Protein Engineering and Reactivity Control in Multi-functional Chimeric Metalloproteins

多功能嵌合金属蛋白的蛋白质工程和反应控制

基本信息

批准号：
08458175
负责人：
ISHIMORI Koichiro
金额：
$ 4.22万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458175/
关键词：
Chimeric Proteins Gene Regulation CooA Protein

项目摘要

This research project includes the following two subjects.(1)Structural Characterization of Natural Chimeric Protein, CooA Protein, and Its Molecular MechanismTo investigate structural characterization of CooA protein, which has a myoglobin-like heme binding domain and helix-turn-helix type DNA binding domain, NMR, resonance Raman spectra and ligand binding were utilized. The resonance Raman spectrum for the ferrous CO adduct of CooA protein confirmed the ligation of a histidine residue to the heme iron, while, in the absence of CO, the ligation of an arginine or lysine residue instead of CO were suggested by the NMR spectrum, which is quite unusual in hemoproteins. Although the association rate for the CO rebinding in CooA protein was comparable to that in myoglobin or hemoglobin, it was characterized by its large fraction of the very fast (<ns) rebinding process in the CO recombination. Such a large fraction of the very fast rebinding process can be ascribed to the steric hindrance around the CO binding site and suggest that the ligation of CO kicks or pushes out some amino acid residues near the binding site, inducing the large structural rearrangements for the efficient DNA binding.(2)Protein Engineering for New Chimeric Metalloproteins by Module Substitution.Systematic module substitutions for globins in this research project have shown that the previously proposed module is not a minimum structural unit, but can be further divided into two "sub-modules". Particularly, I focused on one of the "sub-modules", "heme binding module", which consists of the iron-liganded histidine and about 20 amino acid residues in the heme proximal side.On the basis of various spectroscopic data including NMR and resonance Raman spectra, it can be concluded that the "heme binding module" would be a primary determinant for the electronic state of the heme and configuration of the liganded histidine in globins.

本研究项目包括以下两个课题：(1)天然嵌合蛋白CooA蛋白的结构表征及其分子机制研究具有肌红蛋白样血红素结合结构域和螺旋-转角-螺旋型DNA的CooA蛋白的结构表征利用结合域、NMR、共振拉曼光谱和配体结合。 CooA蛋白的亚铁CO加合物的共振拉曼光谱证实了组氨酸残基与血红素铁的连接，而在不存在CO的情况下，NMR光谱表明精氨酸或赖氨酸残基而不是CO的连接，这在血红素蛋白中很不寻常。尽管 CooA 蛋白中 CO 重新结合的关联率与肌红蛋白或血红蛋白中的关联率相当，但其特征在于 CO 重组中非常快 (< ns) 重新结合过程的很大一部分。如此快速的重结合过程的很大一部分可以归因于CO结合位点周围的空间位阻，并且表明CO的连接踢出或推出了结合位点附近的一些氨基酸残基，从而诱导了大的结构重排，以有效地实现重结合。 DNA结合。（2）通过模块替换对新型嵌合金属蛋白进行蛋白质工程。本研究项目中球蛋白的系统模块替换表明，先前提出的模块不是最小结构单元，而是可以进一步分为两个“子模块”。特别是，我重点关注了“子模块”之一“血红素结合模块”，它由铁配体组氨酸和血红素近侧约 20 个氨基酸残基组成。基于包括 NMR 和通过共振拉曼光谱，可以得出结论，“血红素结合模块”将是血红素的电子状态和球蛋白中配体组氨酸的构型的主要决定因素。