ZINC COORDINATION AND REGULATION OF ZINC METALLOPROTEINS

锌金属蛋白的锌配位和调节

• 批准号: 6385937
• 负责人: DAVID P. GIEDROC
• 金额: $ 24.56万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385937
• 关键词: DNA binding protein; analytical ultracentrifugation; atomic absorption spectrometry; binding sites; biological signal transduction; chemical kinetics; chemical stability; gene expression; gene induction /repression; metal complex; metal poisoning; metalloproteins; metallothionein; molecular site; nuclear magnetic resonance spectroscopy; protein structure function; sedimentation equilibrium; stoichiometry; thermodynamics; thermostability; transcription factor; virus protein; zinc

This proposal requests continued support for ongoing studies of the structure, bioinorganic chemistry and allosteric regulation by zinc of selected zinc metalloregulatory transcription factors derived from mammalian and prokaryotic organisms which play roles in zinc homeostasis. Human metal-responsive element (MRE)-binding transcription factor-1 (MTF-1) possesses six Cys2-His2 zinc finger domains and activates the expression of metallothionein genes in a zinc-dependent manner. Previous studies led the PI to hypothesize that the N-terminal four fingers play a structural role in mediating high affinity binding to the MRE while the C-terminal finger domains perform a zinc sensor and/or transducer role. The following experiments on MTF-1 are proposed: 1) Define the solution structure of a C-terminal zinc finger fragment (finger domains 4-6) of MTF-1 using multi-dimensional NMR spectroscopy. These studies will be complemented by equilibrium and kinetic metal binding studies to delineate the unusual metal binding properties of these metalloregulatory domains; and 2) Test the metalloregulatory model of MTF-1 function in vivo by determining the metal-dependent transcriptional activation efficacy of broken-finger mutants of intact MTF-1 by transfection experiments in mammalian and yeast cells. Comparative studies of three evolutionarily related prokaryotic zinc metalloregulatory repressors, Synechococcus SmtB, Synechocystis ZiaR, S. aureus CzrA and a cadmium repressor, S. aureus pI258 CadC are also proposed. These studies will provide a critical test of the metalloregulatory hypothesis which holds that the biological specificity and mechanism of regulation is encoded in the bioinorganic chemistry (metal coordination chemistry, specificity, affinity and stoichiometry) and the thermodynamic linkage relationships between metal binding, DNA binding and protein self-association of these metal switch proteins. Results of the proposed studies will provide new molecular insights into the relationships between structure, coordination chemistry and regulation by metal ions in metalloregulatory transcription factors, which, over the longer term, will permit their systematic re-engineering as templates for metal-site redesign and metal sensors with novel properties, tailored to biomedical and bioremediation uses.

该提案要求继续支持持续研究 锌的结构，生物无机化学和变构调节 源自哺乳动物的锌金属调节转录因子 以及在锌稳态中扮演角色的实质性生物。人类 金属响应元件（MRE）结合转录因子1（MTF-1）拥有 六个Cys2-His2锌指域，并激活 金属硫硫蛋白基因以锌的依赖性方式。先前的研究领导了PI 假设N末端四根手指在 介导高亲和力与MRE的高亲和力结合，而C-末端指域则介导 执行锌传感器和/或换能器角色。以下实验 提出了MTF-1：1）定义C末端锌的溶液结构 MTF-1使用多维NMR的手指碎片（手指域4-6） 光谱法。这些研究将通过平衡和动力学补充 金属结合研究描绘了不寻常的金属结合特性 这些金属调节域； 2）测试的金属调节模型 通过确定金属依赖性转录，MTF-1在体内功能 通过转染完整的MTF-1的破碎手指突变体的激活功效 哺乳动物和酵母细胞的实验。三个比较研究 进化相关的核锌金属调节阻遏物， Sychococcus SMTB，Synechocystis Ziar，S。金黄色葡萄球菌和镉阻遏物， 还提出了金黄色葡萄球菌PI258 CADC。这些研究将提供关键 金属调节假设的测试，该假设认为生物学 调节的特异性和机制在生物无机中编码 化学（金属协调化学，特异性，亲和力和 化学计量学）和金属之间的热力学连接关系 这些金属开关的结合，DNA结合和蛋白质自我关联 蛋白质。拟议研究的结果将提供新的分子见解 进入结构，协调化学和调节之间的关系 在金属调节转录因子中，金属离子在 长期，将允许其系统重新设计为模板 金属站点的重新设计和具有新型特性的金属传感器，量身定制 生物医学和生物修复用途。