HEME ENZYME PROTEIN ENGINEERING

血红素酶蛋白质工程

• 批准号: 3301312
• 负责人: THOMAS L POULOS
• 金额: $ 3.99万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1991-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301312
• 关键词: Escherichia coli; X ray crystallography; aminoacid; catalase; cytochrome P450; cytochrome b; cytochrome c; cytochrome oxidase; electron transport; enzyme mechanism; heme; hemerythrin; mutant; peroxidases; protein engineering; protein structure; site directed mutagenesis; Escherichia coli; X ray crystallography; aminoacid; catalase; cytochrome P450; cytochrome b; cytochrome c; cytochrome oxidase; electron transport; enzyme mechanism; heme; hemerythrin; mutant; peroxidases; protein engineering; protein structure; site directed mutagenesis

The ability to clone, express, and manipulate the sequences of DNA molecules has provided a powerful new set of tools for studying the relationship between protein structure and function. One can now study the effects of specific amino acid replacements of function and more precisely determine the specific role of individual residues in an enzyme mechanisms, stability, and folding properties of proteins. The goal of this proposal is to apply these protein engineering techniques to the study of heme enzyme structure and function. Heme proteins have provided some valuable insights into the relationship between protein structure and function for the following reason. Most well studied heme proteins (globins, c- and b-type cytochromes, peroxidases, P450) all have exactly the same heme prosthetic group yet each exhibits very different and clearly defined functions. These difference are dictated by the interaction between protein and heme much of which has been deciphered through a variety of spectral probes and x-ray crystallography. Therefore, there are some specific questions that can be asked regarding the relationship between structure and function in heme proteins. The research in this proposal will focus on yeast cytochrome c peroxidase (CCP) where the reaction mechanism with peroxides, the interaction between CCP and cyt.c, and the stability of the two CCP structural domains will be studied using mutagenesis methods. CCP was chosen for the following reasons: 1) CCP has been cloned and expressed at high levels in E. coli; 2) the crystal structure is known; 3) CCP forms a tight, 1:1 electrostatic complex with cyt.c and has provided some of the most detailed structural information on electrostatic interprotein electron transfer complexes; and 4) the PI's laboratory has had extensive experience working with CCP and related enzymes.

克隆，表达和操纵DNA序列的能力 分子提供了一套强大的新工具来研究 蛋白质结构与功能之间的关系。现在可以学习 特定氨基酸替代功能的影响以及更多 精确确定个体残基在酶中的特定作用 蛋白质的机理，稳定性和折叠特性。目标 该建议是将这些蛋白质工程技术应用于 血红素酶结构和功能的研究。血红素蛋白提供了 对蛋白质结构之间关系的一些有价值的见解 和功能以下原因。最精心研究的血红素蛋白 （Globins，C-和B型细胞色素，过氧化物酶，P450）都完全具有 相同的血红素假肢群体但每个人的表现都大不相同， 明确定义的功能。这些差异由 蛋白质与血红素之间的相互作用已被解密 通过各种光谱探针和X射线晶体学。 因此，可以问一些有关的特定问题 血红素蛋白中的结构与功能之间的关系。这 该提案中的研究将重点放在酵母菌细胞色素C过氧化物酶上 （CCP）其中与过氧化物的反应机理相互作用 在CCP和CYT.C之间，以及两个CCP结构的稳定性 将使用诱变方法研究域。选择了CCP 以下原因：1）CCP已被克隆并以高水平表示 大肠杆菌； 2）已知晶体结构； 3）CCP形成一个紧密的1：1 用Cyt.C进行静电复合物，并提供了一些最多的 有关静电解释蛋白电子的详细结构信息 转移复合物； 4）PI的实验室已经广泛 使用CCP和相关酶的经验。