Genetic and biophysical causes of historical protein evolution

我的实验室在分子进化和蛋白质的界面上开发和应用了新的方法 生物化学。我们在开发祖先蛋白重建(APR)方面发挥了主导作用 分子实验是决定性地识别遗传、生物物理和进化的有力策略 现存蛋白质进化新功能的机制。我们最近通过以下方式扩展了这种方法 进行第一次研究，使用大量蛋白质文库的深度突变扫描来表征 在祖先蛋白质周围的序列空间中的功能分布；这使我们能够比较 历史上可能采取的大量替代道路的轨迹，因此 深入了解职能优化、中性机会、上位主义和历史 在塑造蛋白质进化轨迹和结果方面的偶然性。在未来5年，我们将 进一步发展这些方法，并将其应用于两个主要问题领域： 1)复杂蛋白质特征的进化。许多蛋白质组装成特定的多聚体 并通过与变构效应器的结合进行功能调节。这些功能通常有许多 相互作用的残基，所以很难确定进化的遗传和生化机制 它们是在进化过程中起源的。我们将使用APR和脊椎动物血红蛋白作为理想的模型 系统来剖析特定的历史突变和随后的物理属性变化， 使这种必需的蛋白质从一个更简单的前体获得多聚化和变构。 2)功能、适合性和上位性影响的综合评估 在蛋白质长期进化过程中的替代。有针对性的实验表明，突变 经常上位性地修改同一蛋白质中其他突变的影响；理论和案例研究表明 这些依赖关系可能使进化路径和结果取决于偶发事件。有 然而，还没有全面的研究来表征在整个集合中上位的程度 历史上发生的替换，它们对进化过程的影响，或时间动力学 这些效应就是通过这种方式出现的。我们将使用高通量蛋白质文库分析祖先蛋白质来 测量发生在以下位置的所有替代的功能和适应性效应以及上位性交互作用 长期的、解决得很好的历史轨迹，决定了这些效应随时间的变化如何影响 进化过程，并分析潜在的生物物理机制如何调节这些影响。 与我们过去的工作一样，我们希望这些新战略能够产生强有力的新知识 关于推动蛋白质进化的机制和力量，以及我们的方法将被采用 以加深我们对其他蛋白质家族的进化和生化理解。

My lab develops and applies new approaches at the interface of molecular evolution and protein biochemistry . We have played a lead role in developing ancestral protein reconstruction (APR) with molecular experiments as a powerful strategy to decisively identify the genetic, biophysical, and evolutionary mechanisms by which extant proteins evolved new functions. We recently expanded this approach by conducting the first studies to use deep mutational scanning of massive protein libraries to characterize the distribution of functions in the sequence space around ancestral proteins; this allows us to compare the trajectories taken during history to the vast number of alternative paths that could have been taken, thus providing insight into the roles of functional optimization, neutral chance, epistasis, and historical contingency in shaping the trajectories and outcomes of protein evolution. In the next 5 years, we will further develop these approaches and apply them to two major problem areas: 1) Evolution of complex protein features. Many proteins assemble into specific multimeric complexes and are functionally regulated by binding to allosteric effectors. These features usually many interacting residues, so it has been difficult to identify the evolutionary genetic and biochemical mechanisms by which they originate during evolution. We will use APR and vertebrate hemoglobin as an ideal model system to dissect the particular historical mutations and consequent changes in physical properties that cause this essential protein to acquire multimerization and allostery from a simpler precursor. 2) Comprehensive assessment of the functional, fitness, and epistatic effects of substitutions during long-term protein evolution. Targeted experiments have shown that mutations often epistatically modify the effects of other mutations in the same protein; theory and case studies suggest these dependencies can make evolutionary paths and outcomes contingent on chance events. There have been no comprehensive studies, however, to characterize the extent of epistasis among the full set of substitutions that occurred during history, their effects on evolutionary processes, or the temporal dynamics by which these effects emerge. We will use high-throughput protein library assays on ancestral proteins to measure the functional and fitness effects and epistatic interactions of all substitution that occurred across long, well-resolved historical trajectories, determine how shifts in these effects over time affected evolutionary processes, and analyze how underlying biophysical mechanisms mediated these effects. As in our past work, we expect these new strategies to generate strongly supported new knowledge concerning the mechanisms and forces that drive protein evolution, and that our approaches will be adopted by other groups to deepen our evolutionary and biochemical understanding of other protein families.