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.

项目总结/摘要 自然地，酶的特征在于它们能够按以下顺序加速化学反应： 其强度和特异性远大于在水溶液中观察到的。然而，这些催化剂 在合成的化学或生物系统中还没有实现这些性质。虽然有很多 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， 酶催化所必需的官能团仍然是一个长期存在的挑战， 进化和高通量筛选以改进设计。活性位点的第一性原理设计 能够以与天然酶相似的速率和亲和力结合底物和催化 将代表我们对蛋白质结构-功能关系和起源的理解的突破 （dys）函数。最近在小分子结合蛋白的从头设计方面的进展表明， 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， 以亚微米的精度。基于这种方法，假设如果非共价相互作用可以 合理设计的结合，那么它们可以直接实现从头酶的设计， 优先TS稳定化以实现快速反应速率。为了验证这一假设，酶能够 模型肯普消除反应将从头设计使用自下而上的方法，安装一个通用的 基础和调整其反应性，组装一个能够与底物和TS类似物结合的活性位点， 一个消极的设计策略，优先TS稳定。这些基本的见解将被导向 第一个从头金属-β-内酰胺酶，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的和高极性的β-内酰胺抗生素的催化周转提供了一种 这是对我们设计非共价相互作用能力的一个敏感测试。这将通过扩大规模来实现， 使用生物信息学和功能指导设计的可设计蛋白质支架的不对称性和拓扑结构 多结构域蛋白质策略，Zn 2+和底物结合位点的从头设计，以及去乙酰化酶的开发 优先过渡态稳定化的新方法。拟开展的调查将使用 计算和实验方法，从硅片方法开始，以基准的折叠， 在设计的蛋白质中结合反应中间体。有前途的设计将被表达，纯化， 使用等温量热法和光学方法对其结合亲和力和催化速率常数进行表征 光谱学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NMR光谱学，以确认设计方法的准确性。成功的从头开始 功能酶的设计将代表我们对结构-功能理解的突破 关系和非共价相互作用在复杂蛋白质功能中的作用。