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Multiparametric high-resolution imaging of native proteins by force-distance curve–based AFM

Nature Protocols volume 9, pages 1113–1130 (2014)Cite this article

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Abstract

A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in force-distance (FD) curve–based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1–3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution.

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Figure 1: Principles of FD curve–based AFM for imaging and mapping multiple properties of biological samples.
Figure 2: High-resolution FD-based AFM images of membrane proteins and fibrillated water-soluble proteins.
Figure 3: Approach and retraction FD curves recorded on supporting surfaces.
Figure 4: FD-based AFM images of purple membrane adsorbed onto mica.
Figure 5: High-resolution FD-based AFM of the extracellular purple membrane surface reveals sub-structural details of bacteriorhodopsin trimers.

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Acknowledgements

We thank D. Alsteens and I. Medalsy for stimulating discussions and advice. This work was supported by the Swiss National Science Foundation, the European Union Marie Curie Actions program through the ACRITAS Initial Training Network (FP7-PEOPLE-2012-ITN, Project 317348), and the European Molecular Biology Organization long-term fellowship 506-2012.

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Authors and Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

    Moritz Pfreundschuh, David Martinez-Martin, Estefania Mulvihill & Daniel J Muller

  2. Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA

    Susanne Wegmann

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  1. Moritz Pfreundschuh
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Contributions

M.P., D.M.-M., S.W. and D.J.M. designed the AFM protocol. M.P., E.M. and S.W. performed and optimized the experimental procedure. M.P., D.M.-M., S.W. and D.J.M. wrote the manuscript.

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Correspondence to Daniel J Muller.

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Pfreundschuh, M., Martinez-Martin, D., Mulvihill, E. et al. Multiparametric high-resolution imaging of native proteins by force-distance curve–based AFM. Nat Protoc 9, 1113–1130 (2014). https://doi.org/10.1038/nprot.2014.070

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