高等学校化学学报 ›› 2009, Vol. 30 ›› Issue (5): 928.

• 研究论文 • 上一篇    下一篇

神经红蛋白突变体Tyr44Phe的制备、表征和稳定性研究

李海丽1, 李连之1,2, 郭玉静1, 田素燕1, 薛泽春1, 姜玉岗1   

    1. 聊城大学化学化工学院, 聊城 252059;
    2. 南京大学配位化学国家重点实验室, 南京 210093
  • 收稿日期:2008-09-22 出版日期:2009-05-10 发布日期:2009-05-10
  • 通讯作者: 李连之, 男, 博士, 教授, 主要从事生物无机化学研究, E-mail: lilianzhi1963@163.com
  • 基金资助:

    国家自然科学基金(批准号: 20471025)资助.

Preparation, Characterization and Stability of Neuroglobin Mutant Tyr44Phe

LI Hai-Li1, LI Lian-Zhi1,2*, GUO Yu-Jing1, TIAN Su-Yan1, XUE Ze-Chun1, JIANG Yu-Gang1   

    1. School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, China;
    2. State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, China
  • Received:2008-09-22 Online:2009-05-10 Published:2009-05-10
  • Contact: LI Lian-Zhi, E-mail: lilianzhi1963@163.com

摘要:

构建了突变体蛋白Tyr44Phe的基因, 进行了蛋白的表达、分离纯化、谱学表征和稳定性研究. 由电喷雾质谱所得突变体蛋白的分子量与理论值一致; UV-Vis吸收光谱、荧光光谱和圆二色光谱表明, Tyr44Phe的点突变虽没有改变血红素的六配位结构, 但对血红素的构象有所影响. 突变体蛋白的热、酸稳定性研究表明, 定点突变降低了血红素与蛋白肽链之间的结合力, 导致血红素易从疏水腔中脱出, 说明Tyr44对蛋白的结构稳定性起一定的作用.

关键词: 神经红蛋白, 突变体Tyr44Phe, 稳定性

Abstract:

Neuroglobin(Ngb) is a recently discovered member of the hemoglobin superfamily in nervous system of vertebrates. Tyr44 nearing bis-histidyl ligand His64 contacts with one of the heme propionates by hydrogen bonds, which may involue in the stabilization for the hexacoordinate state of neuroglobin. In order to investigate the role of Tyr44 in structural stabilization of neuroglobin, the neuroglobin mutant Tyr44Phe gene was constructed by site-directed mutagenesis, and the mutant protein was expressed, purified and characterized spectroscopicaly, and its stability was also studied. The electrospray ionization mass spectroscopy result show that the molecular weight of the mutant Tyr44Phe corresponds with the theoretical value. The UV-Visible spectra, fluorescence spectra and circular dichroism(CD) spectra of mutant Tyr44Phe indicated that the mutagenesis doesn′t change neuroglobin’s bis-histidyl heme hexacoordination, but has an effect on heme configuration. Results of stability towards heat and acid show that the mutagenesis decreases the interaction of heme with peptide chain and the heme’s hydrophobic cavity becomes flexible, which proves that Tyr44 plays an important role in neuroglobin structural stability.

Key words: Neuroglobin, Mutant Tyr44Phe, Stability

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