Insights into rhodopsin molecular evolution from mice with “humanized” Phe-88 to Leu substitution

The function of rod photoreceptors as dim light photon detectors depends critically on the molecular properties of their visual pigment, rhodopsin. The structure of rhodopsin has evolved under selective pressure to light conditions of different spectral composition and overall intensity. One notable example is the switch of mammalian species from nocturnal to diurnal environments. Comparison of the rhodopsins of the nocturnal mouse and the diurnal human reveals high sequence similarity, with only 18 distinct amino acids. Here, we examined the role of one of these, mouse phenylalanine (F) vs. human leucine (L) at position 88, in modulating the molecular properties of rhodopsin and the function of rods by generating F88L rhodopsin knock-in mouse. Our detailed in vitro analysis of the physicochemical properties of this mutant F88L rhodopsin showed a higher conformational stability and more efficient chromophore regeneration compared to the WT mouse pigment. We also found that the decay of metarhodopsin II in the F88L mutant occurred significantly faster than in the WT. However, despite these molecular changes, the visual function of knock-in mutant mice carrying the F88L mutation was not significantly altered by this amino acid change. These findings demonstrate the role of the F88L evolutionary switch in enhancing the stability and regeneration of rhodopsin towards visual function in diurnal human rods over nocturnal mouse rods. Our results provide new insights into the molecular evolution of rhodopsin in vertebrates.