Structure and Dynamics of Heme Protein Active Sites

描述(由申请人提供)：这项建议涉及两种非常不同类型的血红素蛋白的结构和化学特征。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的铁血红素酶，其细胞功能是利用其天然氧化还原伙伴细胞色素c(Cytc)的还原等价物来分解过氧化氢。在这一作用中，它作为细胞毒性保护剂，参与长距离电子传递，也可能是氧化应激信号的重要组成部分。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。这些包括来自慢生根瘤菌(BjFixL)和紫花苜蓿中华根瘤菌(SmFixL)的FixL，以及来自大肠杆菌的直接氧传感器蛋白(EcDos)。FixL蛋白调节各自细菌中NIF和FIX操纵子的表达，进而控制固氮所需所有蛋白质的生物合成。ECDos蛋白被认为参与了大肠杆菌中的好氧/厌氧转换。所有这三种蛋白质都含有一个中心结构域，由一个pAS-血红素结合域(感应域)连接到一个催化域(Fixls的激酶；EcDos的磷酸二酯酶)组成。虽然这三个传感器是细菌起源的，但最近人们注意到，由缺氧引起的触发人类肾脏纤维化的分子事件涉及一种具有基于血红素的氧感应的蛋白质，该蛋白质与蛋白激酶催化有关，类似于FixLS(Norman，J.T.，Clark，J.M.，.和Garcia)。P.L.，“低氧促进人肾成纤维细胞的纤维化形成”，(2000)肾脏国际，58,2351-2366)。我们打算研究的所有四种血红素蛋白(CCP、Fixls、EcDos)都已经在我们的实验室表达和研究。这两种蛋白质的目标是相同的。我们建议对它们的功能、结构和动力学进行综合研究。我们的目标是阐明它们在分子基础上是如何发挥作用的，以及哪些结构特征对这一功能至关重要。我们将继续使用现代蛋白质工程方法，结合X射线结晶学、动力学、光热方法、平衡动力学方法和核磁共振光谱。

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.