Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores.
Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores.
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DOI：
10.1085/jgp.118.5.481
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发表时间：
2001-11
期刊：
影响因子：
--
通讯作者：
Bayley H
中科院分区：
文献类型：
--
作者：
Gu LQ;Cheley S;Bayley H
非共价分子衔接子（如环糊精）在进入α-溶血素（αHL）孔腔时可作为通道阻滞剂的结合位点，从而为以αHL作为传感元件检测各种有机分子提供了基础。β-环糊精（βCD）驻留在野生型αHL孔中数百微秒。通过调节pH值和跨膜电位，可以将停留时间延长到几毫秒。在这里，我们描述了能够容纳βCD数十秒的突变同源七聚体αHL孔。突变体通过在位置113（其是位于跨膜β桶的内腔中的缩窄部附近的残基）处的定点诱变获得，并且分为两类。紧密结合类的成员M113 D、M113 N、M113 V、M113 H、M113 F和M113 Y与βCD的结合强度是其余αHL孔（包括WT-αHL）的104倍。这些突变体的较低Kd值主要由koff值降低。突变的主要影响很可能是位置113附近βCD结合位点的重塑。此外，还有一个较小的结合电压敏感组分，它也受到113位残基的影响，可能是中性βCD分子通过电渗流转运的结果。βCD停留时间延长的突变孔可以作为随机传感器的改进组件。
Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼104-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K d values of these mutants are dominated by reduced values of koff. The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors.
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影响因子：
3
作者：
Bayley, H
通讯作者：
Bayley, H
影响因子：
46.9
作者：
Movileanu, L;Howorka, S;Bayley, H
通讯作者：
Bayley, H
影响因子：
64.8
作者：
Bayley, H;Cremer, PS
通讯作者：
Cremer, PS
影响因子：
3.5
作者：
Bezrukov, SM;Kullman, L;Winterhalter, M
通讯作者：
Winterhalter, M
DOI：
10.1073/pnas.78.9.5475
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BHAKDI, S;FUSSLE, R;TRANUMJENSEN, J
通讯作者：
TRANUMJENSEN, J