Voltage dependent closure of PorB class II porin from Neisseria meningitidis investigated using impedance spectroscopy in a tethered bilayer lipid membrane interface

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 depositing1,2-dioleoyl-sn-glycero-3-phoshocholine (DOPC) liposomes. At 0 mV bias DC potential, incorporation of PorBII decreased the membrane resistance (Rm) from 2.5 MΩ cm2 to 0.6 MΩ cm2, giving a ΔRm of 1.9 MΩ cm2 and a normalized ΔRm (ΔRm divided by the Rm of the tBLM without PorBII) of 76%. When the bias DC potential was increased to 200 mV, the normalized ΔRm value decreased to 20%. The effect of applied voltage on ΔRm 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.

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► A tBLM platform was used to study voltage-dependent closure of the PorBII porin.
► Single channel studies using traditional pBLM technique corroborated tBLM results.
► Impedance spectroscopy was used for functional characterization of protein.