STRUCTURE/FUNCTION CORRELATES OF PROTEIN OXIDATION

蛋白质氧化的结构/功能相关性

• 批准号: 2154583
• 负责人: PAUL M HOROWITZ
• 金额: $ 14.67万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2154583
• 关键词: chemical binding; chemical fingerprinting; circular dichroism; conformation; enzyme mechanism; enzyme substrate; fluorescent dye /probe; free radical oxygen; gel electrophoresis; high performance liquid chromatography; hydrogen peroxide; light scattering; oxidation reduction reaction; posttranslational modifications; protein sequence; protein structure function; proteolysis; site directed mutagenesis; sulfur group transferase; ultraviolet spectrometry

Proteins are targets of reactive oxygen species (ROS) that are produced in the environment and in the course of normal and xenobiotic metabolism. Protein oxidation is often detrimental, but it may be involved in normal biological processes. Hydrogen peroxide is a common ROS produced in the environment and in the cell, e.g. the mitochondrion. Hydrogen peroxide is the mildest of the ROS and is the most likely to act at sites distant from it's generation. Rhodanese, a sulfur-transfer enzyme in the mitochondrial matrix, can regulate iron-sulfur centers, and it may be controlled by oxidation. Rhodanese can be reversibly and irreversibly modified by hydrogen peroxide. Properties of modified rhodanese are those reported to be involved in damage, aging and turnover. This research program will characterize the altered forms and will determine the mechanism for conformational changes triggered by oxidation. Analyses can be pursued in unprecedented detail because of extensive kinetic and X-ray information already available for rhodanese. Our working hypotheses are: a) oxidation can be considered as a post- translational protein modification; b) oxidative damage is progressive and can be initiated by reaction at specific sites; c) oxidative effects can be propagated to distant regions of the protein; and d) oxidation and conformational changes are reciprocally linked, each facilitating the other. The specific approaches would be as follows: I. Oxidized amino acids will be located in the sequence of reversibly and irreversibly oxidized rhodanese. Oxidation states of sulfhydryl groups will be determined. II. Sequencing methods will be used to determine regions of the protein, not directly oxidized, that respond to oxidation. This is possible because oxidation reveal new antigenic determinants and exposes sites of proteolysis. III. Non-covalent conformational consequences of oxidation will be probed by physico-chemical methods. Changes in conformation, flexibility and domain interactions will be measured using: fluorescence, circular dichroism, tritium exchange, light scattering and hydrophobic chromatography. IV. Directed mutagenesis will modify regions of rhodanese suggested to either trigger or respond to oxidative changes. Site directed mutagenesis will change cys to ser and remove prolines in the interdomain tether. The N-terminal residues 1-23 will be deleted. C-terminal deletions will be produced by treating recombinant rhodanese with carboxypeptidase. These species will be studied as above.

蛋白质是产生的活性氧（ROS）的靶标 环境以及正常和异种生物代谢的过程。 蛋白质氧化通常是有害的，但可能与正常有关 生物过程。 过氧化氢是在 环境和细胞中，例如线粒体。 过氧化氢是 ROS中最温和的，最有可能在远离地点作用 这是一代。 罗丹尼，线粒体中的硫 - 转移酶 矩阵，可以调节铁硫心中心，并且可以控制 氧化。 罗丹尼人可以通过可逆性和不可逆转地修改 过氧化氢。 修改的罗丹尼人的特性是据报道 参与损害，衰老和营业额。 该研究计划将 表征改变的形式，并将确定 氧化触发的构象变化。 可以在 由于广泛的动力学和X射线信息，前所未有的细节 已经可用于罗达尼人。 我们的工作假设是：a）氧化可以被视为后 翻译蛋白修饰； b）氧化损伤是进行性的，并且 可以在特定部位通过反应引发； c）氧化作用可以是 传播到蛋白质的遥远区域； d）氧化和 构象变化是相互链接的，每种变化都促进 其他。 具体方法将如下： I.氧化的氨基酸将位于可逆的序列中， 不可逆转地氧化的罗丹尼人。 亚硫赖尔基团的氧化态 将确定。 ii。 测序方法将用于确定蛋白质的区域 未直接氧化，对氧化有反应。 这是可能的，因为 氧化揭示了新的抗原决定因素并暴露了 蛋白水解。 iii。 将探测氧化的非共价构象后果 通过物理化学方法。 构象，灵活性和 域相互作用将使用：荧光，圆形 二分性能，trip换，光散射和疏水 色谱法。 iv。 定向诱变将修改建议的罗丹尼人的区域 触发或响应氧化变化。 站点定向诱变 将更改Cys以SER并去除域间系绳中的脯氨酸。 这 N末端残基1-23将被删除。 C端缺失将是 通过用羧肽酶处理重组的罗丹尼人产生。 这些 物种将如上所述研究。