DYNAMICS OF HEME PROTEIN ACTIVE SITES

血红素蛋白活性位点的动力学

• 批准号: 2185111
• 负责人: James D. Satterlee
• 金额: $ 17.49万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185111
• 关键词: Saccharomyces cerevisiae; chemical binding; cytochrome c; enzyme mechanism; enzyme structure; gene mutation; genetic manipulation; heme; hemoglobin; hemoprotein structure; ligands; molecular site; nuclear magnetic resonance spectroscopy; nucleic acid sequence; peroxidases; protein engineering; protein reconstitution; protein sequence; protein structure function; stable isotope

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.

该提案涉及过氧化物酶，典型的是 酵母细胞色素c过氧化物酶和单体氧- 结合蛋白，以 Glycera dibranchiata 单体为代表 血红蛋白。 该提案的目标包括使用隔离的、 天然蛋白、重组野生型蛋白和重组蛋白 突变蛋白最大化 NMR 表征并量化血红素 现场化学。 就细胞色素 C 过氧化物酶而言，我们是 提议在我们过去取得的重大成功的基础上再接再厉 年使用各种 2D NMR 进行质子 NMR 分配 方法来扩展这些任务。 特别创建 位点特异性突变蛋白将在这项工作中发挥作用。 同位素标记（15N 和 13C）将用作分配工具。 此外，还可以对个体的特征进行描述。 突变蛋白将通过NMR进行。 在这种情况下 Glycera dibranchiata 单体血红蛋白，其结构为 事实上，我们可以与抹香鲸的肌红蛋白叠加 建议使用同位素标记的 NMR 来实现 “总计”作业。 我们打算演示核磁共振方法 分析特定突变引起的结构变化。 我们 建议使用五种特定位点突变体来修饰配体 以可预测的方式结合动力学，并设计突变体 预计会以预期的方式改变血红素-珠蛋白的相互作用。 此外，我们打算使用这些结果和其他结果，以便 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。