Protein Engineering and Reactivity Control in Multi-functional Chimeric Metalloproteins

本研究课题包括以下两个课题。（1）天然嵌合蛋白CooA蛋白的结构表征及其分子机制为了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结合结构域的CooA蛋白的结构表征，利用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，通过NMR光谱建议连接精氨酸或赖氨酸残基而不是CO，这在血红素蛋白中是非常不寻常的。虽然CooA蛋白中CO再结合的结合率与肌红蛋白或血红蛋白中的结合率相当，但其特征在于CO再结合中的非常快（<ns）的再结合过程的大部分。这种非常快速的再结合过程的大部分可以归因于CO结合位点周围的空间位阻，并表明CO的连接踢出或推出结合位点附近的一些氨基酸残基，诱导大的结构重排以实现有效的DNA结合。（2）通过模块替换实现新型嵌合金属蛋白的蛋白质工程本研究项目对球蛋白进行的系统性模块替换表明，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”。特别是其中的一个“子模块”--血红素结合模块，它由铁配体组氨酸和血红素近端的大约20个氨基酸残基组成，基于包括NMR和共振拉曼光谱在内的各种光谱数据，可以得出结论，“血红素结合模块”是血红素电子状态和球蛋白中配体组氨酸构型的主要决定因素。

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.