rhodopsin activation are currently under investigation.

Rhodopsin activation is influenced by membrane soft matter

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