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Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording

Nature Chemical Biology volume 2, pages 314–318 (2006)Cite this article

Abstract

Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process1. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, α-helix bundles and β barrels, which are represented respectively by a K+ channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.

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Figure 1: Screening membrane proteins expressed in bacteria.
Figure 2: Transfer of K+ channels from bacteria to a planar bilayer using the probe technique.
Figure 3: Screening colonies for the M113F/K147N αHL mutant in a wild-type (WT) αHL background with the probe technique.
Figure 4: Concept for the Luk screen.
Figure 5: Rapid screening of 35 Luk mutants for binding of TRIMEG.

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Acknowledgements

O.D. is a Junior Research Fellow of Christ Church, Oxford. H.B. is the holder of a Royal Society–Wolfson Research Merit Award. This work was supported by grants from the Medical Research Council and the Office of Naval Research. We thank M. Wallace for assistance with the probe and bilayer movie.

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Author notes

  1. Lakmal Jayasinghe and Oliver Daltrop: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK

    Matthew A Holden, Lakmal Jayasinghe, Oliver Daltrop, Amy Mason & Hagan Bayley

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  1. Matthew A Holden
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  2. Lakmal Jayasinghe
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Correspondence to Matthew A Holden.

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Supplementary information

Supplementary Fig. 1

Typical measurement of the capacitance during the engagement and withdrawal of a glass probe. (PDF 20 kb)

Supplementary Fig. 2

Single pore recording of WT leukocidin in the presence of TRIMEG. (PDF 30 kb)

Supplementary Fig. 3

SDS-PAGE of whole bacterial colonies expressing WT KcsA or WT αHL.SDS-PAGE of whole bacterial colonies expressing WT KcsA or WT αHL. (PDF 56 kb)

Supplementary Video 1

A planar bilayer was formed across a 120-μm Teflon aperture prior to the start of the video. A glass probe approaches from behind (cis chamber) and passes through the plane of the bilayer into the trans chamber. Because the aperture (which appears elliptical but is circular) is viewed from above at an angle of 45°, the probe initially appears to be thick and conical, however it is approximately 20 μm in radius at the tip and tapers slightly outward along the length used. The probe comes into focus only as it passes through the plane of the bilayer; the tip is out of focus when it enters the trans chamber. After the first insertion/withdrawal cycle, a dark region is visible in the center of the bilayer at the spot from which the tip of the probe was withdrawn. We attribute this to a mass of hexadecane and lipid which is shed from the probe surface as it is pulled from the bilayer. Note that the mass rises due to buoyancy and subsequently fuses with the annulus of the planar bilayer. The deposition of this material does not destabilize the membrane. In total, three insertion/withdrawal cycles are shown, the last withdrawal being especially slow to emphasize the ability of the planar bilayer to withstand mechanical perturbation without rupturing. The video is shown in real time. (MOV 2571 kb)

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Holden, M., Jayasinghe, L., Daltrop, O. et al. Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording. Nat Chem Biol 2, 314–318 (2006). https://doi.org/10.1038/nchembio793

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