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

生物大分子中未配对的电子自旋密度可以 大大限制了核磁共振作为一种独立技术的应用 结构工具，即使当其他参数(溶解度、尺寸 有利于核磁共振结构研究。尽管方法已经被 它在许多情况下允许识别共振， 被超精细相互作用移动和/或加宽，它通常是 很难获得完整的顺序共振分配 结合顺磁中心的蛋白质。最近，我们有 报告了广泛的顺序1H共振分配，并描述了 2-Fe-恶臭还蛋白(PDX)的二级结构元素 从恶臭假单胞菌中分离出含S-2的铁氧还蛋白。虽然 在精炼氧化的溶液结构方面取得了进展 PDX(Fe+3-Fe+3)，1H共振的展宽由于靠近 金属团簇导致光谱信息的大量损失。 在氧化的PDX中，约14%的质子共振是看不到的 由于超精细相互作用。金属簇需要用于 折叠，因为脱辅基蛋白实际上是无结构的。最近， Rubredosin的锌重组，这是一种通常含有一个 四个半胱氨酸硫配位的单铁离子 配体，已有报道。然而，两种“无机物”的存在 2-Fe S-2团簇中铁原子间的硫化物离子 引入了一个更复杂的问题。据我们所知，没有Fe2S2 铁氧还蛋白已成功地与非天然金属重组 离子。我们目前的研究表明，Ga+3对重构是有用的 得到了一种结构类似于PDX的折叠蛋白 天然(含离子)形式。质谱学正在提供一种 蛋白质镓络合物组成的重要探索。 一篇描述这项工作的论文发表在《J.Am》杂志上。化学。SoC。117(1995) 6625号。计划进行实验以研究蛋白质锌络合物。 在我们对镓的实验过程中，我们发现 发现分子是在硫醇上共价修饰的 功能。建立了修改的地点，并 被发现的修饰来源是硫醇 用于确保减少污染的环境。将还原剂改为 二硫苏糖醇去掉了不想要的修饰，打开了 高分辨X射线结晶学的成功之路 铁氧还蛋白，其结构以前对X射线不敏感 分析。

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.