STUDIES OF THE DYNAMICS OF HEME PROTEIN ACTIVE SITES

这项提议涉及过氧化物酶，如下图所示 由酵母菌细胞色素C过氧化物酶和单体氧- 结合蛋白，以二枝甘油单体为代表 血红蛋白。这项提议的目标包括使用孤立的、 天然蛋白、重组野生型蛋白和重组蛋白 突变蛋白质最大限度地进行核磁共振表征并量化血红素- 现场化学。在细胞色素c过氧化物酶的情况下，我们 提议在我们过去取得的重大成功的基础上再接再厉 一年使用各种2D核磁共振进行质子核磁共振任务的经验 方法，以扩展这些赋值。专门创建的 定点突变蛋白将在这一努力中发挥作用。 将使用同位素标记(15N和13C)作为分配工具。 此外，对个体属性的表征 突变的蛋白质将通过核磁共振进行检测。在这种情况下 二枝甘油单体血红蛋白，其结构为 几乎可以与抹香鲸的肌红蛋白重叠，我们 建议以同位素标记核磁共振为目标 “全部”作业。我们打算演示核磁共振方法，用于 分析由特定突变引起的结构变化。我们 建议使用五个特定的位点突变体来修饰配体 以可预测的方式结合动态，并设计突变体 预计将以预期的方式改变血红素-珠蛋白的相互作用。 此外，我们打算利用这些结果和其他结果来 测试我们设计和创造突变体的能力 特定目的，基于晶体结构的推论。

This proposal involves the peroxidase enzymes, typified in this case by yeast cytochrome c peroxidase, and the monomeric oxygen- binding proteins, typified by the Glycera dibranchiata monomer hemoglobins. Goals of this proposal include using isolated, natural proteins, recombinant wild-type proteins and recombinant mutant proteins maximize NMR characterization and quantify heme- site chemistry. In the case of cytochrome c peroxidase, we are proposing to build upon our significant success during the past year in making proton NMR assignments using a variety of 2D NMR methods in order to expand these assignments. Specifically created site-specific mutant proteins will be useful in this effort. Isotope labeling (15N & 13C) will be used as an assignment tool. In addition, characterization of the properties of the individual mutant proteins will be carried out by NMR. In the case of the Glycera dibranchiata monomer hemoglobins, whose architecture is virtually superimposable to that of sperm-whale myoglobin, we propose to NMR with isotope labelling for the goal of making "total" assignments. We intend to demonstrate NMR methods for assaying structural changes induced by specific mutations. We propose to use five specific site-mutants designed to modify ligand binding dynamics in a predictable manner, and design mutants predicted to modify heme--globin interactions in anticipated ways. In addition we intend to use these results and others in order to test our ability to design and create mutants designed for a specific purpose, based upon inferences from the crystal structure.