【目的】研究延长的N端肽链对铁蛋白结构稳定性及蛋白自组装的影响,为在N端融合生物分子构建功能性纳米材料奠定基础。【方法】在大肠杆菌铁蛋白N端添加6个组氨酸残基,所得重组蛋白采用体积排阻色谱、非变性凝胶电泳和圆二色谱等手段与野生型铁蛋白的聚合态、二级结构及热力学稳定性进行对比分析。【结果】体积排阻色谱表明野生型铁蛋白在溶液中以24聚体和二聚体混合物形式存在,但突变蛋白仅以二聚体的形式存在; 非变性凝胶电泳实验结果与体积排阻色谱结果吻合,即突变蛋白仅显示1条带,且与野生型铁蛋白的2聚体迁移位置基本相同; 圆二色谱结果表明突变蛋白的二级结构与野生型类似,呈典型的α-螺旋结构,突变蛋白的Tm值比野生型降低了1.1 ℃。【结论】在大肠杆菌铁蛋白亚基的N端增加6个组氨酸残基所得突变蛋白的二级结构没有发生明显变化,但却无法形成24聚体壳状结构,在溶液中仅以二聚体的形式存在,且热力学稳定性有所降低。N端的延长可能导致铁蛋白单体构型发生改变,因而无法进一步组装成壳状结构。
Abstract
【Objective】Investigate the effect of N-terminal elongation on the structural stability and protein self-assembly of the nano-cage bacterioferritin to provide the basis for the construction of functional nanomaterials by incorporating biomolecules at the N-terminus of bacterioferritin.【Method】Escherichia coli bacterioferritin protein was genetically engineered by elongating the N-terminus with six histidine residues. The mutant oligomerization state, secondary structure and thermo-stability were characterized and compared with that of the wild type using size exclusion chromatography, native gel electrophoresis and circular dichroism spectroscopy.【Result】The wild type formed a mixture of 24-mer and dimer in solution, while the mutant formed solely dimer. Consistent with the size exclusion chromatography experiment, only one band was observed corresponding to the wild-type dimer according to the native gel electrophoresis. Characterization of the mutant using circular dichroism demonstrated that it still folded into α-helical structure, exhibiting similar secondary structure as compared to the wild type. The melting temperature of the mutant was found to be 1.1 ℃ lower than that of the wild type. 【Conclusion】Although it still folded into the α-helical structure, the protein with an elongated N-terminus completely lost the ability to form the nano-cage structure, and formed dimer solely in solution with reduced thermo-stability compared with that of the parent protein. This study indicates that N-terminal elongation might result in a conformational change of bacterioferritin subunits, preventing the protein from self-assembling into the nano-cage structure.
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基金
基金项目:国家自然科学基金项目(31200564); 江苏高校品牌专业建设工程资助项目; 江苏省大学生实践创新训练计划项目(2013)
第一作者:张瑜(yuzhang@njfu.edu.cn),副教授,博士。
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