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 0mV bias DC potential, incorporation of PorBII decreased the membrane resistance (R(m)) from 2.5 MΩc m(2) to 0.6 MΩ cm(2), giving a ΔR(m) of 1.9 MΩ cm(2) and a normalized ΔR(m) (ΔR(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 ΔR(m) value decreased to 20%. The effect of applied voltage on ΔR(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.
Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USA.
Sachin R
SR
Jadhav
Kota Sreenivasa
KS
Rao
Yi
Y
Zheng
R Michael
RM
Garavito
R Mark
RM
Worden
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D058266:Dielectric Spectroscopy; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry*; D009345:Neisseria meningitidis / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D018272:Porins / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D018272:Porins; C057699:porin protein, Neisseria; C021016:1,2-diphytanoylphosphatidylcholine