RECONSTITUTION OF ION FERREDOXIN W/ DIAMAGNETIC METAL ION: GALLIUM PUTIDAREDOXIN

用抗磁性金属离子重建离子铁氧还蛋白：镓腐铁氧还蛋白

• 批准号: 6307603
• 负责人: Thomas Charles Pochapsky
• 金额: $ 0.82万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6307603
• 关键词: biological products; biomedical resource; proteins; spectrometry

Unpaired electron spin density in biological macromolecules can significantly limit the application of NMR as an independent structural tool, even when other parameters (solubility, size are favorable for NMR structural studies. Although methods have been developed which in many cases allow identification of resonances which are shifted and/or broadened by hyperfine interactions, it is often difficult to obtain complete sequential resonance assignments for proteins which incorporate paramagnetic centers. Recently, we have reported extensive sequential 1H resonance assignments and described the secondary structural elements of putidaredoxin (Pdx), a 2-Fe S-2-containing ferredoxin isolated from Pseudomonas putida. Although progress has been made in refining the solution structure of oxidized Pdx (Fe+3-Fe+3), broadening of 1H resonances due to proximity to the metal cluster results in a considerable loss of spectral information. with ca. 14% of all proton resonances i n oxidized Pdx unobservable due to hyperfine interactions. The metal cluster is required for folding, as the apoprotein is virtually structureless. Recently, the zinc reconstitution of rubredoxin, a protein which normally contains a single iron ion tetrahedrally coordinated by four cysteinyl sulfur ligands, has been reported. However, the presence of two "inorganic" sulfide ions which bridge the iron atoms in a 2-Fe S-2 cluster introduces a further complication. To our knowledge, no Fe2S2 ferredoxins have been successfully reconstituted with non-native metal ions. We are presently shown that Ga+3 is useful for reconstitution of Pdx, giving a folded protein which is structurally similar to the native (ion-containing) form. Mass spectrometry is providing an important probe of the composition of the protein gallium complexes. A paper describing this work appeared in J. Am. Chem. Soc. 117 (1995) 6625. Experiments are planned to investigate protein Zn complexes. During the course of our experiments with gallium putidaredoxin, we di scovered that the molecule is co-valently modified at the thiol functionalities. The sites of modification were established and the source of the modification found to be the mercaptoethanol that was used to ensure a reducing environment. Changing the reducing agent to dithiothreitol got rid of the unwanted modification and opened up the way to successful high resolution X-ray crystallography of certain ferredoxins whose structure had previously been refractory to X-ray analysis.

生物大分子中未配对的电子自旋密度可以 显着限制了NMR作为独立的应用 结构工具，即使其他参数（溶解度，大小为 有利于NMR结构研究。 尽管方法已经 开发在许多情况下允许识别共振 被超精美相互作用转移和/或拓宽，通常是 难以获得完整的顺序共振分配 蛋白质的蛋白质融合了顺磁中心。 最近，我们有 报告了广泛的顺序1H共振分配，并描述了 putidaredoxin（PDX）的二级结构元素，2-FE 从假单胞菌分离的含S-2的含S-2的铁氧还蛋白。 虽然 在完善氧化的溶液结构方面取得了进展 PDX（Fe+3-Fe+3），由于靠近距离的1H共振扩大 金属簇导致光谱信息的大量丢失。 与大约 所有质子共振的14％不可观察 由于超细相互作用。 需要金属簇 折叠，因为载脂蛋白实际上是没有结构的。 最近， Rubredoxin的锌重建，一种通常包含一个的蛋白质 单铁离子四面体由四个半胱氨酸硫协调 配体已有报道。 但是，存在两个“无机” 在2-FE S-2簇中桥接铁原子的硫化物离子 引入了进一步的并发症。 据我们所知，没有FE2S2 铁蛋白已成功地用非本地金属重构 离子。 目前，我们表明GA+3对于重组有用 PDX的折叠蛋白，在结构上与 天然（含离子）形式。 质谱法正在提供 对蛋白质危险复合物组成的重要探针。 一篇描述这项工作的论文出现在J. Am中。化学 Soc。 117（1995） 6625。计划研究蛋白质Zn复合物。 在我们使用甘油putidaredoxin的实验过程中，我们可以 Scover认为该分子是在硫醇中共价修饰的 功能。 建立了修改的地点，并 修改的来源被认为是墨塔乙醇 用于确保还原环境。 将还原剂更改为 Dithiothreitol摆脱了不需要的修改，并打开了 成功的高分辨率X射线晶体学的方法 铁蛋白的结构以前是X射线的难治性的 分析。