| 光谱学与光谱分析 |
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| Probe of the Effect of Asp44 on the Stability of Myoglobin by Circular Dichroism Spectropolarimeter |
| WANG Jing-yun1,LIU Dan1,TANG Qian1,2,BAO Yong-ming1,ZHENG Xue-fang2,AN Li-Jia1 |
1. School of Environmental & Life Science, Dalian University of Technology, Dalian 116024, China
2. Liaoning Key Lab of Bioorganic Chemistry, Dalian University, Dalian 116622, China |
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Abstract 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 ℃, from 71.9 to 75.1 ℃, but shows little effect on its resistance to acid denaturation.
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Received: 2006-11-18
Accepted: 2007-02-26
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Corresponding Authors:
WANG Jing-yun
E-mail: wangjingyun@dl.cn
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