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工程改造的耻垢分枝杆菌孔蛋白A(MspA)纳米孔中硬-软-酸-碱(HSAB)相互作用的单分子观察
Single molecule observation of hard–soft-acid–base (HSAB) interaction in engineered Mycobacterium smegmatis porin A (MspA) nanopores†

Chemical Science(中文翻译) ( IF 9.346 ) Pub Date : 2019-12-10 , DOI: 10.1039/c9sc05260g
ShaWang, JiaoCao, WendongJia, WeimingGuo, ShuanghongYan, YuqinWang, PankeZhang, Hong-YuanChen, ShuoHuang

在天然金属蛋白中金属离子和氨基酸之间形成配位相互作用时,结合的金属离子对于稳定蛋白质结构或作为酶辅因子至关重要。尽管尺寸很小,但当金属离子与工程改造的生物纳米孔的受限环境结合时,其会在单通道记录期间导致可检测到的扰动。所有此类报道的工作都是使用工程改造的α-溶血素纳米孔进行的,观察到的事件振幅极小(约1-3 pA)。作者推测,α-溶血素的圆柱形孔限制可能不适用于探测极小的分析物。耻垢分枝杆菌孔蛋白A(MspA)是一个圆锥形的纳米孔,经过工程设计可与Ca2+、Mn2 +、Co2 +、Ni2+、Zn2+、Pb2+和Cd2+相互作用,并且观察到较大的事件振幅(最高10 pA)。测得的速率常数表明,单个离子与氨基酸的配位遵循硬-软-酸-碱理论,该理论从未在单分子情况下得到系统验证。通过将测量pH值从6.8调节到8.0,可使单个离子结合事件的持续时间延长约46倍。因此,MspA可作为探测各种极小分析物(例如单原子和多原子离子、小分子或化学中间体)的出色模板,而硬-软-酸-碱相互作用的原理可能对孔设计具有指导意义。


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In the formation of coordination interactions between metal ions and amino acids in natural metalloproteins, the bound metal ion is critical either for the stabilization of the protein structure or as an enzyme co-factor. Though extremely small in size, metal ions, when bound to the restricted environment of an engineered biological nanopore, result in detectable perturbations during single channel recordings. All reported work of this kind was performed with engineered α-hemolysin nanopores and the observed events appear to be extremely small in amplitude (∼1–3 pA). We speculate that the cylindrical pore restriction of α-hemolysin may not be optimal for probing extremely small analytes. Mycobacterium smegmatis porin A (MspA), a conical shaped nanopore, was engineered to interact with Ca2+, Mn2+, Co2+, Ni2+, Zn2+, Pb2+ and Cd2+ and a systematically larger event amplitude (up to 10 pA) was observed. The measured rate constant suggests that the coordination of a single ion with an amino acid follows hard–soft-acid–base theory, which has never been systematically validated in the case of a single molecule. By adjusting the measurement pH from 6.8 to 8.0, the duration of a single ion binding event could be modified with a ∼46-fold time extension. The phenomena reported suggest MspA to be a superior engineering template for probing a variety of extremely small analytes, such as monatomic and polyatomic ions, small molecules or chemical intermediates, and the principle of hard–soft-acid–base interaction may be instructive in the pore design.
更新日期:2019-12-11

 

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