'Mutational pleiotropy, epistasis, and the adaptive evolution of hemoglobin funct

描述(由申请人提供)： 进化遗传学中的一个基本问题涉及突变多效性和上位性在塑造蛋白质进化轨迹中的作用。解决这个问题的一个强有力的方法包括使用定点突变来探索序列空间中实验定义的区域中蛋白质功能的突变图景。在这里，我们描述了一项计划，以评估在哺乳动物血红蛋白(Hb)的适应性功能进化过程中，多效性权衡和上位性相互作用如何影响替代突变途径的选择性可获得性。使用祖先蛋白质复活和基于定点突变的组合蛋白质工程方法，我们将检查顺序突变步骤在所有可能的途径中的结构和功能影响，这些途径导致适应环境低氧的哺乳动物Hb-O2亲和力增强的进化。为了评估多效性和上位性对适应性蛋白质进化的影响，我们将在进化出不同Hb-O2亲和力的哺乳动物类群对之间的几个不同水平上考察Hb功能适应性变化的分子基础。该实验方法结合了对重组表达的HBs中特定突变的影响的生化和生物物理检查。本项目的主要创新之一是我们开发了一种表达载体系统，使我们能够在大肠杆菌宿主细胞中合成重组Hb。本研究旨在实现以下目标：(1)确定导致Hb功能进化变化的特定突变，并确定加性效应和上位性效应的相对贡献，(2)识别和表征导致观察到的多效性效应和上位性相互作用的生化/生物物理机制。实现这两个目标将揭示有助于蛋白质功能适应性修饰的特定突变，并将阐明多效性和上位性影响进化路径选择性可及性的特定生化/生物物理机制。通过使用Hb作为模型分子，我们可以利用关于结构-功能关系的极其丰富的信息来源来深入了解机理。通过直接测量致病突变的结构和功能影响，我们的实验结果将为有关分子适应和蛋白质进化机制的基本问题提供答案。

DESCRIPTION (provided by applicant): A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. A powerful means of addressing this question involves the use of site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here we describe a plan to evaluate how pleiotropic trade-offs and epistatic interactions influence the selective accessibility of alternative mutational pathways during the adaptive functional evolution of mammalian hemoglobin (Hb). Using ancestral protein resurrection in conjunction with a combinatorial protein-engineering approach based on site-directed mutagenesis, we will examine the structural and functional effects of sequential mutational steps in all possible pathways that lead to the evolution of an increased Hb-O2 affinity in mammals that have adapted to environmental hypoxia. To evaluate the influence of pleiotropy and epistasis on adaptive protein evolution, we will examine the molecular basis of adaptive changes in Hb function at several different levels of divergence between pairs of mammalian taxa that have evolved different Hb-O2 affinities. The experimental approach integrates biochemical and biophysical examinations of the effects of specific mutations in recombinantly expressed Hbs. One of the primary innovations of this project is that we have developed an expression vector system that allows us to synthesize recombinant Hb in E. coli host cells. The research is designed to accomplish the following aims: (1) Identify the specific mutations that contribute to evolutionary changes in Hb function, and determine the relative contributions of additive and epistatic effects, and (2) Identify and characterize the biochemical/biophysical mechanisms responsible for observed pleiotropic effects and epistatic interactions. Accomplishing these two aims will reveal the specific mutations that have contributed to adaptive modifications of protein function, and will elucidate the specific biochemical/biophysical mechanisms by which pleiotropy and epistasis affect the selective accessibility of evolutionary pathways. By using Hb as a model molecule, we can leverage extremely rich sources of information about structure-function relationships to gain insights into mechanism. By directly measuring the structural and functional effects of causative mutations, our experimental results will provide answers to fundamental questions about molecular adaptation and mechanisms of protein evolution.