Comprehensive analysis of fitness effects and epistasis along a billion-year evolutionary trajectory

原则上，蛋白质内部的上位相互作用可以决定进化的路径和结果。 取决于偶然事件;它们也可以用似乎是最佳的残基来巩固蛋白质， 都是历史的偶然上位性在多大程度上影响了 分子进化取决于取代在历史上发生时的适合度效应 与它们在时间上或早或晚的潜在影响以及相互作用的时间顺序相比， 发生了替换。深入的突变扫描研究揭示了 大量的可能的突变，但没有研究直接评估如何健身的影响， 在历史上发生的替换随着蛋白质的进化而改变。我们将执行 第一次全面实验分析了所有氨基酸的健身效果 在长期系统发育轨迹中，蛋白质中进化的状态，无论是在 它们发生的时间，以及它们是否发生在历史上的其他时间点。这些数据将 在蛋白质发生的有序时间背景下进行分析，并辅以生物化学 实验，使上位的原因和后果的深刻特点， 偶然性和在一个必需蛋白质的数十亿年历史中的巩固。 我们的模型系统是实现这一目标的理想选择。热休克蛋白90，所有真核生物中必不可少的分子伴侣- 耳细胞在蛋白质折叠和成熟、细胞信号传导和广泛的疾病中起关键作用。 强大的系统发育信号使我们能够有信心地重建数十亿年的进化历史， 从动物、真菌和相关原生生物的最后共同祖先到 现在的酿酒酵母。我们将生成包含所有蛋白质的靶向蛋白质文库， 在这个系统发育轨迹中发生的祖先和衍生状态，单独地和在每一个可能的情况下， 在所有30个重建的祖先蛋白质的背景下，沿着轨迹沿着。使用高- 分辨率散装竞争试验在酵母中，我们将精确地测量选择系数和上位性 相互作用，并量化这些属性如何随时间变化。这将揭示健身效果， 每一种替代在其发生的大致时间的相互作用，以及其影响和 如果它发生在更早的任何时候（或恢复到祖先状态）， 后来在轨道上。我们还将应用生物物理和结构技术来阐明 驱动这些遗传和进化现象的潜在生化机制。这项工作 将为蛋白质的遗传和物理结构的方式提供新的见解， 影响，并受到影响，他们演变的过程;它也将加强我们的 理解生物学必需蛋白质中的序列-结构-功能关系。

Epistatic interactions within proteins can, in principle, make the paths and outcomes of evolution contingent on chance events; they can also entrench proteins with residues that appear to be optimal but are accidents of history. The extent to which epistasis actually affected the trajectory and outcomes of molecular evolution depends on the fitness effects of substitutions when they occurred in history compared to their potential effects earlier or later in time and on the temporal order in which interacting substitutions occurred. Deep mutational scanning studies have revealed pervasive epistasis among the huge number of possible mutations, but no studies have directly assessed how the fitness effects of substitutions that happened during history changed over time as the protein evolved. We will perform the first comprehensive experimental analysis of the fitness effects of all amino acid states that evolved in a protein during a long-term phylogenetic trajectory, both at the time they occurred and if they had occurred at other points in history. These data will be analyzed in the ordered temporal context of the protein's phylogeny and supplemented with biochemical experiments, enabling a deep characterization of the causes and consequences of epistasis, contingency and entrenchment across the billion-year history of an essential protein. Our model system is ideal for this purpose. Hsp90, the essential molecular chaperone in all eukary- otic cells, plays key roles in protein folding and maturation, cell signaling, and a wide range of diseases. Strong phylogenetic signal allows confident reconstruction of the billion-year evolutionary history of Hsp90's protein sequence from the last common ancestor of animals, fungi and related protists to present-day Saccharomyces cerevisiae. We will generate targeted protein libraries containing every ancestral and derived state that occurred during this phylogenetic trajectory, singly and in every possible pair, in the background of all 30 reconstructed ancestral proteins along the trajectory. Using a high- resolution bulk competition assay in yeast, we will precisely measure selection coefficients and epistatic interactions and quantify how these properties changed over time. This will reveal the fitness effects and interactions of every substitution at the approximate time it occurred, as well as the effects and interactions it would have had if it happened (or reverted to the ancestral state) at any point earlier or later during the trajectory. We will also apply biophysical and structural techniques to elucidate the underlying biochemical mechanisms that drove these genetic and evolutionary phenomena. This work will provide deep new insight into the ways in which proteins' genetic and physical architecture influences, and is influenced by, the processes by which they evolve; it will also strengthen our understanding of sequence-structure-function relationships in a biologically essential protein.