De novo design of functional metallocofactor-binding proteins

功能性金属辅因子结合蛋白的从头设计

• 批准号: 10478225
• 负责人: Samuel I Mann
• 金额: $ 9.4万
• 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: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10478225
• 关键词: Active Sites; Affinity; Amino Acids; Anti-Inflammatory Agents; Binding; Binding Proteins; Binding Sites; Bioinformatics; Catalysis; Chemicals; Chemistry; Complex; Computing Methodologies; Covalent Interaction; Crystallization; Development; Diclofenac; Dioxygen; Distant; Engineering; Ensure; Environment; Foundations; Geometry; Glutamine; Goals; Health; Hemeproteins; Human; Hydrogen Bonding; Hydroxylation; Individual; Ions; Learning; Libraries; Ligands; Link; MPP1 gene; Metalloproteins; Metals; Methods; Modeling; Molecular Biology; Mutagenesis; Mutation; Oxidants; Oxides; Pathway interactions; Pharmaceutical Preparations; Porphyrins; Positioning Attribute; Protein Engineering; Proteins; Reaction; Research; Rest; Role; Scaffolding Protein; Structure; Structure-Activity Relationship; Substrate Specificity; Sulfoxide; Surface; System; Techniques; Temperature; Testing; Training; Vertebral column; Water; Work; cofactor; design; electronic structure; frontier; high throughput screening; interest; metalloenzyme; mutant; nanomolar; oxidation; programs; scaffold; small molecule; thioether

Project Summary/Abstract Metalloproteins perform chemical transformations with rates and selectivites that have yet to be achieved in synthetic or designed systems. These differences in reactivity are directly linked to the environment produced by the protein matrix. To test our understanding of how metalloproteins function, I aim to design de novo metalloenzymes from scratch. Proteins that bind porphyrin-like cofactors are of particular interest, as heme proteins are known to perform a variety of reactions. Recently, I designed a protein to bind the abiological porphyrin, Mn-diphenylporphyrin (MnDPP), that provided the first crystallographic structure of a de novo designed porphyrin-binding protein (MPP1). MPP1 was also capable of stabilizing a Mn(V)-oxo species, a powerful oxidant that can perform sulfoxidation of thioether substrates. The proposed research seeks to elucidate design features necessary to control the reactivity/stability of this high-valent species through rational mutagenesis of my designed protein. This will allow direct correlation of changes in reactivity to changes in structure. To gain greater control of substrate orientation and, therefore, product distribution, I will design a 5-helix bundle that has a large pocket for substrate binding. Using the design strategy for MPP1 and in-house developed computational methods, the 5-helix bundle will be parameterized from scratch and designed to bind MnDPP. A library of sequences will be expressed and screened using high-throughput methods for binding and sulfoxidation activity. Promising scaffolds will then be redesigned to include substrate-specific interactions to bind the anti-inflammatory drug, diclofenac. Using COMBS, a recently developed bioinformatics method for designing backbone specific polar interactions, I will design two proteins to control the orientation of diclofenac to direct the hydroxylation to yield 5-hydroxydiclofenac or 4’- hydroxydiclofenac. This work would be a breakthrough in protein design and will directly impact the fundamental understanding of the effects of protein environments on the function of metal centers in metalloproteins.

项目总结/摘要 金属蛋白质进行化学转化的速度和选择性，尚未达到在 合成或设计的系统。这些反应性的差异与所产生的环境直接相关 被蛋白质基质所覆盖。为了测试我们对金属蛋白功能的理解，我的目标是重新设计 从零开始结合卟啉样辅因子的蛋白质特别令人感兴趣，如血红素 已知蛋白质进行多种反应。最近我设计了一种蛋白质 卟啉，锰-二苯基卟啉（MnDPP），提供了从头开始的第一个晶体结构， 设计的卟啉结合蛋白（MPP 1）。MPP 1还能够稳定Mn（V）-氧代物种， 可以对硫醚底物进行磺基氧化的强氧化剂。拟议的研究旨在 阐明控制这种高价物质的反应性/稳定性所需的设计特征， 我设计的蛋白质的合理突变。这将允许反应性变化与 结构的变化。为了更好地控制基板方向，从而更好地控制产品分布， 设计一个5-螺旋束，具有一个大的口袋，用于基板结合。使用MPP 1的设计策略， 内部开发的计算方法，5螺旋束将从头开始参数化， 设计用于结合MnDPP。将表达序列文库，并使用高通量 结合和磺基氧化活性的方法。有前途的脚手架将被重新设计， 底物特异性相互作用结合抗炎药物双氯芬酸。使用COMBS，最近 开发了设计骨架特异性极性相互作用的生物信息学方法，我将设计两个蛋白质 以控制双氯芬酸的取向，从而引导羟基化，得到5-羟基双氯芬酸或4 '-羟基双氯芬酸。 羟基双氯芬酸。这项工作将是蛋白质设计的一个突破，并将直接影响蛋白质的合成。 基本了解蛋白质环境对金属中心功能的影响， 金属蛋白