血红素对大肠杆菌铁蛋白稳定性及自组装的影响

Effect of Heme on the Stability and Self-assembly of Escherichia coli Bacterioferritin

大肠杆菌铁蛋白(bacterioferritin,BFR)是由同源亚基24聚体组成的高度对称的八面体壳状结构,其中处在C2对称轴的每两个亚基之间即存在一个血红素分子。为了探讨血红素对细菌铁蛋白结构稳定性及在蛋白质自组装过程中的影响,通过定点突变将第52位蛋氨酸突变为丙氨酸以去除血红素分子,并应用体积排阻色谱、透射电镜、紫外-可见光吸收光谱及圆二色谱等手段对突变蛋白M52A的聚合态、二级结构及热力学性质等进行分析。结果表明:去除血红素虽然对铁蛋白的自组装没有明显影响,但是对其热稳定性产生较大影响,突变后蛋白质的T_m值为54.3℃,远小于野生型铁蛋白T_m值69.9℃,且经过氯化血红素与同浓度下该突变体M52A化学诱导结合后,发现其T_m值重新恢复至67.7℃。由此可知,血红素对大肠杆菌铁蛋白稳定性影响很大,且具有调控铁蛋白稳定性的作用。

Bacterioferritin （BFR） from Escherichia coli is composed of 24 subunits that assemble into a large spherical cage with octahedral symmetry, with one heme molecule located at each C2 symmetry interface. To study the effect of heme on the protein stability and self-assembly, heme molecules were removed through site-directed mutagenesis. The mutant protein M52A was expressed, purified and characterized using size exclusion chromatography, transmission electron microscopy, UV-Vis spectroscopy and circular dichroism. Results indicated that although little effect was observed on the self-assembly of protein structure, removal of heme showed great impact on its thermostability. The melting temperature （Tm） of mutant M52A was 54.3℃, which was much lower than that of its wild type 69.9 ℃. Interestingly, when mutant M52A was incubated with hemin chloride, the melting temperature was restored to 67.7 ℃. Hence, heme molecules located at C2 symmetry interfaces in the bacterioferritin has a regulatory effect and plays a key role in the protein thermostability.