影响大肠埃希菌 FtsZ 功能和自身组装的重要氨基酸分析

Analysis of the key amino acids involved in the function and cellular self-assembly of FtsZ protein in Escherichia coli strains

目的：探索大肠埃希菌（ Escherichia coli， E．coli） FtsZ 突变体 FtsZP74R、FtsZG77D和FtsZA81R对FtsZ功能和胞内自身组装的影响。方法利用分子克隆和定点突变技术，常规构建带His或YFP标签的FtsZ及其突变体融合表达质粒，亲和纯化得到相应的蛋白；采用同源重组技术构建FL37（△ftsZ-Cat）菌株；活细胞成像观察FtsZ及其突变体在胞内定位模式；免疫共沉淀试验检测FtsZ与其突变体间的相互作用；光扫描检测定点突变对FtsZ组装特性的影响；高压液相色谱法检测FtsZ突变体GTPase酶活性的变化。结果 FtsZP74R、FtsZG77D和FtsZA81R突变体在E．coli内不能正确定位和形成功能性Z环、FtsZ-FtsZ倡单体间的相互作用减弱或消失、突变体体外聚合效率减少及FtsZA81R的GTPase酶活性显著降低。结论 P74、G77和A81是影响FtsZ功能和胞内正确组装的重要氨基酸；A81可同时影响FtsZ的侧面相互作用和GTPase活性。

Objective To investigate the self-assembly and cellular localization patterns of fila-mentous temperature-sensitive protein Z （FtsZ） in Escherichia coli （E.coli） strains by using FtsZP74R, FtsZG77D and FtsZA81R mutants.Methods YFP or His labeled FtsZ proteins and the plasmids of FtsZ mu-tants were constructed by using molecular clone and site-directed mutagenesis methods.The targeted proteins were purified by affinity chromatography.FL37（△ftsZ-Cat） strains were constructed via linear DNA homol-ogous recombination.Living cell imaging was performed to observe the cellular localization patterns of FtsZ protein and its mutants in E.coli strains.The interactions between FtsZ-FtsZ/FtsZ mutants were examined by coi-mmunoprecipitation assay . The polymerization properties of FtsZ mutants were analyzed by light scattering.The activities of GTPase were monitored by using high performance liquid chromatography.Re-sults The P74, G77 and A81 amino acids were respectively replaced by different polar amino acids to change the amphipathicity of the helix within the domain of FtsZ （ 74-82 ） .The YFP-labeled FtsZP74R , FtsZG77D and FtsZA81R mutants failed to assemble into functional Z-ring structure in E.coli strains.The inter-actions between FtsZ protein and its mutants were weakened or completely disappeared.In addition, in vitro experiments showed that P74R, G77D and A81R mutations caused a decrease in the polymerization efficien-cy of FtsZ monomer.The activity of GTPase was significantly decreased in the FtsZA81R mutant. Conclusion The P74, G77 and A81 were critical amino acids in the function and assembly of FtsZ protein in E.coli strains.Moreover, A81 amino acid regulated the lateral interaction of FtsZ monomer and the activity of GTPase.