Structure and Dynamics of Heme Protein Active Sites

描述（由申请人提供）：该提案涉及表征两种非常不同类型的血红素蛋白的结构和化学。其中第一种是酵母细胞色素c过氧化物酶（CcP），它自然存在于酵母线粒体中，是一种典型的过氧化物酶。它是一种-34KD铁血红素酶，其细胞功能是利用其天然氧化还原伙伴细胞色素c （cytc）的还原等量物来分解过氧化氢。在这种作用下，它作为细胞毒性保护剂，参与远距离电子转移，也可能对氧化应激信号传导很重要。我们计划研究的第二组蛋白质是一组基于血红素的生物氧传感器。其中包括来自日本慢生根瘤菌（BjFixL）和Sinorhizobium meliloti （SmFixL）的固定蛋白，以及来自大肠杆菌（EcDos）的直接氧传感器蛋白。FixL蛋白在各自的细菌中调节nif和fix操纵子的表达，这反过来又控制了固氮所需的所有蛋白质的生物合成。EcDos蛋白被认为参与了大肠杆菌的好氧/厌氧转换。这三种蛋白质都含有一个中心结构域结构，由pas -血红素结合结构域（传感结构域）连接到催化结构域（fixl的激酶；ecdo的磷酸二酯酶）。虽然这三种传感器都是细菌来源，但最近人们注意到，缺氧引发人肾纤维化的分子事件涉及一种与蛋白激酶催化相关的血红素氧传感蛋白，类似于fixl （Norman， J. T., Clark， J. M .,）。加西亚。P. L.，“缺氧促进人类肾成纤维细胞的纤维形成”，（2000）肾国际杂志。农业学报，58,2351-2366)。我们打算研究的所有四种血红素蛋白（CcP, fixl, EcDos）都已经在我们的实验室中表达和研究。这两种蛋白质的目标是相同的。我们建议综合研究它们的功能、结构和动态。目标是阐明它们如何在分子基础上起作用，以及哪些结构特征对该功能至关重要。我们将继续使用现代蛋白质工程方法结合x射线晶体学，动力学，光热方法，平衡动力学方法和核磁共振波谱。

DESCRIPTION (provided by applicant): This proposal involves characterizing the structures and chemistry of two very different types of heme proteins. The first of these is yeast cytochrome c peroxidase (CcP), which occurs naturally in yeast mitochondria and is a prototypical peroxidase. It is a -34KD ferriheme enzyme whose cellular function is to use reducing equivalents from its natural redox partner, cytochrome c (cytc), to decompose hydrogen peroxide. In this role it acts as a cytotoxic protective agent, participates in long-distance electron transfer and may also be important for oxidative stress signaling. The second group of proteins that we plan to study are a group of heme-based biological oxygen sensors. These include the FixLs from Bradyrhizobium japonicum (BjFixL) and Sinorhizobium meliloti (SmFixL) and the Direct Oxygen Sensor protein from E. coli (EcDos). The FixL proteins regulate expression of the nif and fix operons in their respective bacteria, which, in turn, control the biosynthesis of all proteins needed for nitrogen fixation. The EcDos protein is thought to participate in the aerobic/anaerobic switch in E. coli. All three of these proteins contain a central domain structure consisting of a PAS-heme binding domain (sensing domain) linked to a catalytic domain (kinase for the FixLs; phosphodiesterase for EcDos). While these three sensors are of bacterial origin, it has recently been noted that molecular events triggering human renal fibrosis, resulting from hypoxia involves a protein with heme-based oxygen sensing linked to protein kinase catalysis, similar to the FixLs (Norman, J. T., Clark, J. M,., and Garcia. P. L., "Hypoxia Promotes Fibrogenesis in Human Renal Fibroblasts," (2000) Kidney Int., 58, 2351-2366). All four of the heme proteins that we intend to study (CcP, FixLs, EcDos) are already being expressed and studied in our laboratory. The goals for both protein types are the same. We propose an integrated effort to study their function, structure and dynamics. The goal is to elucidate how they function on a molecular basis, and what structural features are critical to that function. We shall proceed using modern protein engineering methods combined with x-ray crystallography, kinetics, photothermal methods, equillibrium dynamics methods and NMR spectroscopy.