NADH荧光探针FrexH的结构模建与功能优化  

Structure modelling and functional optimization of NADH fluorescent probe FrexH

作　　者：陈强[1] 

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《计算机与应用化学》2015年第6期651-656,共6页Computers and Applied Chemistry

基　　金：国家自然科学基金(31171275)

摘　　要：FrexH是一种由枯草芽孢杆菌烟酰胺腺嘌呤二核苷酸(NADH)感受性转录抑制因子(B-Rex)的NADH结合结构域和循环排列黄色荧光蛋白(cpYFP)结构域嵌合而成的检测活细胞中NADH的新型蛋白荧光探针,因其荧光强度受体系中pH值波动变化影响而使其应用受到限制,因此该探针的结构需要进一步的改造。本文采用氨基酸位点特异性突变和添加柔性区段的改造方法,构建改造结构并利用同源模建和分子对接方法对比分析结构改造后功能的变化。经建模得到了真实度较高的cpYFP结构域模拟结构。经分子对接获得了NADH与FrexH的结合信息并分析了改造对NADH结合的影响。改造后的cpYFP结构域两端的loop区弯曲程度加强并更为靠近而使稳定性加强,邻近二级结构空间位置和桶状结构弯曲程度发生改变;改造后的B-Rex结合结构域氢键和原子间距离减小,空间位阻增加,范德华力加强,对接后的NADH配体更加内缩进受体结构。与外界环境接触面变小,质子交换程度下降。研究结果为类似的基于荧光蛋白核心的蛋白质工具构建提供了理论依据和优化思路,针对FrexH的改造有助于令其更有效地应用于高特异性高灵敏度NADH检测,以检测细胞代谢途径的特征并有助于相关的基础研究,增进对代谢途径的认知。Frex H is a new live-cell NADH fluorescent protein probe composed by NADH binding domain of Bacillus subtilis redox-sensing transcriptional repressor（B-Rex） and circular permuted yellow fluorescent protein（cp YFP） domain. Given its application is limited by its sensitivity to environmental p H fluctuation, the structure improvement is necessary. Site-direct mutagenesis and loop insertions were introduced into Frex H to build up an improved structure. Homology modelling and molecular docking were applied to comparison and analysis of structural and functional modification. Simulated structure of cp YFP domain with high fidelity is acquired by modelling. Binding of NADH to Frex H is acquired by molecular docking and analyzed for the effect of modification on NADH binding. Improved cp YFP domain has more twisted and tight lid regions which improves its stability. The β-barrel structure transforms as well as surrounding secondary structure. Modified B-Rex domain has less hydrogen bonds. Atomic distance is reduced with the strengthened van der Waals force and steric hindrance. Docked NADH ligand is trapped into B-Rex domain deeper and has less contact with environment and proton transfer. The result has provided theoretical basis and thought for optimization for construction of protein tools with fluorescent protein as core. Modification of Frex H could make it be applied in highly specific and sensitive NADH assay more effectively, which could reflect characters of cell metabolism pathway and be beneficial to relevant fundamental study to enhance recognition about metabolism pathway.

关 键 词：NADH FrexH 同源模建 分子对接 结构优化 

分 类 号：O6-41[理学—化学]