神经红蛋白突变体(F28Y,F106Y)的构建、表达与表征

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

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.

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 phenylalanines（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 Sorer 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 heine disassociate from the protein chain more easily.