De novo design of functional metallocofactor-binding proteins

项目摘要/摘要 金属蛋白以尚未实现的速率和选择性进行化学转化。 合成的或设计的系统。这些反应性的差异与所产生的环境直接相关。 通过蛋白质矩阵。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 金属酶从无到有。与类似于卟啉的辅因子结合的蛋白质特别令人感兴趣，如血红素。 众所周知，蛋白质可以进行各种反应。最近，我设计了一种蛋白质来结合非生物 二苯基卟啉(MnDPP)，提供了从头算起的第一个晶体结构 设计了卟啉结合蛋白(MPP1)。MPP1还能够稳定Mn(V)-oxo物种，a 强大的氧化剂，可对硫醚底物进行亚磺氧化。这项拟议的研究旨在 阐明控制这种高价物种的反应性/稳定性所必需的设计特征 对我设计的蛋白质进行合理的突变。这将允许将反应性的变化与 结构上的变化。为了更好地控制衬底取向，从而获得产品分布，我将 设计一种5-螺旋束，它有一个大口袋用于底物结合。使用MPP1的设计策略和 内部开发的计算方法，5-螺旋束将从头开始和 旨在约束MnDPP。将使用高通量来表达和筛选序列文库 结合活性和亚硫氧化活性的方法。有希望的支架将被重新设计，以包括 底物特异性相互作用，以结合抗炎药双氯芬酸。使用Combs，这是最近的一个 发展了生物信息学方法来设计骨架特定的极性相互作用，我将设计两个蛋白质 控制双氯芬酸的取向，以指导羟化反应生成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突破，并将直接影响到 蛋白质环境对金属中心功能影响的基本认识 金属蛋白。

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.