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Two-chamber AFM: probing membrane proteins separating two aqueous compartments

Nature Methods volume 3, pages 1007–1012 (2006)Cite this article

Abstract

Biological membranes compartmentalize and define physical borders of cells. They are crowded with membrane proteins that fulfill diverse crucial functions. About one-third of all genes in organisms code for, and the majority of drugs target, membrane proteins. To combine structure and function analysis of membrane proteins, we designed a two-chamber atomic force microscopy (AFM) setup that allows investigation of membranes spanned over nanowells, therefore separating two aqueous chambers. We imaged nonsupported surface layers (S layers) of Corynebacterium glutamicum at sufficient resolution to delineate a 15 Å–wide protein pore. We probed the elastic and yield moduli of nonsupported membranes, giving access to the lateral interaction energy between proteins. We combined AFM and fluorescence microscopy to demonstrate the functionality of proteins in the setup by documenting proton pumping by Halobacterium salinarium purple membranes.

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Figure 1: Membrane adsorption on holey Si(001) surfaces.
Figure 2: High-resolution imaging of nonsupported membranes.
Figure 3: Membrane piercing.
Figure 4: Proton pumping of nonsupported purple membranes.

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Acknowledgements

We thank F. Oesterhelt for assistance in force curve analysis, A. Martinez-Gil for technical assistance, C. Gueudry for help with fluorescence microscopy and S. Lesko for spring-constant determination. This study was supported by the INSERM and INSERM Avenir, a 'Ministère de l'Education Nationale' scholarship (to R.P.G.) and an Action Concertée Incitative Nanosciences 2004 grant (NR206).

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Authors and Affiliations

  1. Institut Curie, UMR168-CNRS, 26 Rue d'Ulm, Paris, 75248, France

    Rui Pedro Gonçalves & Simon Scheuring

  2. IEF, MMS, Université Paris-Sud, Bat. 220, Orsay, 91405, France

    Guillaume Agnus & Bernard Bartenlian

  3. ESPCI, CNRS-UMR 7083, 10 rue Vauquelin, Paris, 75231, France

    Pierre Sens

  4. LBMII, IGM, Université Paris-Sud, Bat. 360, Orsay, 91405, France

    Christine Houssin

Authors
  1. Rui Pedro Gonçalves
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  2. Guillaume Agnus
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  4. Christine Houssin
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  5. Bernard Bartenlian
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  6. Simon Scheuring
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Contributions

R.P.G. performed AFM imaging, S-layer preparation with C.H., fluorescence imaging and fluorescence data analysis. G.A. and B.B. prepared the nanopatterned Si(001) surfaces. P.S. and S.S. performed force-curve analysis and physical interpretation. S.S performed image and data analysis, conceived the project and prepared the manuscript.

Corresponding author

Correspondence to Simon Scheuring.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Schematic representation of the 2-chamber AFM setup. (PDF 663 kb)

Supplementary Fig. 2

AFM characterization of the holey Si(001) support wafers. (PDF 1512 kb)

Supplementary Video 1

Proton pumping of non-supported purple membranes. Sequence of green fluorescence images over a time-range of 500 seconds documenting fluorescence intensity variations in chambers. (MOV 467 kb)

Supplementary Note

Digital image treatment (PDF 48 kb)

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Gonçalves, R., Agnus, G., Sens, P. et al. Two-chamber AFM: probing membrane proteins separating two aqueous compartments. Nat Methods 3, 1007–1012 (2006). https://doi.org/10.1038/nmeth965

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