NMR SPECTROSCOPY OF APO-SUPEROXIDE DISMUTASE

载脂蛋白超氧化物歧化酶的核磁共振波谱

• 批准号: 8361261
• 负责人: JAMES W WHITTAKER
• 金额: --
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361261
• 关键词: Behavior; Binding; Enzymes; Escherichia coli; Eubacterium; Funding; Grant; Growth; Heating; High temperature of physical object; In Vitro; Iron; Manganese; Manganese Superoxide Dismutase; Metal Ion Binding; Metals; NMR Spectroscopy; National Center for Research Resources; Physiological; Principal Investigator; Process; Property; Proteins; Research; Research Infrastructure; Resources; Salts; Source; Superoxide Dismutase; Temperature; Thermus thermophilus; United States National Institutes of Health; Vanadium; cold temperature; cost; metal complex; protein misfolding; protein structure; uptake

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Manganese superoxide dismutase from the extremely thermophilic eubacterium Thermus thermophilus has been cloned and expressed at high levels in a mesophilic host (Escherichia coli) as a soluble tetrameric protein mainly present as the metal-free apo-enzyme. Incubation of the purified apo-enzyme with manganese salts at ambient temperature did not restore superoxide dismutase activity, but reactivation could be achieved by heating the protein with Mn(II) at higher temperatures, approaching the physiological growth temperature for T. thermophilus. Heat annealing followed by incubation with manganese at lower temperature fails to reactivate the enzyme, demonstrating that a simple misfolding of the protein is not responsible for the observed behavior. The in vitro metal uptake is nonspecific, and manganese, iron, and vanadium all bind, but only manganese restores catalytic activity. Bound metal ions do not exchange during heat treatment, indicating that the formation of the metal complex is effectively irreversible under these conditions. The metallation process is strongly temperature-dependent, suggesting that substantial activation barriers to metal uptake at ambient temperature are overcome by a thermal transition in the apo-protein structure. We will study these properties by NMR spectroscopy.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 来自极端嗜热真细菌嗜热栖热菌的锰超氧化物歧化酶已在嗜温宿主（大肠杆菌）中克隆并高水平表达，作为可溶性四聚体蛋白，主要以不含金属的脱辅基酶形式存在。在环境温度下将纯化的脱辅基酶与锰盐一起孵育不会恢复超氧化物歧化酶活性，但可以通过在更高温度下加热蛋白质与 Mn(II) 来实现重新激活，接近嗜热链球菌的生理生长温度。热退火，然后在较低温度下与锰一起孵育，无法重新激活酶，这表明蛋白质的简单错误折叠并不是造成观察到的行为的原因。体外金属吸收是非特异性的，锰、铁和钒都结合，但只有锰恢复催化活性。结合的金属离子在热处理过程中不会交换，表明金属配合物的形成在这些条件下实际上是不可逆的。金属化过程强烈依赖于温度，这表明脱辅基蛋白结构中的热转变克服了环境温度下金属吸收的大量活化障碍。我们将通过核磁共振波谱研究这些特性。