Single channel recordings of α-hemolysin reconstituted in S-layer-supported lipid bilayers

doi:10.1016/S1567-5394(01)00148-7

Bioelectrochemistry

Volume 55, Issues 1–2, January 2002, Pages 5-7

https://doi.org/10.1016/S1567-5394(01)00148-7 Get rights and content

Abstract

Previous studies demonstrated that lipid membranes attached to a proteinaceous crystalline surface-layer (S-layer) revealed a prolonged lifetime and showed a reduced tendency to rupture in the presence of membrane active molecules. In addition, comparative studies on folded and S-layer-supported lipid membranes (SsLM) revealed an uniform capacitance of 0.64±0.04 μF/cm² for both composite membranes. In the present study, the feasibility to reconstitute the channel-forming protein α-hemolysin (αHL) into SsLM at single channel resolution was investigated. Single αHL channels could be recorded and the intrinsic properties like unitary conductance, current–voltage characteristics, and closure was found to be similar at both membranes. Thus, the tightly attached S-layer allowed complete reconstitution of αHL channels in SsLM.

Introduction

Crystalline monomolecular bacterial cell surface layers (S-layers) represent the outermost cell envelope component of organisms of almost every taxonomic group of walled bacteria and archaea [1], [2]. S-layers are isoporous structures, composed of a single protein or glycoprotein species. Isolated S-layer subunits are able to assemble into monomolecular arrays at many interfaces and particularly on lipid films, mimicking the supramolecular architecture of archaeal cell envelopes. [2], [3]. Certain protein domains of the S-layer protein interact with some headgroups of the adjacent lipid layer, resulting in a lipid bilayer membrane with an enhanced stability [4], [5]. Interestingly, the tight lipid–protein interaction has minute impact on the hydrophobic part of the lipid membrane as determined by X-ray [6] and capacitance measurements [4]. These intrinsic features make the composite S-layer/lipid membranes attractive for the use at biosensors based on transmembrane channel proteins as sensing elements. The aim of the present work was to study the feasibility of single channel measurements in S-layer-supported lipid membranes (SsLM). For this purpose, the staphylococcal toxin α-hemolysin (αHL) has been reconstituted in SsLM (Fig. 1).

Section snippets

Experimental

Growth, cell wall preparations, and extraction of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 (Czech Collection of Microorganisms) were performed as described elsewhere [7]. For the formation of SsLM, a lipid membrane was generated as described below and after half an hour, 0.5 ml of a SbpA solution (1.8 mg protein/ml) was injected into the trans chamber. The recrystallization was completed after 3 h as determined by transmission electron microscopy as described previously [4].

Results and discussion

Electron microscopical studies demonstrated the recrystallization of SbpA into large continuous lattices on the planar lipid bilayer. The attachment of the proteinaceous lattice caused no significant change in the specific capacitance of the lipid membrane, which was 0.64±0.04 μF/cm² for both the folded and the SsLM. Thus, the attached S-layer obviously has no impact on the area, thickness, or the dielectric properties of the hydrophobic part of the membrane. The capacitance is also close to

Conclusion

The present results demonstrated the feasibility to use SsLM for the investigation of unitary αHL channels. Since SsLM reveal a decreased tendency to rupture [4], these composite structures represent an appealing biomimetic system for studying structural and functional properties of membrane proteins. In addition, a great variety of solid surfaces can be completely covered with a closed ultrathin S-layer [2]. This water-containing layer, separating the lipid membrane and reconstituted proteins

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Bioelectrochemistry

Abstract

Introduction

Section snippets

Experimental

Results and discussion

Conclusion

References (17)

S-layer-supported lipid membranes

Rev. Mol. Biotechnol.

Self-assembled α-hemolysin pores in an S-layer-supported lipid bilayer

Biochim. Biophys. Acta

Bacterial S-layer protein coupling to lipids: X-ray reflectivity and grazing incidence diffraction studies

Biophys. J.

Nanopores and nucleic acids: prospects for ultrarapid sequencing

Trends Biotechnol.

S-layer proteins

J. Bacteriol.

Crystalline bacterial cell surface layers (S-Layers): from supramolecular cell structure to biomimetics and nanotechnology

Angew. Chem., Int. Ed.

Probing the stability of S-layer-supported planar lipid membranes

Eur. Biophys. J.

Structural and chemical characterization of S-layer of selected strains of Bacillus stearothermophilus and Desulfotomaculum nigrificans

Arch. Microbiol.

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Tethered bilayer lipid membranes with giga-ohm resistances