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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 极端嗜热真细菌Thermus thermophilus的锰超氧化物歧化酶已被克隆，并以可溶性四聚体蛋白的形式在中温宿主(E.colia.)中高效表达，主要以不含金属的脱辅酶形式存在。纯化的脱辅酶与锰盐在常温下孵育不能恢复超氧化物歧化酶的活性，但可以通过在更高的温度下将蛋白与锰(II)一起加热来重新激活，接近嗜热链霉菌的生理生长温度。热处理，然后在较低的温度下与锰孵化，未能重新激活该酶，表明蛋白质的简单错误折叠不是观察到的行为的原因。体外金属摄取是非特异性的，锰、铁和钒都结合在一起，但只有锰才能恢复催化活性。结合的金属离子在热处理过程中不交换，表明在这些条件下金属络合物的形成实际上是不可逆的。金属化过程强烈依赖于温度，这表明在常温下金属吸收的实质性激活障碍可以通过脱辅蛋白结构中的热转变来克服。我们将用核磁共振波谱来研究这些性质。