Generation of artificial channels by multimerization of β-strands from natural porin

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Marco Lolicato ◽  
Simona Reina ◽  
Angela Messina ◽  
Francesca Guarino ◽  
Mathias Winterhalter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Voltage Dependence ◽  
Structural Elements ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Conserved Motif ◽  
Basic Module ◽  
Transmembrane Channels ◽  
Recombinant Technology ◽  
Bacterial Porins

Abstract General diffusion porins are passive transmembrane channels. We have explored the possibility to create artificial nanopores starting from natural β-barrel structures. Structural elements of bacterial porins were used to build a series of artificial nanopores. The basic module was selected by multi-alignment of general diffusion porins. The sequence corresponded to a highly conserved motif containing two β-strands, which was obtained from Escherichia coli OmpF. Dimeric to octameric repeats were obtained through cDNA recombinant technology. The hexameric repeat was used to test its properties. This protein was expressed, purified and reconstituted in the planar bilayer membranes. It was able to form channels in membranes with a conductance of 300 pS in 150 mm KCl and did not show any relevant voltage-dependence.

scholarly journals The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

1995 ◽  
Vol 106 (5) ◽  
pp. 783-802 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
F S Cohen
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Pore Formation ◽  
Surface Proteins ◽  
Fusion Pore ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Pore Evolution ◽  
Fusion Pores ◽  
Kinetics Of

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

Fusion of Large Unilamellar Liposomes Containing Hemocyanin with Planar Bilayer Membranes

1984 ◽  
Vol 39 (1-2) ◽  
pp. 147-155 ◽  
Author(s):  
Flavia Pasquali ◽  
Gianfranco Menestrina ◽  
Renzo Antolini
Keyword(s):  
Electrical Properties ◽  
Ionic Channels ◽  
Planar Bilayer ◽  
Artificial Membrane ◽  
Bilayer Membranes ◽  
Large Unilamellar Vesicles ◽  
Strong Current ◽  
Voltage Curve ◽  
Current Voltage ◽  
Current Voltage Curve

Abstract Large unilamellar vesicles were prepared by detergent removal from micelles containing phosphatidylcholine, phosphatidylethanolam ine and phosphatidylserine. Liposomes were then interacted with Megathura crenulata hemocyanin, a well studied channel former. Incubation of the resulting proteoliposomes on one side of a phosphatidylserine-containing planar bilayer under fusion conditions yielded strong current increases. Such increase is due to insertion of ionic channels from the liposomes into the planar bilayer. Studying the effects of Ba2+ on the electrical properties of the channel we could show that the protein is always inserted into a bilayer during this process, i.e. fusion of proteoliposomes with the artificial membrane occurs. The strong non linearity of the current-voltage curve of the hemocyanin pore could be used as a probe of the extent to which fusion preserves the orientation of the channel through the bilayer.

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