De Novo Design of Functional Metalloporphyrin-Containing Proteins

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 通过蛋白质基质，不能很容易地复制在合成结构。来测试我们对 我们的目标是从头开始设计新的金属蛋白。结合的蛋白质 卟啉样辅因子是特别令人感兴趣的，因为已知血红素蛋白质执行多种功能， 反应.直到最近，我们才能设计出能以低于100%的精确度结合辅因子的蛋白质.这 通过将合成的无机辅因子结合到蛋白质中， 缺乏进化前功能的蛋白质。拟议的研究策略旨在阐明设计特点 需要将M-四苯基卟啉（M-TPP; M=Fe，Mn）络合物紧密地结合到基底 装订口袋。第一和第二壳层的相互作用将被设计成控制方向，电子 结构和辅因子与底物的反应途径。装订口袋将设计樟脑 和苯乙烯，并且将测试这些从头金属蛋白对羟基化的活性， 环氧化这些金属酶的选择性也将被测试，蛋白质支架将被测试。 重新设计以引发区域选择性反应（即仅与两种可能的可官能化基团中的一种反应）。 将使用光学光谱、电子顺磁共振（EPR） 光谱法、NMR光谱法和X射线衍射（XRD）法。这项工作将是一个突破 在蛋白质设计中，将直接影响对蛋白质作用的基本理解， 环境对金属蛋白中金属中心功能的影响。

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 that cannot be easily reproduced in synthetic constructs. To test our understanding of how metalloproteins function, we aim to design de novo metalloproteins from scratch. Proteins that bind porphyrin-like cofactors are of particular interest, as heme proteins are known to perform a variety of reactions. Only recently have we been able to design proteins that bind a cofactor with sub-Å accuracy. This opens the door to expand on the utility of natural proteins by incorporating synthetic inorganic cofactors into proteins that lack pre-evolved function. The proposed research strategy seeks to elucidate the design features necessary to bind M-tetraphenylporphyrin (M-TPP; M=Fe, Mn) complexes in close proximity to a substrate binding pocket. First- and second-shell interactions will be engineered to control orientation, electronic structure, and reaction pathway of the cofactor and substrate. Binding pockets will be designed for camphor and styrene and these de novo metalloproteins will be tested for their activity towards hydroxylation and epoxidation. The selectivity of these metalloenzymes will also be tested and the protein scaffold will be redesigned to elicit regioselective reactions (i.e. reacting with only one of two possible functionalizable groups). The proteins will be characterized using optical spectroscopies, electron paramagnetic resonance (EPR) spectroscopy, NMR spectroscopy, and by X-ray diffraction (XRD) methods. 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.