Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range.
Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range.
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DOI：
10.1021/acs.biochem.5b00449
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发表时间：
2015-08-18
期刊：
影响因子：
2.9
通讯作者：
Webb MR
中科院分区：
文献类型：
--
作者：
Solscheid C;Kunzelmann S;Davis CT;Hunter JL;Nofer A;Webb MR
重新设计了一种基于大肠杆菌PstS磷酸盐结合蛋白的无荧光试剂无机磷生物传感器，以允许在较高的无机磷浓度和低亚化学计量比的生物传感器浓度下进行测量。这是通过削弱先前生物传感器的PI结合来实现的，并且描述了能够使这种性质改变的不同方法。读数由四甲基罗丹明荧光传递，提供对PI浓度的响应。除了罗丹明标记在第17和197位的两个半胱氨酸突变外，最终的变异体在组成蛋白质的两个叶之间的铰链区域有一个I76G突变。PI结合后，叶在这个铰链上旋转，铰链上的突变使亲和力∼降低200倍，根据溶液条件的不同，解离常数现在在几十到数百微摩尔范围内。根据pH的不同，PI结合的信号变化高达9倍。结果表明，该生物传感器具有较低的生物传感器使用量和较强的抗PI污染能力，适用于稳态的ATPase检测。
A fluorescent reagentless biosensor for inorganic phosphate (Pi), based on the E. coli PstS phosphate binding protein, was redesigned to allow measurements of higher Pi concentrations and at low, substoichiometric concentrations of biosensor. This was achieved by weakening Pi binding of the previous biosensor, and different approaches are described that could enable this change in properties. The readout, providing response to the Pi concentration, is delivered by tetramethylrhodamine fluorescence. In addition to two cysteine mutations for rhodamine labeling at positions 17 and 197, the final variant had an I76G mutation in the hinge region between the two lobes that make up the protein. Upon Pi binding, the lobes rotate on this hinge and the mutation on the hinge lowers affinity ∼200-fold, with a dissociation constant now in the tens to hundreds micromolar range, depending on solution conditions. The signal change on Pi binding was up to 9-fold, depending on pH. The suitability of the biosensor for steady-state ATPase assays was demonstrated with low biosensor usage and its advantage in ability to cope with Pi contamination.