RECONSTRUCION OF TWO ION FERREDOXIN W/ DIAMAGNETIC METAL ION

用抗磁性金属离子重建双离子铁氧还蛋白

• 批准号: 6118277
• 负责人: Thomas Charles Pochapsky
• 金额: $ 0.09万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-10 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6118277
• 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 resonanc es 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 discovered that the molecule is co-valently modified at the thiol functionalities. The sties of modification were established and the source of the modifications found to be the mercaptoethanol that was used to ensure a reducing environment. Changin g 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.

在生物大分子中，不成对的电子自旋密度可以