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Tuning membrane protein mobility by confinement into nanodomains

Nature Nanotechnology volume 12, pages 260–266 (2017)Cite this article

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Abstract

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

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Figure 1: Platform for transmembrane protein observation.
Figure 2: Membrane protein mobility depends on the extent of chemical fixation of the SA crystal.
Figure 3: Confined lateral mobility of membrane proteins.
Figure 4: Characterization of membrane proteins embedded in SA crystal SLBs.

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References

  1. Cho, N.-J., Frank, C. W., Kasemo, B. & Höök, F. Quartz crystal microbalance with dissipation monitoring of supported lipid bilayers on various substrates. Nat. Protoc. 5, 1096–1106 (2010).

    Article  CAS  Google Scholar 

  2. Patching, S. G. Surface plasmon resonance spectroscopy for characterisation of membrane protein–ligand interactions and its potential for drug discovery. Biochim. Biophys. Acta 1838, 43–55 (2014).

    Article  CAS  Google Scholar 

  3. 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  Google Scholar 

  4. Giocondi, M.-C., Seantier, B., Dosset, P., Milhiet, P.-E. & Le Grimellec, C. Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM. Pflugers Arch. 456, 179–188 (2008).

    Article  CAS  Google Scholar 

  5. Yilmaz, N. et al. Real-time visualization of assembling of a sphingomyelin-specific toxin on planar lipid membranes. Biophys. J. 105, 1397–1405 (2013).

    Article  CAS  Google Scholar 

  6. Preiner, J. et al. IgGs are made for walking on bacterial and viral surfaces. Nat. Commun. 5, 4593 (2014).

    Article  Google Scholar 

  7. Casuso, I. et al. Characterization of the motion of membrane proteins using high-speed atomic force microscopy. Nat. Nanotech. 7, 525–529 (2012).

    Article  CAS  Google Scholar 

  8. Casuso, I., Sens, P., Rico, F. & Scheuring, S. Experimental evidence for membrane-mediated protein–protein interaction. Biophys. J. 99, L47–L49 (2010).

    Article  CAS  Google Scholar 

  9. Johnson, C. P. et al. Structural studies of the neural-cell-adhesion molecule by X-ray and neutron reflectivity. Biochemistry 44, 546–554 (2005).

    Article  CAS  Google Scholar 

  10. Miller, C. E., Majewski, J., Gog, T. & Kuhl, T. L. Characterization of biological thin films at the solid–liquid interface by X-ray reflectivity. Phys. Rev. Lett. 94, 238104 (2005).

    Article  CAS  Google Scholar 

  11. Nováková, E., Giewekemeyer, K. & Salditt, T. Structure of two-component lipid membranes on solid support: An x-ray reflectivity study. Phys. Rev. E 74, 051911 (2006).

    Article  Google Scholar 

  12. Przybylo, M. et al. Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions. Langmuir 22, 9096–9099 (2006).

    Article  CAS  Google Scholar 

  13. Preiner, J. et al. High-speed AFM images of thermal motion provide stiffness map of interfacial membrane protein moieties. Nano Lett. 15, 759–763 (2014).

    Article  Google Scholar 

  14. Horner, A. et al. The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues. Sci. Adv. 1, e1400083 (2015).

    Article  Google Scholar 

  15. Yamashita, H. et al. Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy. J. Struct. Biol. 167, 153–158 (2009).

    Article  CAS  Google Scholar 

  16. Yamamoto, D. et al. Chapter twenty-High-Speed atomic force microscopy techniques for observing dynamic biomolecular processes. Methods Enzymol. 475, 541–564 (2010).

    Article  CAS  Google Scholar 

  17. Chan, Y.-H. M. & Boxer, S. G. Model membrane systems and their applications. Curr. Opin. Chem. Biol. 11, 581–587 (2007).

    Article  CAS  Google Scholar 

  18. Castellana, E. T. & Cremer, P. S. Solid supported lipid bilayers: From biophysical studies to sensor design. Surf. Sci. Rep. 61, 429–444 (2006).

    Article  CAS  Google Scholar 

  19. Park, E. et al. Structure of the SecY channel during initiation of protein translocation. Nature 506, 102–106 (2014).

    Article  CAS  Google Scholar 

  20. Antonenko, Y. N., Horner, A. & Pohl, P. Electrostatically induced recruitment of membrane peptides into clusters requires ligand binding at both interfaces. PloS ONE 7, e52839 (2012).

    Article  CAS  Google Scholar 

  21. Tanaka, M. & Sackmann, E. Polymer-supported membranes as models of the cell surface. Nature 437, 656–663 (2005).

    Article  CAS  Google Scholar 

  22. Gonçalves, R. P. et al. Two-chamber AFM: probing membrane proteins separating two aqueous compartments. Nat. Methods 3, 1007–1012 (2006).

    Article  Google Scholar 

  23. Frauenfeld, J. et al. Cryo-EM structure of the ribosome–SecYE complex in the membrane environment. Nat. Struct. Mol. Biol. 18, 614–621 (2011).

    Article  CAS  Google Scholar 

  24. Fu, D. et al. Structure of a glycerol-conducting channel and the basis for its selectivity. Science 290, 481–486 (2000).

    Article  CAS  Google Scholar 

  25. Saparov, S. M., Tsunoda, S. P. & Pohl, P. Proton exclusion by an aquaglyceroprotein: a voltage clamp study. Biol. Cell 97, 545–550 (2005).

    Article  CAS  Google Scholar 

  26. Reviakine, I. & Brisson, A. Streptavidin 2D crystals on supported phospholipid bilayers: toward constructing anchored phospholipid bilayers. Langmuir 17, 8293–8299 (2001).

    Article  CAS  Google Scholar 

  27. Yamamoto, D., Nagura, N., Omote, S., Taniguchi, M. & Ando, T. Streptavidin 2D crystal substrates for visualizing biomolecular processes by atomic force microscopy. Biophys. J. 97, 2358–2367 (2009).

    Article  CAS  Google Scholar 

  28. Calvert, T. L. & Leckband, D. Two-dimensional protein crystallization at solid-liquid interfaces. Langmuir 13, 6737–6745 (1997).

    Article  CAS  Google Scholar 

  29. Schütz, G. J., Schindler, H. & Schmidt, T. Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys. J. 73, 1073–1080 (1997).

    Article  Google Scholar 

  30. Le Trong, I. et al. Streptavidin and its biotin complex at atomic resolution. Acta Crystallogr. D 67, 813–821 (2011).

    Article  CAS  Google Scholar 

  31. Knyazev, D. G. et al. The bacterial translocon SecYEG opens upon ribosome binding. J. Biol. Chem. 288, 17941–17946 (2013).

    Article  CAS  Google Scholar 

  32. Breyton, C., Haase, W., Rapoport, T. A., Kühlbrandt, W. & Collinson, I. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418, 662–665 (2002).

    Article  CAS  Google Scholar 

  33. Zimmer, J., Nam, Y. & Rapoport, T. A. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature 455, 936–943 (2008).

    Article  CAS  Google Scholar 

  34. de Keyzer, J., van der Does, C., Kloosterman, T. G. & Driessen, A. J. M. Direct demonstration of ATP-dependent release of SecA from a translocating preprotein by surface plasmon resonance. J. Biol. Chem. 278, 29581–29586 (2003).

    Article  CAS  Google Scholar 

  35. Lill, R., Dowhan, W. & Wickner, W. The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell 60, 271–280 (1990).

    Article  CAS  Google Scholar 

  36. Wallin, E. & Heijne, G. V. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 7, 1029–1038 (1998).

    Article  CAS  Google Scholar 

  37. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010).

    Article  CAS  Google Scholar 

  38. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333, 755–758 (2011).

    Article  CAS  Google Scholar 

  39. Schwenen, L. L. et al. Resolving single membrane fusion events on planar pore-spanning membranes. Sci. Rep. 5, 12006 (2015).

    Article  Google Scholar 

  40. Knyazev, D. G., Winter, L., Bauer, B. W., Siligan, C. & Pohl, P. Ion conductivity of the bacterial translocation channel SecYEG engaged in translocation. J. Biol. Chem. 289, 24611–24616 (2014).

    Article  CAS  Google Scholar 

  41. Sbalzarini, I. F. & Koumoutsakos, P. Feature point tracking and trajectory analysis for video imaging in cell biology. J. Struct. Biol. 151, 182–195 (2005).

    Article  CAS  Google Scholar 

  42. Tseng, Q. et al. Spatial organization of the extracellular matrix regulates cell–cell junction positioning. Proc. Natl Acad. Sci. USA 109, 1506–1511 (2012).

    Article  CAS  Google Scholar 

  43. Bowman, A. W. & Azzalini, A. Applied Smoothing Techniques for Data Analysis (Clarendon, 2004).

    Google Scholar 

  44. Rankl, C. et al. Multiple receptors involved in human rhinovirus attachment to live cells. Proc. Natl Acad. Sci. USA 105, 17778–17783 (2008).

    Article  Google Scholar 

  45. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E. & Webb, W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069 (1976).

    Article  CAS  Google Scholar 

  46. Klotzsch, E. et al. Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria. Proc. Natl Acad. Sci. USA 112, 130–135 (2015).

    Article  CAS  Google Scholar 

  47. Smith, C. S., Joseph, N., Rieger, B. & Lidke, K. A. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat. Methods 7, 373–375 (2010).

    Article  CAS  Google Scholar 

  48. Gao, Y. & Kilfoil, M. L. Accurate detection and complete tracking of large populations of features in three dimensions. Opt. Express 17, 4685–4704 (2009).

    Article  Google Scholar 

  49. Wieser, S. & Schütz, G. J. Tracking single molecules in the live cell plasma membrane—do's and don't’s. Methods 46, 131–140 (2008).

    Article  CAS  Google Scholar 

  50. Harpaz, Y., Gerstein, M. & Chothia, C. Volume changes on protein folding. Structure 2, 641–649 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Austrian Science Fund (FWF, P25844 to J.P.), the European Fund for Regional Development (EFRE, Regio 13) and the Federal State of Upper Austria. The authors thank H. Gruber for helpful discussion and Q. Beatty for editorial help.

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Authors and Affiliations

  1. Center for Advanced Bioanalysis GmbH, Gruberstrasse 40–42, Linz, 4020, Austria

    Andreas Karner, Benedikt Nimmervoll & Johannes Preiner

  2. Upper Austria University of Applied Sciences, Campus Linz, Garnisonstrasse 21, Linz, 4020, Austria

    Birgit Plochberger

  3. EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia

    Enrico Klotzsch

  4. Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, Linz, 4020, Austria

    Andreas Horner, Denis G. Knyazev, Roland Kuttner, Klemens Winkler, Lukas Winter, Christine Siligan, Nicole Ollinger & Peter Pohl

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  1. Andreas Karner
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Contributions

A.K. and J.P. performed HS-AFM experiments and performed data analysis. B.N., B.P., and E.K. performed fluorescence experiments and did data analysis. A.K., A.H., D.G.K., R.K., K.W., L.W., C.S., N.O, and J.P. developed sample preparation techniques. J.P. and A.K. designed the experiments. A.K., J.P. and P.P. prepared the final manuscript.

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Correspondence to Johannes Preiner.

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The authors declare no competing financial interests.

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Karner, A., Nimmervoll, B., Plochberger, B. et al. Tuning membrane protein mobility by confinement into nanodomains. Nature Nanotech 12, 260–266 (2017). https://doi.org/10.1038/nnano.2016.236

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