Development of a Mechanism and Structure-Guided Methodology for De Novo Enzyme Design

开发从头酶设计的机制和结构引导方法

• 批准号: 10231920
• 负责人: Samuel H. Schneider
• 金额: $ 6.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231920
• 关键词: Acceleration; Active Sites; Address; Affinity; Agreement; Antibiotic Resistance; Benchmarking; Binding; Binding Proteins; Binding Sites; Bioinformatics; Biological Models; Biophysics; Calorimetry; Catalysis; Chemicals; Chemistry; Circular Dichroism; Complement; Complex; Development; Directed Molecular Evolution; Docking; Enzymes; Evolution; Exhibits; Foundations; Health; Human; Hydrogen Bonding; Hydrolysis; Hydrophobicity; In Vitro; Individual; Investigation; Kinetics; Lactamase; Lactams; Ligand Binding; Ligands; Maintenance; Measures; Metals; Methodology; Methods; Modeling; Monobactams; NMR Spectroscopy; Optics; Positioning Attribute; Property; Protein Engineering; Proteins; Protons; Reaction; Role; Scaffolding Protein; Site; Specificity; Spectrum Analysis; Structure; Structure-Activity Relationship; Substrate Specificity; Tertiary Protein Structure; Testing; Therapeutic; Work; X-Ray Crystallography; analog; aqueous; base; beta-Lactamase; beta-Lactams; bioinformatics tool; biological systems; biophysical techniques; chemical reaction; design; functional group; high throughput screening; improved; in silico; insight; iterative design; novel; prediction algorithm; protein complex; protein function; protein structure; protein structure function; reaction rate; simulation; small molecule

Project Summary/Abstract Naturally enzymes are characterized by their ability to accelerate chemical reactions by orders of magnitude and with far greater specificity than those observed in aqueous solution. However, these catalytic properties have yet to be realized in synthetic chemical or biological systems. While there have been many recent advances in the de novo design of proteins, achieving the exquisite control of individual atoms and functional groups necessary for enzyme catalysis remains a long-standing challenge and has relied on directed evolution and high-throughput screening to improve upon designs. The first principles design of an active site capable of substrate binding and catalysis with rates and affinities similar to those found in natural enzymes would represent a breakthrough in our understanding of structure-function relationships and the origins of protein (dys)function. A recent advance in the de novo design of small-molecule-binding proteins demonstrates the ability to position non-covalent interactions, such as hydrogen bonds from a protein to ligand functional groups, with sub-Å accuracy. Based on this methodology, it’s hypothesized that if non-covalent interactions can be rationally designed for binding, then they can be directed towards achieving de novo enzyme design through preferential TS-stabilization to achieve fast reaction rates. In order to test this hypothesis, enzymes capable of the model Kemp elimination reaction will be de novo designed using a bottom-up approach to install a general base and tune its reactivity, assemble an active site capable of substrate- and TS-analog-binding, and develop a negative design strategy for preferential TS-stabilization. These fundamental insights will be directed towards the first de novo metallo-β-lactamase, a model system for studying antibiotic resistance and protein evolution with human health implications. Catalytic turnover of the large and highly polar β-lactam antibiotics provides a sensitive test of our ability to design non-covalent interactions. This will be achieved by expanding upon the size, asymmetry, and topology of designable protein scaffolds using a bioinformatic and function-guided design strategy for multi-domain proteins, de novo design of a Zn2+- and substrate-binding site, and development of de novo methods for preferential transition-state stabilization. The proposed investigations will be achieved using both computational and experimental methods, starting from in silico approaches to benchmark the folding and binding of reaction intermediates in designed proteins. Promising designs will then be expressed, purified, and characterized in terms of their binding affinity and catalytic rate constants using isothermal calorimetry and optical spectroscopies. These functional studies will be complemented by structural characterization using X-ray crystallography and NMR spectroscopy to confirm the accuracy of the design methodology. Successful de novo design of functional enzymes would represent a breakthrough in our understanding of structure-function relationships and the role of non-covalent interaction in complex protein function.

项目概要/摘要 自然地，酶的特点是能够按以下顺序加速化学反应： 其幅度和特异性远高于在水溶液中观察到的结果。然而，这些催化 其特性尚未在合成化学或生物系统中实现。虽然已经有很多 蛋白质从头设计的最新进展，实现了对单个原子的精确控制 酶催化所需的官能团仍然是一个长期存在的挑战，并且依赖于定向 进化和高通量筛选以改进设计。活动站点设计的首要原则 能够以与天然酶相似的速率和亲和力进行底物结合和催化 将代表我们对结构-功能关系和蛋白质起源的理解的突破 （dys）功能。小分子结合蛋白从头设计的最新进展表明 定位非共价相互作用的能力，例如从蛋白质到配体官能团的氢键， 精度亚 Å 级​​。基于这种方法，假设如果非共价相互作用可以 合理设计结合，然后它们可以通过以下方式直接实现从头酶设计 优先 TS 稳定以实现快速反应速率。为了验证这一假设，酶能够 Kemp 消除反应模型将使用自下而上的方法从头设计，以安装通用的 基础并调整其反应性，组装能够结合底物和 TS 类似物的活性位点，并开发 优先 TS 稳定的负设计策略。这些基本见解将针对 第一个从头金属-β-内酰胺酶，用于研究抗生素耐药性和蛋白质进化的模型系统 对人类健康有影响。大且高极性的 β-内酰胺抗生素的催化转化提供了 对我们设计非共价相互作用的能力的敏感测试。这将通过扩大规模来实现， 使用生物信息学和功能引导设计的可设计蛋白质支架的不对称性和拓扑结构 多结构域蛋白策略、Zn2+-和底物结合位点的从头设计以及 de 用于优先过渡态稳定的新方法。拟议的调查将通过使用 计算和实验方法，从计算机方法开始对折叠和 设计蛋白质中反应中间体的结合。然后，有前途的设计将得到表达、纯化和 使用等温量热法和光学对其结合亲和力和催化速率常数进行表征 光谱检查。这些功能研究将通过使用 X 射线的结构表征来补充 晶体学和核磁共振波谱来确认设计方法的准确性。成功从头开始 功能酶的设计将代表我们对结构功能理解的突破 非共价相互作用在复杂蛋白质功能中的关系和作用。