Data from: Epistatic interactions influence terrestrial-marine functional shifts in cetacean rhodopsin

Like many aquatic vertebrates, whales have blue-shifting spectral tuning substitutions in the dim-light visual pigment, rhodopsin, that are thought to increase photosensitivity in underwater environments. We have discovered that known spectral tuning substitutions also have surprising epistatic effects on another function of rhodopsin, the kinetic rates associated with light-activated intermediates. By using absorbance spectroscopy and fluorescence-based retinal release assays on heterologously expressed rhodopsin, we assessed both spectral and kinetic differences between cetaceans (killer whale) and terrestrial outgroups (hippo, bovine). Mutation experiments revealed that killer whale rhodopsin is unusually resilient to pleiotropic effects on retinal release from key blue-shifting substitutions (D83N and A292S), largely due to a surprisingly specific epistatic interaction between D83N and the background residue, S299. Ancestral sequence reconstruction indicated that S299 is an ancestral residue that predates the evolution of blue-shifting substitutions at the origins of Cetacea. Based on these results, we hypothesize that intramolecular epistasis helped to conserve rhodopsin’s kinetic properties while enabling blue-shifting spectral tuning substitutions as cetaceans adapted to aquatic environments. Trade-offs between different aspects of molecular function are rarely considered in protein evolution, but in cetacean and other vertebrate rhodopsins, may underlie multiple evolutionary scenarios for the selection of specific amino acid substitutions.

与诸多水生脊椎动物一样，鲸类的弱光视觉色素视紫红质（rhodopsin）中存在蓝移光谱调节替换（blue-shifting spectral tuning substitutions），这类替换被认为可提升水下环境中的光敏感性。我们发现，已知的光谱调节替换对视紫红质的另一项功能——与光激活中间体相关的动力学速率——还存在出人意料的上位效应（epistatic effects）。我们通过对异源表达的视紫红质开展吸收光谱学与基于荧光的视黄醛释放实验，评估了鲸类（虎鲸）与陆地外类群（河马、牛）视紫红质在光谱与动力学层面的差异。突变实验显示，虎鲸视紫红质对关键蓝移替换（D83N与A292S）带来的视黄醛释放多效性异常耐受，这在很大程度上源于D83N与背景残基S299之间一种出人意料且特异性极强的上位相互作用。祖先序列重建结果表明，S299是一处祖先残基，其出现早于鲸目起源时蓝移光谱调节替换的演化。基于上述结果，我们提出假说：在鲸类适应水生环境的过程中，分子内上位效应在允许蓝移光谱调节替换发生的同时，帮助保留了视紫红质的动力学特性。蛋白质演化研究中极少考虑分子功能不同维度间的权衡，但在鲸类与其他脊椎动物的视紫红质中，这类权衡或许是特定氨基酸替换受到选择的多种演化场景背后的潜在机制。