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.

与多数水生脊椎动物类似，鲸类在弱光视觉色素视紫红质（rhodopsin）中存在蓝移光谱调谐替换（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，已知的光谱调谐替换还会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——产生出人意料的上位效应（epistatic effects）。本研究通过对异源表达（heterologously expressed）的视紫红质开展吸收光谱法（absorbance spectroscopy）检测与基于荧光的视黄醛释放检测（fluorescence-based retinal release assays），评估了鲸类（虎鲸）与陆生外类群（河马、牛）之间的光谱与动力学差异。突变实验表明，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效应（pleiotropic effects）具有异常强的耐受性，这主要源于D83N与背景残基S299之间一种出人意料的特异性上位相互作用。祖先序列重建（Ancestral sequence reconstruction）结果显示，S299是一类祖先残基，其出现早于鲸类起源时蓝移替换的演化过程。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，分子内上位性（intramolecular epistasis）在允许蓝移光谱调谐替换出现的同时，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取舍，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被选择的多种演化场景的核心成因。