Outer membrane protein G: Engineering a quiet pore for biosensing
Outer membrane protein G: Engineering a quiet pore for biosensing
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DOI：
10.1073/pnas.0711561105
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发表时间：
2008-04-29
影响因子：
11.1
通讯作者：
Bayley, Hagan
中科院分区：
文献类型：
--
作者：
Chen, Min;Khalid, Syma;Bayley, Hagan
细菌外膜孔蛋白具有强大的P-桶结构，因此显示出作为基于单分子检测的随机传感器的潜力。与多亚基蛋白相比，单体孔蛋白OmpG特别有吸引力，因为可以通过诱变容易地实现孔的适当修饰。然而，OmpG的门控导致单通道记录中的瞬态电流阻断，这将干扰分析物检测。为了消除这种自发的门控活动，我们使用分子动力学模拟来确定涉及门控的OmpG区域。基于我们的研究结果，两种方法被用来提高稳定性的开放构象的定点突变。首先，通过在链β 12和β 13的细胞外末端之间引入二硫键来降低环6的移动性。其次，通过缺失残基D215优化链β 11和β 12之间的链间氢键。具有两种稳定突变的OmpG孔蛋白表现出门控活性降低95%。我们使用这种突变体的检测腺苷二磷酸在单分子水平上，配备后的孔蛋白与环糊精分子适配器，从而证明其潜在的随机传感应用。
Bacterial outer membrane porins have a robust P-barrel structure and therefore show potential for use as stochastic sensors based on single-molecule detection. The monomeric porin OmpG is especially attractive compared with multisubunit proteins because appropriate modifications of the pore can be easily achieved by mutagenesis. However, the gating of OmpG causes transient current blockades in single-channel recordings that would interfere with analyte detection. To eliminate this spontaneous gating activity, we used molecular dynamics simulations to identify regions of OmpG implicated in the gating. Based on our findings, two approaches were used to enhance the stability of the open conformation by site-directed mutagenesis. First, the mobility of loop 6 was reduced by introducing a disulfide bond between the extracellular ends of strands beta 12 and beta 13. Second, the interstrand hydrogen bonding between strands beta 11 and beta 12 was optimized by deletion of residue D215. The OmpG porin with both stabilizing mutations exhibited a 95% reduction in gating activity. We used this mutant for the detection of adenosine diphosphate at the single-molecule level, after equipping the porin with a cyclodextrin molecular adapter, thereby demonstrating its potential for use in stochastic sensing applications.