DYNAMICS OF HEME PROTEIN ACTIVE SITES

血红素蛋白活性位点的动力学

• 批准号: 2185112
• 负责人: James D. Satterlee
• 金额: $ 18.32万
• 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/2185112
• 关键词: DYNAMICS; HEME; PROTEIN; ACTIVE; SITES

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.

这个建议涉及过氧化物酶，典型的在这个 酵母细胞色素c过氧化物酶的情况下，和单体氧- 结合蛋白，以二鳃甘油脂单体为代表 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 突变蛋白质最大化NMR表征和量化血红素， 网站化学 在细胞色素c过氧化物酶的情况下， 建议在我们过去取得的重大成功的基础上， 一年使用各种2D NMR进行质子NMR分配 方法，以扩展这些分配。 专门创建 位点特异性突变蛋白将在这一努力中有用。 同位素标记（15 N和13 C）将用作分配工具。 此外，个人属性的表征 突变蛋白质将通过NMR进行。 的情况 二鳃海鞘单体血红蛋白，其结构是 几乎可以与抹香鲸肌红蛋白的性质重叠， 建议核磁共振与同位素标记的目标， “总”任务。 我们打算证明核磁共振方法， 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力学， 预计会以预期的方式改变血红素-球蛋白的相互作用。 此外，我们打算利用这些结果和其他结果， 测试我们设计和创造突变体的能力， 根据晶体结构的推断，确定特定的目的。