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

生物大分子中的不成对电子自旋密度可以 这大大限制了NMR作为独立的 结构工具，即使当其它参数（溶解度、尺寸 有利于核磁共振结构研究。 虽然方法已经 在许多情况下允许识别共振， 由于超精细相互作用， 很难获得完整的顺序共振分配， 含有顺磁性中心的蛋白质。 最近我们 报道了广泛的顺序1H共振分配，并描述了 putidaredoxin（Pdx）的二级结构元件， 从恶臭假单胞菌中分离出含有S-2的铁氧还蛋白。 虽然 在细化氧化物的溶液结构方面取得了进展， Pdx（Fe+3-Fe+3），由于靠近1H共振峰， 金属簇导致光谱信息的相当大的损失。 用约 氧化Pdx中所有质子共振的14%不可观察 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。 近日 锌重建的rubredoxin，一种蛋白质，通常含有 单铁离子四面体配位四个半胱氨酸硫 配体，已经报道。 然而，两种“无机”的存在 在2-Fe S-2簇中桥接铁原子的硫离子 带来了进一步的复杂性。 据我们所知，没有Fe 2S 2 铁氧还蛋白已经成功地用非天然金属 离子。 我们目前表明，Ga+3是有用的重建 的Pdx，得到折叠的蛋白质，其在结构上类似于 天然（含离子）形式。 质谱分析提供了 蛋白质镓复合物组成的重要探针。 一篇论文描述了这项工作出现在J。Chem.Soc.117（1995） 6625. 实验计划研究蛋白质锌络合物。 在我们用镓putidaredoxin进行实验的过程中， 发现该分子在巯基上进行了共修饰， 功能。 确定了修饰位点， 发现修饰的来源是巯基乙醇， 用于确保还原环境。 将还原剂改为 二硫苏糖醇摆脱了不必要的修饰， 成功的高分辨率X射线晶体学的方法 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.