A protein pore with a single polymer chain tethered within the lumen
A protein pore with a single polymer chain tethered within the lumen
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DOI：
10.1021/ja993221h
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发表时间：
2000-03-22
影响因子：
15
通讯作者：
Bayley, H
中科院分区：
文献类型：
--
作者：
Howorka, S;Movileanu, L;Bayley, H
一个跨膜蛋白孔与一个单一的5000 Da聚（乙二醇）（PEG）分子共价连接在通道内腔已被构建从七个葡萄球菌α-溶血素亚基。修饰的七聚体是稳定的，并且可以通过在十二烷基硫酸钠中电泳来纯化，而不解离亚基。用单通道电流记录法研究了修饰孔的性质。的PEG分子减少了18%的平均电导率的孔，将预测从PEG对本体电解质的电导率的影响。记录还揭示了各种低幅度的电流波动的时间尺度上的秒，这是暂时归因于重组的PEG分子内的通道内腔和相关的运动的多肽链。另一类事件，包括持续时间为毫秒的电流的均匀高振幅负波动，归因于PEG分子进入通道入口之一的运动，从而产生更广泛的通道阻断。相反，当3000 Da PEG连接在通道内腔内时，单通道特性随着聚合物的较低质量而改变。例如，高振幅波动更频繁地发生并且持续时间更短，表明3000 Da PEG比5000 Da链更移动的。随着进一步的发展，这里采取的方法应该是有用的，在单分子水平上的聚合物动力学的间接监测。通过使用对分析物有反应的聚合物，也应该可以从共价修饰的孔制造生物传感器。
A transmembrane protein pore with a single 5000 Da poly(ethylene glycol) (PEG) molecule attached covalently within the channel lumen has been constructed from seven staphylococcal alpha-hemolysin subunits. The modified heptamer is stable and can be purified by electrophoresis in sodium dodecyl sulfate, without dissociation of the subunits. The properties of the modified pore were studied by single channel current recording. The PEG molecule reduces the mean conductance of the pore by 18%, as would be predicted from the effects of PEG on the conductivity of bulk electrolytes. The recordings also reveal a variety of low amplitude current fluctuations on a time scale of seconds, which are tentatively ascribed to the reorganization of the PEG molecule within the channel lumen and associated movements of the polypeptide chain. Another class of events, comprising uniform high-amplitude negative fluctuations in current with durations of milliseconds, is ascribed to motions of the PEG molecule into one of the channel entrances, thereby producing more extensive channel block. When instead a 3000 Da PEG is attached within the channel lumen, the single channel properties are changed in keeping with the lower mass of the polymer. For example, the high-amplitude fluctuations occur more frequently and are of shorter duration suggesting that the 3000 Da PEG is more mobile than the 5000 Da chain. With further development, the approach taken here should be useful for the indirect monitoring of polymer dynamics at the single molecule level. By using polymers that respond to analytes, it should also be possible to make biosensors from the covalently modified pores.