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

原则上，蛋白质内部的上位性相互作用可以决定进化的路径和结果 视偶然事件而定；它们也可以用看似最理想的残基来巩固蛋白质，但 都是历史的巧合。上位性实际影响运动轨迹和结果的程度 分子进化取决于取代在历史上发生时的适合度效应。 相对于它们在时间上更早或更晚的潜在影响，以及在相互作用的时间顺序上 发生了替换。深度突变扫描研究显示，上位性基因在 大量可能的突变，但还没有研究直接评估 随着蛋白质的进化，历史上发生的替换会随着时间的推移而改变。我们将表演 首次对所有氨基酸的健身效果进行全面的实验分析 在长期的系统发育轨迹中进化成蛋白质的状态，无论是在 它们发生的时间，以及它们是否发生在历史的其他时间点。这些数据将是 在蛋白质系统发展的有序时间背景下进行分析，并补充生化 实验，使得能够对上位主义的原因和后果进行深入的表征， 一种必需蛋白质在数十亿年的历史上的偶然性和根深蒂固。 我们的模型系统非常适合这一目的。HSP90，所有真核生物中必不可少的分子伴侣- 听觉细胞在蛋白质折叠和成熟、细胞信号转导以及多种疾病中起着关键作用。 强大的系统发育信号使人们能够自信地重建人类数十亿年的进化史 动物、真菌及相关原生生物最后一个共同祖先的热休克蛋白90‘S蛋白序列 今天的酿酒酵母。我们将生成目标蛋白质文库，其中包含 在这个系统发展轨迹中出现的祖先和派生状态，在任何可能的情况下 一对，在所有30个重建的祖先蛋白的背景中沿着轨迹。使用高- 在酵母菌的拆分竞争试验中，我们将精确测量选择系数和上位性 并量化这些属性随时间的变化情况。这将揭示健身的效果和 每一替代在其发生的大致时间的相互作用，以及影响和 如果它在更早或更早的任何时候发生(或恢复到祖先状态)，它会有相互作用 后来在弹道上。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。这部作品 将为蛋白质的遗传和物理结构提供深刻的新见解 影响它们发展的过程，并受其影响；它还将加强我们的 理解生物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.