Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Atomic force microscopy and spectroscopy of native membrane proteins


Membrane proteins comprise 30% of the proteome of higher organisms. They mediate energy conversion, signal transduction, solute transport and secretion. Their native environment is a bilayer in a physiological buffer solution, hence their structure and function are preferably assessed in this environment. The surface structure of single membrane proteins can be determined in buffer solutions by atomic force microscopy (AFM) at a lateral resolution of less than 1 nm and a vertical resolution of 0.1–0.2 nm. Moreover, single proteins can be directly addressed, stuck to the AFM stylus and subsequently unfolded, revealing the molecular interactions of the protein studied. The examples discussed here illustrate the power of AFM in the structural analysis of membrane proteins in a native environment.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: High-resolution atomic force microscopy (AFM) imaging.
Figure 2: High-resolution atomic force microscopy (AFM) of purple membrane.
Figure 3: High-resolution atomic force microscopy (AFM) topographs of native membrane proteins.
Figure 4: Unfolding single membrane proteins by single-molecule force spectroscopy.


  1. Müller, D.J., Fotiadis, D., Scheuring, S., Müller, S.A. & Engel, A. Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. Biophys. J. 76, 1101–1111 (1999).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Hoogenboom, B.W. et al. Quantitative dynamic-mode scanning force microscopy in liquid. Appl. Phys. Lett. 88, 193109 (2006).

    Article  Google Scholar 

  3. Putman, C.A., van der Werf, K.O., de Grooth, B.G., van Hulst, N.F. & Greve, J. Tapping mode atomic force microscopy in liquid. Appl. Phys. Lett. 64, 2454–2456 (1994).

    Article  CAS  Google Scholar 

  4. Hansma, P.K. et al. Tapping mode atomic force microscopy in liquids. Appl. Phys. Lett. 64, 1738–1740 (1994).

    Article  CAS  Google Scholar 

  5. Engel, A. & Müller, D.J. Observing single biomolecules at work with the atomic force microscope. Nat. Struct. Biol. 7, 715–718 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Yokokawa, M. et al. Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL. EMBO J. 25, 4567–4576 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Müller, D.J., Büldt, G. & Engel, A. Force-induced conformational change of bacteriorhodopsin. J. Mol. Biol. 249, 239–243 (1995).

    Article  PubMed  Google Scholar 

  9. Viani, M.B. et al. Small cantilevers for force spectroscopy of single molecules. J. Appl. Phys. 86, 2258–2262 (1999).

    Article  CAS  Google Scholar 

  10. Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl. Acad. Sci. USA 98, 12468–12472 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Yasumura, K.Y. et al. Quality factors in micron- and submicron-thick cantilevers. J. Microelectromech. Syst. 9, 117–125 (2000).

    Article  CAS  Google Scholar 

  12. Chon, J.W.M., Mulvaney, P. & Sader, J.E. Experimental validation of theoretical models for the frequency response of atomic force microscope cantilever beams immersed in fluids. J. Appl. Phys. 87, 3978–3988 (2000).

    Article  CAS  Google Scholar 

  13. Sader, J.E., Chon, J.W.M. & Mulvaney, P. Calibration of rectangular atomic force microscope cantilevers. Rev. Sci. Instrum. 70, 3967–3969 (1999).

    Article  CAS  Google Scholar 

  14. Czajkowsky, D.M., Hotze, E.M., Shao, Z. & Tweten, R.K. Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane. EMBO J. 23, 3206–3215 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fotiadis, D. et al. Structural analysis of the reaction center light-harvesting complex I photosynthetic core complex of Rhodospirillum rubrum using atomic force microscopy. J. Biol. Chem. 279, 2063–2068 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Scheuring, S. & Sturgis, J.N. Chromatic adaptation of photosynthetic membranes. Science 309, 484–487 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Müller, D.J. & Engel, A. Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy. J. Mol. Biol. 285, 1347–1351 (1999).

    Article  PubMed  Google Scholar 

  18. Müller, D.J., Amrein, M. & Engel, A. Adsorption of biological molecules to a solid support for scanning probe microscopy. J. Struct. Biol. 119, 172–188 (1997).

    Article  PubMed  Google Scholar 

  19. Butt, H.J. et al. Scan speed limit in atomic force microscopy. J. Microsc. 169, 75–84 (1993).

    Article  Google Scholar 

  20. Engel, A., Schoenenberger, C.A. & Müller, D.J. High-resolution imaging of native biological sample surfaces using scanning probe microscopy. Curr. Opin. Struct. Biol. 7, 279–284 (1997).

    Article  CAS  PubMed  Google Scholar 

  21. Schwarz, U.D., Haefke, H., Reimann, P. & Guntherodt, H.J. Tip artefacts in scanning force microscopy. J. Microsc. 173, 183–197 (1994).

    Article  CAS  Google Scholar 

  22. Möller, C., Allen, M., Elings, V., Engel, A. & Müller, D.J. Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces. Biophys. J. 77, 1150–1158 (1999).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Stark, M., Möller, C., Müller, D.J. & Guckenberger, R. From images to interactions: high-resolution phase imaging in tapping-mode atomic force microscopy. Biophys. J. 80, 3009–3018 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Garcia, R. & Perez, R. Dynamic atomic force microscopy methods. Surf. Sci. Rep. 47, 197–301 (2002).

    Article  CAS  Google Scholar 

  25. Kedrov, A., Janovjak, H., Sapra, K.T. & Muller, D.J. Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy. Annu. Rev. Biophys. Biomol. Struct. 36, 233–260 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Seelert, H. et al. Structural biology. Proton-powered turbine of a plant motor. Nature 405, 418–419 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Stahlberg, H. et al. Bacterial Na+-ATP synthase has an undecameric rotor. EMBO Rep. 2, 229–233 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fotiadis, D. et al. Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 421, 127–128 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Yu, J., Bippes, C.A., Hand, G.M., Muller, D.J. & Sosinsky, G.E. Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannels. J. Biol. Chem. 282, 8895–8904 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references


The authors acknowledge support by the Deutsche Forschungsgemeinschaft, Volkswagenstiftung, European Union, the Free State of Saxony, the Swiss National Research Foundation, the M.E. Müller Foundation, the Swiss National Center of Competence in Research (NCCR) 'Structural Biology', the NCCR 'Nanoscale Science' and the NANOMOT project of the EU (grant NEST2004 PathSYS29084).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Andreas Engel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Müller, D., Engel, A. Atomic force microscopy and spectroscopy of native membrane proteins. Nat Protoc 2, 2191–2197 (2007).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing