圆二色谱研究Asp44在稳定肌红蛋白结构中的作用

为了了解肌红蛋白Mb分子表面44位天冬氨酸对稳定肌红蛋白结构的影响，用PCR定点突变的技术将肌红蛋白(Mb)基因上的第44位天冬氨酸的密码子GAT突变成赖氨酸的密码子AAA，获得突变体D44K基因，将突变体基因克隆在肌红蛋白表达质粒pGYM上，转化大肠杆菌BL21，突变蛋白D44K在大肠杆菌中大量表达，收集菌体。酶法裂解细菌，依次用硫酸铵沉淀、离子交换柱层析、凝胶柱层析分离纯化得突变肌红蛋白D44K。用圆二色谱分别研究野生型肌红蛋白及其突变体(D44K)的耐热及耐酸的变性过程。实验结果表明：用碱性氨基酸Lys取代酸性氨基酸Asp44残基，增强了肌红蛋白耐热变性能力，导致蛋白质的热变性中点温度T_m由71.9 ℃增加到75.1 ℃，但对肌红蛋白耐酸变性的能力影响不大。

Probe of the Effect of Asp44 on the Stability of Myoglobin by Circular Dichroism Spectropolarimeter

To characterize the roles played by surface-charged residue Asp44 in the structure stability of horse heart myoglobin, the code of Asp44, GAT, in the gene of horse heart myoglobin was changed into AAA for Lys by PCR site-directed mutagenesis. The mutant gene was ligated into PstI/BamHI-cut pGYM and the resulting plasmid was transformed into E. coli BL21. The mutant protein (D44K) was expressed in BL21 successfully. The bacteria containing mutant myoglobin were treated with lysozyme. Then the mutant protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration. Circular dichroism spectra were employed to monitor the kinetic behaviors of wild-type and mutant myoglobins' denaturation at different pHs or upon heating, and the "two-state" model was used to simulate the kinetic process of wild-type and mutant myoglobins' denaturation upon heating to determine the unfolding thermodynamic parameters of Mb and its mutant (D44K). The results show that the mutation of the surface-charged residue Asp44 to Lys44 can increase the protein's stability on its resistance to heat, resulting in the increase in the protein's denaturing mid-temperature by about 4 degrees C, from 71.9 to 75.1 degrees C, but shows little effect on its resistance to acid denaturation.