REACTIVITY AND FUNCTIONS OF LOBSTER METALLOTHIONEIN

龙虾金属硫蛋白的反应性和功能

• 批准号: 6195807
• 负责人: C Frank SHAW
• 金额: $ 2.19万
• 依托单位: EASTERN KENTUCKY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-25 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6195807
• 关键词: Malacostraca; X ray crystallography; cadmium; chemical kinetics; chemical models; computer simulation; copper; electrophoresis; enzyme activity; hydrogen peroxide; hydroxyl radical; intermolecular interaction; ligands; metal metabolism; metallothionein; nuclear magnetic resonance spectroscopy; protein biosynthesis; protein sequence; protein structure function; sulfur; superoxides; zinc

Metallothioneins (MT) are ubiquitous, cysteine-rich metal-binding protein which are present in most animal tissues. MTs have been associated with [1] essential metal metabolism (zn, Cu); [2] sequestration of environmentally accumulated toxic metals (cd, Hg); [3] scavenging of reactive oxygen species (ROS); and [4] cellular metabolism of electrophilic alkylating agents and metallodrugs (e.g., melphalan and chlorambucil cisplatin and aurothiomalate). Crustacean and mammalian MTs both have two- domain structures in which each domain uses 9-11 cysteine residues to create a metal-thiolate cluster which binds 3-6 metal ions (Zn, Cd, Cu). Our proposal is based on the hypothesis that the two domain structure, coupled with strong preferences for certain reactions in each domain, is the chemical basis that allows different functions to occur simultaneously and with minimal interference. To test this, we will determine the structure of lobster MT-1 by 2D and 3D NMR methods; differences in protein folding between lobster and mammalian MTs indicate that lobster MT is an important structure-function model. We will synthesize peptides of each domain (betaN and betac formed at the N- and C-termini) and reconstitute them with metal ions for kinetic and thermodynamic studies of their reactions. The proclivity of each domain for reactions with metal ions (Cu, Zn, Cd), apoZn-enzymes, electrophiles and metal-chelates and ROS will be compared to the holo-protein reactions. We anticipate that the differential (relative) reactivity of the domains (alpha more beta or beta more alpha) reverses for specific reactions. Some reactions (e.g. metal sequestration) are expected to occur under thermodynamic control and others (e.g. ROS scavenging and many electrophile reactions) under kinetic control. Molecular modeling will be used to provide a structural basis for domain reactions. The results of this proposal should move MT research from the moot question of which is the "real" function of MT to asking how these functions co-exist in a single protein. They will provide a better basis for considering the competing effects of normal metabolic states, pathological conditions and drug therapies that converge via MT-dependent reactions.

金属霉素（MT）是普遍存在的，富含半胱氨酸的金属结合蛋白 大多数动物组织中存在。 MT与 [1]必需金属代谢（Zn，Cu）； [2]隔离 环境积累的有毒金属（CD，HG）； [3]清除 活性氧（ROS）；和[4]亲电的细胞代谢 烷基化剂和金属糖（例如Melphalan和Chlorambucil 顺铂和aurothiomalate）。 甲壳类动物和哺乳动物MTS都有两个 每个域使用9-11半胱氨酸残基的结构结构 创建一个结合3-6个金属离子（Zn，CD，Cu）的金属硫醇簇簇。 我们的建议是基于以下假设。 结合每个域中某些反应的强烈偏好，IS 允许同时发生不同功能的化学基础 并减少干扰。 为了测试这一点，我们将确定 2D和3D NMR方法的龙虾MT-1结构；蛋白质的差异 龙虾和哺乳动物MT之间的折叠表明龙虾MT是 重要的结构功能模型。 我们将合成每个肽 域（在N-和C-Termini形成的Betan和Betac）和重组 它们与金属离子进行动力学和热力学研究 反应。 每个域与金属离子反应的倾向 （Cu，Zn，CD），Apozn-酶，电力和金属芝麻和ROS将 将其与全蛋白反应进行比较。 我们预计 域的差异（相对）反应性（alpha更多beta或beta 更多的α）逆转特定反应。 一些反应（例如金属 预计将在热力学对照和其他情况下发生固换） （例如，ROS清除和许多亲电反应）在动力学下 控制。 分子建模将用于为结构基础提供 域反应。 该提案的结果应移动MT研究 从moot的问题中，哪个是MT的“真实”功能到询问如何 这些功能在单个蛋白质中共存。 他们将提供更好的 考虑正常代谢状态的竞争影响的基础， 通过MT依赖性收敛的病理状况和药物疗法 反应。