Functional characterization of PorB class II porin from Neisseria meningitidis using a tethered bilayer lipid membrane.
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 (DPPTE) 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 2.5MOmegacm(2) and 0.7 microF/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 0.6MOmegacm(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.
Jadhav
Sachin R
SR
Department of Chemical Engineering and Materials Science, Michigan State University, 2527 Engineering Building, East Lansing, MI 48824, USA.
Zheng
Yi
Y
Michael Garavito
R
R
Mark Worden
R
R
D015374:Biosensing Techniques / Q000379:methods*; D004553:Electric Conductivity; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D009345:Neisseria meningitidis / Q000378:metabolism*; D018272:Porins / Q000737:chemistry*; D016062:Porosity
D008051:Lipid Bilayers; D018272:Porins; C057699:porin protein, Neisseria