Functional characterization of PorB class II porin from Neisseria meningitidis using a tethered bilayer lipid membrane
Functional characterization of PorB class II porin from Neisseria meningitidis using a tethered bilayer lipid membrane
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DOI：
10.1016/j.bios.2008.07.010
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发表时间：
2008-12-01
影响因子：
12.6
通讯作者：
Worden, R. Mark
中科院分区：
文献类型：
--
作者：
Jadhav, Sachin R.;Zheng, Yi;Worden, R. Mark
脑膜炎奈瑟氏球菌的PorB II类是一种成孔外膜蛋白，在奈瑟氏球菌感染期间可移位至宿主细胞膜。本报告描述了栓系双层脂质膜（tBLM）系统的发展，以测量PorB电导特性。通过在金电极上沉积1，2-二棕榈酰-sn-甘油-3-磷酸硫乙醇（DPFTE）系留脂质的自组装单层，然后使用1，2-二油酰-sn-甘油-3-磷酸胆碱（DOPC）脂质体存款上部tBLM小叶来制备tBLM。电化学阻抗谱（EIS）和循环伏安法（CV）用于监测tBLM的形成和随后的PorB掺入。高度绝缘的tBLM表现出的膜电阻和电容分别为2.5 M Ω cm（2）和0.7 μ F/cm（2）。PorB被纳入tBLM的活性构象，证明其调解的离子通道和膜阻抗的降低。PorB掺入后，膜电阻降低至0.6 M Ω cm（2）。正如预期的那样，PorB通道被发现是非选择性的，允许阳离子和阴离子的运输。循环伏安法表明，铁氰化物离子也可以通过孔隙。本研究中开发的含PorB的仿生界面可能用于筛选调节PorB活性的化合物。(C)2008 Elsevier B. V.保留所有权利。
PorB class II from Neisseria meningitidis is a pore-forming, outer-membrane protein that can translocate to the host-cell membrane during Neisserial infections. This report describes development of tethered bilayer lipid membrane (tBLM) system to measure PorB conductance properties. The tBLM was fabricated by depositing a self-assembled monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPFTE) tethering lipid on a gold electrode and then using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes to deposit the upper tBLM leaflet. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor tBLM formation and subsequent PorB incorporation. The highly insulating tBLM exhibited a membrane resistance and capacitance of 2.5 M Omega cm(2) and 0.7 mu F/cm(2), respectively. PorB was incorporated into the tBLM in an active conformation, as evidenced by its mediation of ion passage and the decrease in membrane impedance. After PorB incorporation, the membrane resistance decreased to 0.6 M Omega cm(2). As expected, the PorB channel was found to be non-selective, allowing the transport of both cations and anions. Cyclic voltammetry indicated that ferricyanide ions can also pass through the pores. The PorB-containing biomimetic interface developed in this study could potentially be used to screen for compounds that modulate PorB activity. (C) 2008 Elsevier B.V. All rights reserved.