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

突变多效性、上位性和血红蛋白功能的适应性进化

• 批准号: 8902245
• 负责人: Jay Storz
• 金额: $ 34.69万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-22 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8902245
• 关键词: Accounting; Address; Affect; Affinity; Amino Acids; Biochemical; Biophysical Process; Cells; Dependence; Escherichia coli; Evolution; Genetic; Genetic Epistasis; Goals; Health; Hemoglobin; Hypoxia; Lead; Light; Mammals; Measures; Mediating; Medicine; Modeling; Modification; Molecular; Mutation; Pathology; Pathway interactions; Phenotype; Population; Prevalence; Probability; Property; Protein Engineering; Proteins; Recombinants; Relative (related person); Research; Research Project Grants; Role; Shapes; Sister; Site; Site-Directed Mutagenesis; Source; Structure; Structure-Activity Relationship; Suppressor Mutations; System; Taxon; Transfusion; base; combinatorial; design; expression vector; innovation; insight; mutant; oxygen transport; pleiotropism; protein function; protein structure function; public health relevance; sample fixation

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.

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毒在塑造蛋白质进化轨迹中的作用。解决这个问题的一种强大方法涉及使用定位的诱变来探索实验定义的序列空间区域中蛋白质功能的突变景观。在这里，我们描述了一项计划，以评估多效性权衡和上皮相互作用如何影响哺乳动物血红蛋白（HB）自适应功能进化期间替代突变途径的选择性可访问性。使用基于位置定向诱变的组合蛋白质工程方法结合使用祖先蛋白质复活，我们将研究在所有可能导致HB-O2亲和力在已经适应环境低氧的乳腺癌中增加HB-O2亲和力进化的顺序突变步骤的结构和功能效应。为了评估多效性和上毒对自适应蛋白进化的影响，我们将研究Hb功能自适应变化的分子基础，在成对的哺乳动物分类群之间的几个不同水平的差异水平上，这些哺乳动物分类群已经进化出不同的HB-O2亲和力。实验方法整合了重组表达的HBS特异性突变的影响的生化和生物物理检查。该项目的主要创新之一是我们开发了一个表达矢量系统，该系统使我们能够合成大肠杆菌宿主细胞中的重组HB。该研究旨在实现以下目的：（1）确定有助于HB功能进化变化的特定突变，并确定添加剂和上皮效应的相对贡献，以及（2）识别和表征导致观察到的Pleiotiotropic效应和上皮相互作用的生物化学/生物物质机制。实现这两个目标将揭示有助于蛋白质功能自适应修饰的特定突变，并将阐明多效性和上症会影响进化途径的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，我们可以利用有关结构功能关系关系的极丰富的信息来源，以了解机制。通过直接测量因果突变的结构和功能效应，我们的实验结果将为有关分子适应性和蛋白质进化机制的基本问题提供答案。