Deep characterization of the sequence space and evolutionary trajectories of reconstructed ancestral proteins - Resubmission 01

我们首次提出了一个祖先蛋白质周围的序列空间的全面特征。这 工作将1)表征所有可能的突变和突变对功能的影响 蛋白质的全长和关键位点子集上所有可能的突变组合，2)说明了 函数在该多维序列空间中的分布将影响过程 蛋白质进化(分子进化的一个关键目标)，以及3)量化一整套主效和 转录因子DNA专一性的上位性遗传决定因素及其生化性质 原因-蛋白质生物化学和分子基因调控的重要目标。我们使用类固醇 激素受体DNA结合域是一种理想的模型系统，因为它具有很大的生物医学价值 重要；它在实验和系统发育上是容易处理的；它对不同的DNA靶标具有特异性 通过一个众所周知的进化过程，具有一系列已知的历史突变和生物物理 机械装置。这项拟议的工作将揭示为什么这一历史相对于许多其他突变发生 当蛋白质进化出新的特异性时，它可能会采取的轨迹。利用序列空间的映射 另一方面，我们将对祖先蛋白质应用特定的、复制的实验进化，将其放置在 在强选择下探索序列空间并进化出它在 历史演变。通过识别历史轨迹之间的共性和差异， 实验进化轨迹，以及序列空间中的许多其他可能的路径，我们将 深入了解偶然性和决定论在进化中的作用，并阐明 导致这些现象的潜在机械因素。具体问题包括：有多少种方式 有没有进化出DNA的特异性，有多少在选择和漂移下是可获得的？ 历史结果的演变是因为它是最理想的基因型，还是因为它是唯一的？ 可获得的基因分型，还是仅仅是偶然的？如果存在更优的基因类型，是什么阻止了进化 从他们那里得到的蛋白质？新的特性必须在多大程度上通过混杂演变 中间体，以及需要多少突变才能进化出新的特异性？我们还将描述 存在于不同物种的古代受体周围的序列空间和实验进化轨迹 这将揭示蛋白质的进化性和稳健性是如何在 由于上位作用突变而产生的进化时间。最后，通过充分刻画主位和上位位 蛋白质的DNA专一性的遗传决定因素，我们将确定常见的生物物理机制 DNA识别是分子生物学、生物化学、细胞生物学、 和发展。我们开发的方法和概念工具将适用于研究其他 转录因子和许多其他蛋白质家族的进化。

We propose the first comprehensive characterization of sequence space around an ancestral protein. This work will 1) characterize the effects on function of all possible mutations and pairs of mutations across the protein's entire length and of all possible combinations of mutations at a key subset of sites, 2) illuminate how the distribution of function through this multidimensional sequence space would have affected the processes of protein evolution (a key goal in molecular evolution), and 3) quantify the complete set of main-effect and epistatic genetic determinants of DNA specificity in a transcription factor and elucidate their biochemical causes – an important goal for protein biochemistry and molecular gene regulation. We use the steroid hormone receptor DNA-binding domain as an ideal model system, because it is of great biomedical importance; it is experimentally and phylogenetically tractable; and its specificity for DNA targets diversified through a well-understood evolutionary process, with a known set of historical mutations and biophysical mechanisms. The proposed work will reveal why this history occurred relative to the many other mutational trajectories the protein could have taken as it evolved its new specificity. With the map of sequence space in hand, we will then apply locus-specific, replicated experimental evolution to the ancestral protein, placing it under strong selection to explore sequence space and evolve the same novel specificity that it acquired during historical evolution. By identifying commonalities and differences among the historical trajectory, experimental evolution trajectories, and the many other possible pathways through sequence space, we will gain fundamental insight into the roles of contingency and determinism in evolution and illuminate underlying mechanistic factors that caused those phenomena. Specific questions include: how many ways were there to evolve the derived DNA specificity, and how many were accessible under selection and drift? Did the historical outcome evolve because it was the optimal genotype, because it was the best or only accessible genotype, or simply due to chance? If more optimal genotypes exist, what prevented the evolving protein from reaching them? To what extent must new specificities evolve through promiscuous intermediates, and how many mutations does it take to evolve a new specificity? We will also characterize sequence space and experimental evolutionary trajectories around ancient receptors that existed at different times during history; this will reveal how the protein's evolvability and robustness fluctuated over evolutionary time due to epistatically acting mutations. Finally, by fully characterizing the main and epistatic genetic determinants of the protein's DNA specificity, we will identify common biophysical mechanisms that underlie DNA recognition, contributing to an important goal in molecular biology, biochemistry, cell biology, and development. The methods and conceptual tools we develop will be applicable to studying other transcription factors and the evolution of many other protein families.