紫外吸收光谱法研究Asp44在稳定肌红蛋白结构中的作用

摘要

To understand the effect of surface-charged residue Asp44 on 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 sitedirected mutagenesis. The mutant protein was expressed in BL21 （ DE3 ） and purified successfully. UV-Vis spectra of wild-type and mutant myoglobins were measured under different temperatures and pHs. The results showed that the mutation of the surface-charged residue Asp44 to Lys44 increases the protein＇s stability on the resistance to heat and acid denature. This supplies important test gist for the study of superficial amino acid of proteins to affect on structure and function of protein.%为了了解肌红蛋白Mb表面44位天冬氨酸（Asp）残基对稳定蛋白结构的影响，用聚合酶链式反应（PCR）定点突变的技术将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。突变体蛋白在大肠杆菌BL21-DE3中成功表达并且得到纯化。用紫外-可见光谱研究野生型肌红蛋白及其突变体D44K的耐热、耐酸的变性过程。结果表明，用碱性氨基酸赖氨酸（Lys）取代酸性氨基酸Asp44残基，增强了肌红蛋白耐热、耐酸能力，说明Asp44具有稳定肌红蛋白结构的作用。为进一步研究蛋白表面氨基酸对蛋白质结构、功能的影响提供重要的试验依据。