Voltage dependent closure of PorB class II porin from Neisseria meningitidis investigated using impedance spectroscopy in a tethered bilayer lipid membrane interface
Voltage dependent closure of PorB class II porin from Neisseria meningitidis investigated using impedance spectroscopy in a tethered bilayer lipid membrane interface
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DOI：
10.1016/j.jcis.2012.09.031
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发表时间：
2013-01-15
影响因子：
9.9
通讯作者：
Worden, R. Mark
中科院分区：
文献类型：
--
作者：
Jadhav, Sachin R.;Rao, Kota Sreenivasa;Worden, R. Mark
电化学阻抗谱（EIS）被用来表征电压依赖性关闭的PorB II类（PorBII）孔蛋白从脑膜炎奈瑟氏菌纳入栓系双层脂膜（tBLM）。通过在金电极上沉积1，2-二棕榈酰-sn-甘油-3-磷硫乙醇（DPPTE）的自组装单层（SAM）来制造tBLM的下小叶，并且通过沉积1，2-二油酰-sn-甘油-3-磷胆碱（DOPC）脂质体来形成上小叶。在0 mV偏压DC电位下，PorBII的掺入使膜电阻（R-m）从2.5 M Ω cm（2）降低至0.6 M Ω cm（2），得到1.9 M Ω cm（2）的Δ R-m和76%的归一化Δ R-m（Δ R-m除以不含PorBII的tBLM的R-m）。当偏置DC电位增加到200 mV时，归一化Delta R-m值降低到20%。施加电压对Delta R-m的影响是完全可逆的，表明PorBII的电压依赖性关闭。在由1，2-二植烷酰基-sn-甘油基-3-磷酸胆碱（DPhytPC）制成的平面双层脂质膜中进一步研究了PorBII的电压依赖性。一个单一的插入事件后，PorBII表现出多个电导状态，可逆的，电压依赖性关闭的PorBII孔蛋白发生在高跨膜电位。三次孔蛋白在三个离散步骤中闭合，每个步骤对应于一个导电单体单元的闭合。最可能的单通道电导为4.2 nS。与tBLM和pBLM平台获得的结果之间的协议证明了EIS筛选固定在tBLM中的通道蛋白的电压门控行为的实用性。(C)2012 Elsevier Inc. All rights reserved.
Electrochemical impedance spectroscopy (EIS) was used to characterize voltage-dependent closure of PorB class II (PorBII) porin from Neisseria meningitidis incorporated in a tethered bilayer lipid membrane (tBLM). The tBLM's lower leaflet was fabricated by depositing a self assembled monolayer (SAM) of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) on a gold electrode, and the upper leaflet was formed by depositing 1,2-dioleoyl-sn-glycero-3-Phoshocholine (DOPC) liposomes. At 0 mV bias DC potential, incorporation of PorBII decreased the membrane resistance (R-m) from 2.5 M Omega cm(2) to 0.6 M Omega cm(2), giving a Delta R-m of 1.9 M Omega cm(2) and a normalized Delta R-m (Delta R-m divided by the R-m of the tBLM without PorBII) of 76%. When the bias DC potential was increased to 200 mV, the normalized Delta R-m value decreased to 20%. The effect of applied voltage on Delta R-m was completely reversible, suggesting voltage-dependent closure of PorBII. The voltage dependence of PorBII was further studied in a planar bilayer lipid membrane made from 1,2-diphytanoyl-sn-glycero-3-Phosphocholine (DPhytPC). Following a single insertion event, PorBII exhibited multiple conductance states, with reversible, voltage-dependent closure of PorBII porin occurring at high transmembrane potentials. The trimetric porin closed in three discrete steps, each step corresponding to closure of one conducting monomer unit. The most probable single channel conductance was 4.2 nS. The agreement between results obtained with the tBLM and pBLM platforms demonstrates the utility of EIS to screen channel proteins immobilized in tBLM for voltage-gated behavior. (C) 2012 Elsevier Inc. All rights reserved.