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

十亿年进化轨迹上的适应度效应和上位性综合分析

• 批准号: 10212033
• 负责人: DANIEL N BOLON
• 金额: $ 56.58万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10212033
• 关键词: Accidents; Address; Affect; Amino Acid Sequence; Amino Acids; Animals; Architecture; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Biophysical Process; Biophysics; Catalysis; Cells; Chemicals; Client; Complement; Data; Disease; Engineering; Eukaryota; Eukaryotic Cell; Event; Evolution; Future; Gene Proteins; Genetic; Genetic Epistasis; Genetic Variation; Health; Human; Knowledge; Lead; Libraries; Ligand Binding; Measures; Mediating; Methods; Molecular Chaperones; Molecular Conformation; Molecular Evolution; Mutation; Outcome; Pattern; Phylogenetic Analysis; Phylogeny; Play; Process; Property; Protein Engineering; Proteins; Public Health; Recording of previous events; Resolution; Role; Saccharomyces cerevisiae; Sampling; Signal Transduction; Site; Structure; Structure-Activity Relationship; System; Techniques; Technology; Time; Work; Yeasts; biological systems; chemical genetics; experimental analysis; experimental study; fitness; fungus; gene therapy; improved; insight; mutant; mutation screening; new technology; prevent; protein folding; reconstruction; sample fixation; tool

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.

原则上，蛋白质内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；它们还可以用看似最佳的残基来巩固蛋白质，但 都是历史的偶然。上位实际上影响轨迹和结果的程度 分子进化取决于历史上发生的替代的适应性效应 比较它们在时间上早或晚的潜在影响以及相互作用的时间顺序 发生了替换。深度突变扫描研究揭示了普遍的上位性 大量可能的突变，但没有研究直接评估适应性如何影响 随着蛋白质的进化，历史上发生的取代随着时间的推移而发生变化。我们将表演 首次全面实验分析所有氨基酸的健身效果 表明蛋白质在长期系统发育轨迹中进化，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刻。这些数据将 在蛋白质系统发育的有序时间背景下进行分析，并补充生化 实验，能够深入表征上位性的原因和后果， 一种必需蛋白质数十亿年历史中的偶然性和巩固性。 我们的模型系统非常适合此目的。 Hsp90，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、细胞信号传导和多种疾病中发挥着关键作用。 强大的系统发育信号可以自信地重建数十亿年的进化历史 Hsp90 的蛋白质序列从动物、真菌和相关原生生物的最后共同祖先到 现在的酿酒酵母。我们将生成包含每个的目标蛋白质库 在此系统发育轨迹中单独且以各种可能的方式发生的祖先和派生状态 一对，在沿着轨迹的所有 30 个重建的祖先蛋白质的背景中。使用高 在酵母中进行分辨率批量竞争测定，我们将精确测量选择系数和上位系数 相互作用并量化这些属性如何随时间变化。这将揭示健身效果和 每个替换在发生的大致时间之间的相互作用，以及效果和 如果它在更早的任何时刻发生（或恢复到原始状态），它会发生的相互作用 稍后在轨迹中。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的方式提供深入的新见解 影响它们的演化过程，也受其影响；这也将加强我们的 了解生物学必需蛋白质中的序列-结构-功能关系。