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.

该研究项目包括以下两个主题。（1）天然嵌合蛋白，COOA蛋白的结构表征及其分子机制，用于研究COOA蛋白的结构表征，该蛋白具有肌红蛋白样的肌红蛋白样的血红素结合结构域和螺旋 - 螺旋 - 螺旋 - 螺旋 - 螺旋型型DNA使用结合结构域，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，而在没有CO的情况下，NMR Spectrum Spectrum Spectrum，NMR Spectrum Spectrum spectrum spectrum spection of Co的连接。在血红蛋白中这是非常不寻常的。尽管CO在COOA蛋白中的CO重新结合速率与肌红蛋白或血红蛋白的关联率相当，但其特征在于它在CO重组中非常快（<ns）重新固定过程的很大一部分。这么大的一小部分非常快速的重现过程可以归因于CO结合位点周围的空间障碍，并表明CO的连接或将某些氨基酸残基在结合位点附近，从而诱导了大型结构重排，以提高有效的效率DNA结合。（2）通过模块替代的新嵌合金属蛋白的蛋白质工程。该研究项目中全球蛋白的系统模块取代已表明，先前提出的模块不是最小的结构单位，但可以进一步分为两个“子模块” ”。特别是，我专注于其中一个“亚模型”，“血红素结合模块”，该模块由血红素近端的铁辅助组氨酸和约20个氨基酸残基组成。在各种光谱数据的基础上，包括NMR和NMR和共振拉曼光谱，可以得出结论，“血红素结合模块”将是血红素的电子状态的主要决定因素，并且是球蛋白中配体组氨酸的构型。