Synthetic Protein Families by Structure-Guided SCHEMA Recombination

通过结构引导的 SCHEMA 重组合成蛋白质家族

• 批准号: 7528039
• 负责人: FRANCES H ARNOLD
• 金额: $ 32.32万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7528039
• 关键词: Address; Algorithms; Amino Acids; Biological Assay; Biological Models; Cereals; Chimera organism; Chimeric Proteins; Classification; Collaborations; Complement; Complex; Coupled; Coupling; Cytochrome P450; Cytochromes; Data; Distant; Drug Interactions; Drug toxicity; Enzyme Stability; Enzymes; Exhibits; Family; Genetic Recombination; Genetic Variation; Goals; Grant; Health; Heme; Homologous Protein; Homology Modeling; Human; Individual; Interdisciplinary Study; Laboratories; Lead; Libraries; Measurement; Measures; Methodology; Modeling; Molecular Conformation; Mutation; Parents; Pharmaceutical Preparations; Production; Protein Family; Proteins; Publications; Research; Resolution; Resources; Roentgen Rays; Sampling; Specificity; Structural Models; Structure; Structure-Activity Relationship; Students; Substrate Specificity; Surveys; Techniques; Testing; Toxic effect; Vertebral column; Work; base; design; drug discovery; drug metabolism; enzyme structure; family structure; gang; high throughput screening; insight; interdisciplinary collaboration; novel strategies; protein structure prediction; public health relevance; scaffold; synthetic protein; theories; tool; Address; Algorithms; Amino Acids; Biological Assay; Biological Models; Cereals; Chimera organism; Chimeric Proteins; Classification; Collaborations; Complement; Complex; Coupled; Coupling; Cytochrome P450; Cytochromes; Data; Distant; Drug Interactions; Drug toxicity; Enzyme Stability; Enzymes; Exhibits; Family; Genetic Recombination; Genetic Variation; Goals; Grant; Health; Heme; Homologous Protein; Homology Modeling; Human; Individual; Interdisciplinary Study; Laboratories; Lead; Libraries; Measurement; Measures; Methodology; Modeling; Molecular Conformation; Mutation; Parents; Pharmaceutical Preparations; Production; Protein Family; Proteins; Publications; Research; Resolution; Resources; Roentgen Rays; Sampling; Specificity; Structural Models; Structure; Structure-Activity Relationship; Students; Substrate Specificity; Surveys; Techniques; Testing; Toxic effect; Vertebral column; Work; base; design; drug discovery; drug metabolism; enzyme structure; family structure; gang; high throughput screening; insight; interdisciplinary collaboration; novel strategies; protein structure prediction; public health relevance; scaffold; synthetic protein; theories; tool

DESCRIPTION (provided by applicant): Synthetic protein families powerfully complement natural genetic diversity in efforts to understand fundamental sequence-structure-function relationships. We propose to use a unique family of chimeric cytochrome P450s made by structure-guided recombination to make advances in modeling P450 structure, stability and specificity. Differing from one another by many dozens of amino acids yet still likely to fold into highly similar structures, these diverse sequences have different stabilities and exhibit an array of activities and specificities. We seek to maximize the number of accurate structure, stability, and specificity predictions given a set number of experimental measurements that are relatively easily performed on these laboratory-generated sequences. We propose to use this unique library of P450 chimeras to study enzyme structural modularity and exploit that modularity to simplify structure-function predictions. We will determine the X-ray crystal structures for several chimeric P450 heme domains. The crystal structures will assist our concurrent efforts to computationally predict accurate chimera structures. We will test new algorithms that recombine optimized enzyme substructures to sample relevant protein backbone conformations. Our goal is to bridge the gap between low resolution homology models and accurate high resolution structures. The chimeric enzymes are also a rich resource for investigating sequence-structure-stability relationships. The chimeric proteins have diverse stabilities; many are more stable than their parents. We plan to measure stability for selected chimeras and construct and test additive models for protein stability and quantify non- additive coupling effects. Recombination creates sequence intermediates between parent proteins and allows us to reconcile the effects of swapping blocks of sequence with the action of individual mutations. This work will contribute key data and insight into the effects of long-range interactions and coupling. Finally, we plan to explore the functional diversity of the chimeric cytochrome P450s, in the context of their applications in drug discovery and drug metabolism. These soluble, well-expressed chimeric P450s are a viable platform for production of authentic human metabolites of drugs and for lead diversification. Preliminary results indicate that the diverse sequences exhibit activity on a wide range of substrates. We will perform a systematic analysis of the activities of a set of chimeric P450s towards pharmaceutically relevant scaffolds, using clustering and regression to determine whether activity and specificity can be predicted from data on selected chimeras. We will attempt to rationalize the P450 specificity results in terms of structural P450- substrate complex models. This research continues an effective interdisciplinary collaboration that partners powerful experimental and theoretical capabilities. PUBLIC HEALTH RELEVANCE: We propose to demonstrate how a laboratory-generated family of cytochrome P450s can be used to study the basis of P450 structure, stability, and function. We will test a new strategy for predicting the interactions between drugs and P450 enzymes, a potential breakthrough that would allow us to predict drug metabolism and interactions and to efficiently produce drug metabolites for toxicity studies and drug discovery. Finally, this research represents a fundamentally new approach to study protein stability using recombination, with results that can be applied to understanding enzymes in general.

描述（由申请人提供）：合成蛋白质家族在理解基本序列结构功能关系的努力中有力补充自然遗传多样性。我们建议使用由结构引导的重组制成的独特的嵌合细胞色素P450家族，以在建模P450结构，稳定性和特异性上取得进步。这些不同的序列彼此不同，但仍可能折叠成高度相似的结构，这些不同的序列具有不同的稳定性，并且表现出一系列活动和特异性。我们试图最大程度地提高一定数量的实验测量值，这些实验测量值相对容易地进行这些实验室生成的序列。我们建议使用这个独特的P450嵌合体库来研究酶结构模块，并利用该模块化来简化结构 - 功能预测。我们将确定几个嵌合P450血红素结构域的X射线晶体结构。晶体结构将有助于我们同时进行计算预测准确的嵌合体结构的努力。我们将测试新算法，以重组优化的酶亚结构以采样相关的蛋白质主链构象。我们的目标是弥合低分辨率同源模型与准确的高分辨率结构之间的差距。嵌合酶也是研究序列结构稳定关系的丰富资源。嵌合蛋白具有多种稳定性。许多人比父母更稳定。我们计划测量选定的嵌合体的稳定性，并构建和测试蛋白质稳定性的加性模型，并量化非添加剂耦合效应。重组会在母蛋白之间产生序列中间体，并使我们能够调和序列块的影响与单个突变的作用。这项工作将有助于关键数据并深入了解远程相互作用和耦合的影响。最后，我们计划在药物发现和药物代谢中的应用中探索嵌合细胞色素P450的功能多样性。这些可溶性，表达良好的嵌合P450是生产正宗人类药物代谢物和铅多样化的可行平台。初步结果表明，各种序列在广泛的底物上表现出活性。我们将对一组嵌合P450对药物相关的支架的活动进行系统分析，使用聚类和回归来确定是否可以从所选嵌合体的数据中预测活动和特异性。我们将尝试根据结构P450-底物复合模型合理化P450特异性结果。这项研究继续进行了有效的跨学科合作，该合作构成了强大的实验和理论能力。公共卫生相关性：我们建议证明如何使用实验室生成的细胞色素P450家族来研究P450结构，稳定性和功能的基础。我们将测试一种预测药物与P450酶之间相互作用的新策略，这是一个潜在的突破，这将使我们能够预测药物代谢和相互作用，并有效地产生用于毒性研究和药物发现的药物代谢产物。最后，这项研究代表了一种从根本上使用重组研究蛋白质稳定性的新方法，结果可以应用于一般理解酶。