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Modulation of protein properties in living cells using nanobodies

Nature Structural & Molecular Biology volume 17, pages 133–138 (2010)Cite this article

Abstract

Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP–nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

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Figure 1: Identification of nanobodies modulating the fluorescence of GFP.
Figure 2: Structures of the GFP–nanobody complexes.
Figure 3: Nanobody-induced modulation of GFP spectral properties.
Figure 4: Nanobodies modulate GFP fluorescence in living cells.
Figure 5: Nucleocytoplasmic translocation detected by nanobody-mediated fluorescence enhancement.

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References

  1. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. & Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    Article  CAS  Google Scholar 

  2. Ormo, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Article  CAS  Google Scholar 

  3. Yang, F., Moss, L.G. & Phillips, G.N. Jr. The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251 (1996).

    Article  CAS  Google Scholar 

  4. Brejc, K. et al. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc. Natl. Acad. Sci. USA 94, 2306–2311 (1997).

    Article  CAS  Google Scholar 

  5. Heim, R. & Tsien, R.Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182 (1996).

    Article  CAS  Google Scholar 

  6. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  7. Shimomura, O. Discovery of green fluorescent protein. Methods Biochem. Anal. 47, 1–13 (2006).

    PubMed  Google Scholar 

  8. Patterson, G.H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    Article  CAS  Google Scholar 

  9. Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137–141 (2001).

    Article  CAS  Google Scholar 

  10. Akerboom, J. et al. Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J. Biol. Chem. 284, 6455–6464 (2009).

    Article  CAS  Google Scholar 

  11. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).

    Article  CAS  Google Scholar 

  12. Arbabi Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R. & Muyldermans, S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 414, 521–526 (1997).

    Article  CAS  Google Scholar 

  13. Muyldermans, S. Single domain camel antibodies: current status. J. Biotechnol. 74, 277–302 (2001).

    CAS  PubMed  Google Scholar 

  14. Jobling, S.A. et al. Immunomodulation of enzyme function in plants by single-domain antibody fragments. Nat. Biotechnol. 21, 77–80 (2003).

    Article  CAS  Google Scholar 

  15. Rothbauer, U. et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics 7, 282–289 (2008).

    Article  CAS  Google Scholar 

  16. Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).

    Article  CAS  Google Scholar 

  17. Chan, P.H. et al. Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils. Biochemistry 47, 11041–11054 (2008).

    Article  CAS  Google Scholar 

  18. Desmyter, A. et al. Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology. J. Biol. Chem. 277, 23645–23650 (2002).

    Article  CAS  Google Scholar 

  19. Kabat, E.A. & Wu, T.T. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J. Immunol. 147, 1709–1719 (1991).

    CAS  PubMed  Google Scholar 

  20. Tsai, M.J. & O'Malley, B.W. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63, 451–486 (1994).

    Article  CAS  Google Scholar 

  21. Htun, H., Holth, L.T., Walker, D., Davie, J.R. & Hager, G.L. Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. Mol. Biol. Cell 10, 471–486 (1999).

    Article  CAS  Google Scholar 

  22. Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008).

    Article  CAS  Google Scholar 

  23. Kner, P., Chhun, B.B., Griffis, E.R., Winoto, L. & Gustafsson, M.G. Super-resolution video microscopy of live cells by structured illumination. Nat. Methods 6, 339–342 (2009).

    Article  CAS  Google Scholar 

  24. Frauer, C. & Leonhardt, H. A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding. Nucleic Acids Res. (2009).

  25. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  26. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  27. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  Google Scholar 

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Acknowledgements

A. Kirchhofer acknowledges support from the Deutsche Forschungsgemeinschaftgraduate school 1202. U.R., J.H. and K.S. were supported by the GO-Bio program (Bundesministeriums für Bildung und Forschung) and C.F. by the International Doctorate Program 'NanoBioTechnology' of the Elite Network of Bavaria. The authors thank J. Gregor for excellent technical assistance, K. Lammens for help with crystallographic data collection, R. Lewis for fluorescence lifetimes determination and K. Zolghadr and N. Hiller for helpful comments and suggestions. This work was supported by the Center for Integrated Protein Science (CIPSM), the Center for Nanoscience (CeNS), the Nanosystems Initiative Munich (NIM), the BioImaging Network (BIN) and grants from the Deutsche Forschungsgemeinschaft (DFG) to M.C.C., H.L. and K.-P.H. (SFB 684). A. Kirchhofer and K.-P.H. thank K. Römer and the Dr. Klaus Römer Foundation for financial support.

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Authors and Affiliations

  1. Gene Center at the Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany

    Axel Kirchhofer, Sheng Cui, Annette Karcher & Karl-Peter Hopfner

  2. Center for Integrated Protein Science, Munich, Germany

    Axel Kirchhofer, Jonas Helma, Katrin Schmidthals, Carina Frauer, Sheng Cui, Annette Karcher, Heinrich Leonhardt, Karl-Peter Hopfner & Ulrich Rothbauer

  3. Munich Center for Advanced Photonics, Munich, Germany

    Axel Kirchhofer, Sheng Cui, Annette Karcher & Karl-Peter Hopfner

  4. Biocenter at the Department of Biology II, Ludwig-Maximilians University Munich, Planegg-Martinsried, Germany

    Jonas Helma, Katrin Schmidthals, Carina Frauer, Heinrich Leonhardt & Ulrich Rothbauer

  5. Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Brussels, Belgium

    Mireille Pellis & Serge Muyldermans

  6. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium

    Mireille Pellis & Serge Muyldermans

  7. Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany

    Corella S Casas-Delucchi & M Cristina Cardoso

  8. Center for NanoScience, Ludwig-Maximilians University Munich, Munich, Germany

    Heinrich Leonhardt & Ulrich Rothbauer

  9. ChromoTek GmbH, Planegg-Martinsried, Germany

    Ulrich Rothbauer

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  1. Axel Kirchhofer
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Contributions

U.R., M.C.C. and H.L. conceived and initiated the original project; A. Kirchhofer crystallized the GFP–nanobody complexes, determined the crystal structures, carried out life-time measurements and wrote the manuscript; S.C. and A. Karcher assisted in crystallization and structure determination; K.-P.H. helped with interpreting structural and functional data as well as determining crystal structures, designing research and helped with writing the manuscript; M.P. and S.M. provided the nanobodies; J.H. performed in vitro nanobody binding studies; K.S. carried out nanobody and GFP purification; J.H., C.S.C.-D. and M.C.C. carried out the ratio imaging experiments; C.F. performed the in vivo studies and data analysis; H.L. wrote the manuscript; M.C.C., S.M., H.L. and K.-P.H. revised the manuscript and oversaw research. U.R. carried out nanobody purification, in vitro analysis, translocation assays and wrote the manuscript.

Corresponding authors

Correspondence to Heinrich Leonhardt, Karl-Peter Hopfner or Ulrich Rothbauer.

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Competing interests

J.H., K.S., H.L. and U.R. are the founders and shareholders of ChromoTek, which is developing GFP-specific nanobodies for in vitro and in vivo applications.

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Supplementary Methods, Supplementary Figures 1–4 and Supplementary Tables 1–4 (PDF 774 kb)

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Kirchhofer, A., Helma, J., Schmidthals, K. et al. Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol 17, 133–138 (2010). https://doi.org/10.1038/nsmb.1727

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