Leveraging Adaptive Evolution and High-Throughput Techniques to Dissect the Link Between Biochemical Function and Fitness

利用适应性进化和高通量技术来剖析生化功能与健康之间的联系

• 批准号: 10704076
• 负责人: Margaux Pinney
• 金额: $ 40.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-13 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10704076
• 关键词: Active Sites; Address; Affect; Amino Acid Sequence; Antiviral Agents; Appointment; Area; Award; Bar Codes; Basic Science; Benchmarking; Biochemical; Biochemistry; Biological; Biological Assay; Biological Markers; Biology; Biophysics; Bioremediations; Cell Therapy; Cells; Chemicals; Chemistry; Collaborations; Communities; Complex; Data; Data Set; Dedications; Discipline; Doctor of Philosophy; Environment; Enzymes; Evolution; Faculty; Future; Goals; HRK gene; In Vitro; Individual; Industry; Institution; Investigation; Knowledge; Laws; Lead; Learning; Libraries; Life; Link; Maps; Measurement; Measures; Medicine; Mentors; Mentorship; Metabolic Diseases; Methods; Modeling; Molecular; Molecular Evolution; Mutation; Organism; Pathogenesis; Pharmaceutical Preparations; Physics; Positioning Attribute; Postdoctoral Fellow; Predisposition; Property; Proteins; Publications; Regulation; Research; Research Proposals; Specific qualifier value; Specificity; Structure; Techniques; Technology; Temperature; Testing; Therapeutic; Translating; Translational Research; Variant; Virulence; Work; anti-cancer; antimicrobial; biochemical evolution; catalyst; chemical reaction; computing resources; design; enzyme mechanism; experience; faculty mentor; fitness; frontier; genetic variant; graduate student; high throughput technology; human pathogen; improved; in vivo; interest; macromolecule; microfluidic technology; next generation; novel; precision medicine; pressure; protein function; recruit; response; senior faculty; tool; undergraduate student

PROJECT SUMMARY/ABSTRACT Enzymes are the primary functional molecules in cells, providing enormous rate enhancements, specificity and regulation to the diverse chemical reactions that are necessary for life. Enzymes, like all biological macromolecules, are the products of evolution: all enzymes have evolved to operate within the complex environment of the organism/cell in specific environmental niches(s). Thus, an understanding of enzyme function and evolution is fundamental to biology. Enzymes also have tremendous potential in medicine (e.g., as targets for anti-cancer, antimicrobial and antiviral drugs and as therapeutics for metabolic disorders) and in industry (e.g. to make important commodity chemicals and as catalysts for bioremediation). Our central premise is that a quantitative, mechanistic understanding of enzyme function and its relationship to organism fitness is critically needed to precisely manipulate enzymes and to deeply understand biology. To generate this level of understanding, we need: (1) a quantitative, chemical, and physical knowledge of enzyme function, and (2) mechanistic data describing how and when these physical principles contribute to enzyme function within the complex environments where enzymes operate. An enhanced understanding of the relationships between protein sequence, protein function and cellular/organismal fitness will have profound impacts across biology and medicine, from improving our ability to predict how mutations will influence the virulence and drug susceptibility of human pathogens, to enhancing precision medicine by accurately predicting the consequences of allelic variants, to enabling the design of next-generation protein and cellular therapeutics. Achieving this understanding requires new tools and a new conceptual paradigm. Enzymes are highly interconnected, their functions are multifaceted, and their cellular environments are complex. Traditional biochemistry is enormously powerful, allowing for the intensive study of a few individual enzymes in vitro (10s) and providing detailed knowledge of their chemical mechanisms. But identifying the many residues that matter for enzyme function requires investigation of residues beyond the active site at a scale far beyond that of traditional biochemistry. Furthermore, this biochemical information then needs to be translated to organism fitness in vivo in a quantitative manner. Here we will overcome these challenges. We will first use evolutionary sequence information to direct enzyme variant design towards functionally important areas of sequence space. We will adapt high-throughput microfluidic technologies to quantitively measure the biochemical properties (e.g., kcat, Km, Ki, and ∆GFold) of this library of 104 enzyme variants in vitro (Aim 1). Then we will determine how each of these variants affects organismal fitness in vivo using pooled competition and barcode sequencing assays (Aim 2). Finally, we will use this sequence-function-fitness map to test long-standing models in biochemistry and evolution and reveal the biochemical determinants of fitness important for industry and medicine (Aim 3). Such a comprehensive and quantitative mapping of biochemical function to fitness has never been achieved.

项目摘要/摘要 酶是细胞中的主要功能分子，提供了巨大的增强率， 对生命所必需的对潜水员化学反应的特异性和调节。酶，就像所有生物学一样 大分子是进化的产物：所有酶都进化为在复合物中运行 特定环境壁ni的生物/细胞的环境。那是对酶功能的理解 进化对于生物学至关重要。酶在医学方面也具有巨大的潜力（例如，作为目标 用于抗癌，抗菌和抗病毒药物以及作为代谢疾病的治疗学）和工业（例如 制造重要的商品化学物质和作为生物修复的催化剂）。我们的中心前提是 对酶功能的定量，机械理解及其与生物健康的关系至关重要 需要精确操纵酶并深入了解生物学。 为了产生这种理解水平，我们需要：（1）定量，化学和物理知识 酶功能以及（2）描述这些物理原理如何以及何时有助于 酶在酶工作的复杂环境中的功能。对 蛋白质序列，蛋白质功能和细胞/生物适应性之间的关系将具有深刻的 在生物学和医学之间的影响，从提高我们预测突变将如何影响突变的能力 通过准确预测，人类病原体的毒力和药物敏感性，以增强精度医学 Allic变体的后果，以实现下一代蛋白质和细胞疗法的设计。 实现这种理解需要新的工具和新的概念范式。酶高度高 互连，它们的功能是多方面的，并且它们的细胞环境很复杂。传统的 生物化学非常强大，可以在体外对一些单个酶进行深入研究（10s） 并提供有关其化学机制的详细知识。但是确定许多重要的残留物 为了酶功能，需要调查活跃部位以外的残留物，以远远超出 传统生物化学。此外，需要将这些生化信息转换为有机体 体内适应性的定量方式。在这里，我们将克服这些挑战。我们将首先使用进化 序列信息将酶变体设计转向序列空间的功能重要领域。 我们将适应高通量微流体技术，以定量测量生化特性（例如， 该文库的104个酶变体的体外kcat，km，ki和∆gfold）（AIM 1）。然后我们将确定每个 这些变体中的有机健身在体内使用合并的竞争和条形码测序分析 （目标2）。最后，我们将使用此序列功能拟合图来测试生物化学和 进化并揭示了对工业和医学重要的健身生化决定者（AIM 3）。这样的 从未实现生化功能对适应性的全面和定量映射。