高等学校化学学报 ›› 2006, Vol. 27 ›› Issue (4): 641.

• 研究论文 • 上一篇    下一篇

神经红蛋白突变体(F28Y, F106Y)的构建、 表达与表征

赵超1, 李连之1,2, 冀海伟1, 王莉1, 许涛1, 刘金凤1   

  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:2005-08-24 出版日期:2006-04-10 发布日期:2006-04-10
  • 通讯作者: 李连之(1963年出生), 男, 博士, 教授, 主要从事生物无机和配位化学研究. E-mail: lilianzhi1963@163.com
  • 基金资助:

    国家自然科学基金(批准号: 20471025)资助.

Construction, Expression and Characterization of Neuroglobin Mutants(F28Y, F106Y)

ZHAO Chao1, LI Lian-Zhi 1,2* , JI Hai-Wei1, WANG Li1, XU Tao1, LIU Jin-Feng1   

  1. 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:2005-08-24 Online:2006-04-10 Published:2006-04-10
  • Contact: LI Lian-Zhi,E-mail: lilianzhi1963@163.com

摘要:

构建了神经红蛋白F28Y, F106Y的两种突变体, 并进行了表达、 纯化和谱学表征. 电喷雾质谱表明突变体蛋白的分子量与理论值一致. 氧化型和还原型F28Y及F106Y的紫外可见吸收光谱与野生型相似, 仅氧化型F28Y的Soret 带有2 nm的蓝移, 说明这两种突变体蛋白仍保持六配位形式. F28Y的荧光最大发射峰明显红移(340 nm→347 nm), 表明其荧光基团更加暴露于极性环境中. 圆二色光谱表明, 突变体蛋白的α螺旋含量降低且F28Y产生了β折叠, 这是由于F28相对于F106则位于疏水腔外部且更加接近于溶剂表面所致. 热稳定性顺序为NGB>F28Y>F106Y, F106Y最不稳定, 是因为其与血红素间存在着较强的疏水作用, 突变使F106与血红素间的作用力减弱, 从而导致血红素在热变性条件下更容易从蛋白中解离出来.

关键词: 神经红蛋白; 突变体; 表征; 稳定性

Abstract:

Neuroglobin(NGB), a newly discovered member of the hemoglobin superfamily, display a hexacoordination His-Fe-His in the absence of external ligands. In order to investigate the role of phenyla1anines(F) near bishistidyl ligands, the two neuroglobin mutants(F28Y, F106Y) were constructed, expressed, purified and characterized. The electrospray ionization mass spectroscopy results showed that the molecular weight of the two mutants correspond with the theoretical values. The UV-visible spectra of oxidized and reduced F28Y and F106Y were in agreement with the wild NGB, but the oxidized F28Y′s Soret band had a 2 nm blue shift, which suggested that the two mutants still remain hexa-coordinated form. The obvious red shift of F28Y′s fluorescence emission peak(F28Y: 340 nm→347 nm) relative to the wild NGB indicated that the mutant protein′s fluorophores exposed to more polar environment. The circular dichroism spectra results showed that the percentage of α-helix in F28Y, F106Y decreased and the β-sheet occurred in F28Y, this is caused by the specific site of F28 which sites at the outside of hydrophobic pocket and near the solvent surface. Thermal stability is NGB>F28Y>F106Y, which reflects that F106Y is more unstable. This may be explained on the basis of the strong hydrophobic interaction between F106 and heme, and also the decreased interaction, enables the heme disassociate from the protein chain more easily.

Key words: Neuroglobin; Mutant; Characterization; Stability

中图分类号: 

TrendMD: