Highly electrically insulating tethered lipid bilayers for probing the function of Ion channel proteins

A method is presented to form gold-electrode-tethered lipid bilayers with exceptionally high elec. resistances. Elec. impedance spectroscopy is used to monitor the bilayer incorporation of a ligand-gated ion channel protein and the modulation of its channel activity by the selective binding of an antibody. Due to the low defect d. of the tethered membrane, the effect of a few channels can be resolved thus opening the way to single-channel expts. on this highly stable and versatile platform. In turn, a minute quantity of analyte, here antibodies, can be measured, which is of great interest for bioanalytics. [on SciFinder (R)]

Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein

Horst Vogel

Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acid 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer expts., and functionally, by elec. conductance measurements on planar bilayers and release expts. on dye-loaded vesicles. Melittin-TASP and the truncated analog preferentially adopt a-helical structures in methanol (56% and 52%, resp.) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized mols. can be self-assembled and compressed to a compact structure with a mol. area of around 600 .ANG.2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coeff. of 1.5 * 109 M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP mols. are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concn. in the lipid bilayer, addnl., larger conductance states of up to 1 nS were obsd. These states are likely to be formed by aggregated TGASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features on the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye mols. from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation. [on SciFinder (R)]

1994

Pf3 coat protein forms voltage-gated ion channels in planar lipid bilayers

Horst Vogel

The coat protein of bacteriophage Pf3 forms discrete and stable ion channels of uniform size in planar bilayers of asolectin. Its primary sequence suggests a channel formed by a bundle of transmembrane helixes. Since the apparent transmembrane region only consists of strongly hydrophobic residues, it represents a new class of channel-forming proteins. The channel activity is strongly voltage-dependent. The single-channel conductance of 60 pS (at 100 mV) in 0.2 M NaCl is slightly voltage-dependent, indicating conformational changes of the pore upon variation of the transmembrane elec. field. The channel is unselective which suggests that the pore is of aq. character. For the obsd. conductance, a channel diam. of 3.6 .ANG. is consistent with a tetrameric a-helix bundle, as calcd. from a barrel-stave model. A pronounced dependence of the gating kinetics with increasing voltage arises from two opposing effects: an increase in the no. of open channel structures, and a simultaneous, more than 3-fold decrease in the channel lifetime. Thus, a max. activity is reach around 100 mV, a range which corresponds well with physiol. membrane potentials. The channels activate only upon application of a pos. voltage on the side of the membrane to which the protein had been added. The slow relaxation of the mean current upon application of sudden voltage jumps indicates a strong activation barrier in the channel gating process, which may result from the membrane translocation of the charged residues of the peptide ends. A channel-mediated import mechanism is suggested for the bacterial infection by phage DNA. [on SciFinder (R)]

1994

Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein

Horst Vogel

1994