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FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids

Nature Methods volume 3, pages 923–929 (2006)Cite this article

Abstract

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.

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Figure 1: Design of fluorescent non-natural amino acids and incorporation into a protein.
Figure 2: Synthesis and fluorescence properties of labeled calmodulins.
Figure 3: Incorporation of BODIPY558-AF into the N terminus and BODIPYFL-AF into various positions of calmodulin.
Figure 4: FRET analysis of MBP-M13–dependent conformational changes in calmodulin.

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References

  1. Pelton, J.T. & McLean, L.R. Spectroscopic methods for analysis of protein secondary structure. Anal. Biochem. 277, 167–176 (2000).

    Article  CAS  Google Scholar 

  2. Selvin, P.R. The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7, 730–734 (2000).

    Article  CAS  Google Scholar 

  3. Heyduk, T. Measuring protein conformational changes by FRET/LRET. Curr. Opin. Biotechnol. 13, 292–296 (2002).

    Article  CAS  Google Scholar 

  4. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  CAS  Google Scholar 

  5. Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554–10559 (2004).

    Article  CAS  Google Scholar 

  6. Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  Google Scholar 

  7. Hohsaka, T. & Sisido, M. Incorporation of non-natural amino acids into proteins. Curr. Opin. Chem. Biol. 6, 809–815 (2002).

    Article  CAS  Google Scholar 

  8. Hohsaka, T., Ashizuka, Y., Murakami, H. & Sisido, M. Incorporation of nonnatural amino acids into streptavidin through in vitro frame-shift suppression. J. Am. Chem. Soc. 118, 9778–9779 (1996).

    Article  CAS  Google Scholar 

  9. Hohsaka, T., Kajihara, D., Ashizuka, Y., Murakami, H. & Sisido, M. Efficient incorporation of nonnatural amino acids with large aromatic groups into streptavidin in in vitro protein synthesizing systems. J. Am. Chem. Soc. 121, 34–40 (1999).

    Article  CAS  Google Scholar 

  10. Hohsaka, T., Ashizuka, Y., Sasaki, H., Murakami, H. & Sisido, M. Incorporation of two different nonnatural amino acids independently into a single protein through extension of the genetic code. J. Am. Chem. Soc. 121, 12194–12195 (1999).

    Article  CAS  Google Scholar 

  11. Hohsaka, T., Ashizuka, Y., Taira, H., Murakami, H. & Sisido, M. Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system. Biochemistry 40, 11060–11064 (2001).

    Article  CAS  Google Scholar 

  12. Murakami, H., Hohsaka, T., Ashizuka, Y., Hashimoto, K. & Sisido, M. Site-directed incorporation of fluorescent nonnatural amino acids into streptavidin for highly sensitive detection of biotin. Biomacromolecules 1, 118–125 (2000).

    Article  CAS  Google Scholar 

  13. Taki, M., Hohsaka, T., Murakami, H., Taira, K. & Sisido, M. A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe. FEBS Lett. 507, 35–38 (2001).

    Article  CAS  Google Scholar 

  14. Hohsaka, T. et al. Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon. FEBS Lett. 560, 173–177 (2004).

    Article  CAS  Google Scholar 

  15. Hamada, H. et al. Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing. Bioorg. Med. Chem. 13, 3379–3384 (2005).

    Article  CAS  Google Scholar 

  16. Cornish, V.W. et al. Site-specific incorporation of biophysical probes into proteins. Proc. Natl. Acad. Sci. USA 91, 2910–2914 (1994).

    Article  CAS  Google Scholar 

  17. Turcatti, G. et al. Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites. J. Biol. Chem. 271, 19991–19998 (1996).

    Article  CAS  Google Scholar 

  18. Steward, L.E. et al. In vitro site-specific incorporation of fluorescent probes into β-galactosidase. J. Am. Chem. Soc. 119, 6–11 (1997).

    Article  CAS  Google Scholar 

  19. Cohen, B.E. et al. Probing protein electrostatics with a synthetic fluorescent amino acid. Science 296, 1700–1703 (2002).

    Article  CAS  Google Scholar 

  20. Anderson, R.D., III, Zhou, J. & Hecht, S.M. Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase. J. Am. Chem. Soc. 124, 9674–9675 (2002).

    Article  CAS  Google Scholar 

  21. Kajihara, D., Hohsaka, T. & Sisido, M. Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position. Protein Eng. Des. Sel. 18, 273–278 (2005).

    Article  CAS  Google Scholar 

  22. Ikura, M. et al. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–638 (1992).

    Article  CAS  Google Scholar 

  23. Bayley, P.M., Findlay, W.A. & Martin, S.R. Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences. Protein Sci. 5, 1215–1228 (1996).

    Article  CAS  Google Scholar 

  24. Lichtenthaler, F.W., Cuny, E., Morino, T. & Rychlik, I. Synthesis of 3′-homocitrullylamino- and 3′-lysylamino- 3′-deoxy-adenosine and their relation to Cordyceps militaris derived products. Chem. Ber. 112, 2588–2601 (1979).

    Article  CAS  Google Scholar 

  25. dos Remedios, C.G. & Moens, P.D.J. Fluorescence resonance energy transfer spectroscopy is a reliable 'ruler' for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor. J. Struct. Biol. 115, 175–185 (1995).

    Article  CAS  Google Scholar 

  26. Rasnik, I., McKinney, S.A. & Ha, T. Surfaces and orientations: much to FRET about? Acc. Chem. Res. 38, 542–548 (2005).

    Article  CAS  Google Scholar 

  27. Majumdar, Z.K., Hickerson, R., Noller, H.F. & Clegg, R.M. Measurements of internal distance changes of the 30 S ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods. J. Mol. Biol. 351, 1123–1145 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Valvano for the pSCRhaB2 plasmid; the Caenorhabditis Genetics Center for worm strains; and E. Entchev, L. Pelletier, J. Fu and members of the Genomics Department of TU Dresden for discussions. This work was supported by the European Union and Freistaat Sachsen, by Europäischer Fonds für regionale Entwicklung (EFRE) funding (project code 181) through the Sächsisches Staatsministerium für Wissenschaft und Kunst (SMWK) and Technische Universität Dresden (TUD), and by the Bundesminiterium fuer Bildung und Forschung (BMBF) Proteomics Program.

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Authors and Affiliations

  1. School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, 923-1292, Ishikawa, Japan

    Daisuke Kajihara, Issei Iijima & Takahiro Hohsaka

  2. Department of Bioscience and Biotechnology, Okayama University, Tsushimanaka, 3-1-1 700-8530, Okayama, Japan

    Daisuke Kajihara, Ryoji Abe, Chie Komiyama & Masahiko Sisido

  3. PRESTO, Japan Science and Technology Agency, Honcho Kawaguchi, 4-1-8 332-0012, Saitama, Japan

    Takahiro Hohsaka

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  1. Daisuke Kajihara
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Correspondence to Takahiro Hohsaka.

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Supplementary information

Supplementary Fig. 1

CBB-stained SDS-PAGE of labeled calmodulins. (PDF 203 kb)

Supplementary Fig. 2

Fluorescence spectra of double-labeled calmodulins. (PDF 422 kb)

Supplementary Fig. 3

Fluorescence spectra of single-labeled calmodulins. (PDF 417 kb)

Supplementary Fig. 4

Calcium dependence of double-labeled calmodulins. (PDF 426 kb)

Supplementary Fig. 5

Calcium dependence of double-labeled calmodulin-M13 fusion. (PDF 357 kb)

Supplementary Table 1

Fluorescent properties of BODIPYFL and BODIPY558. (PDF 82 kb)

Supplementary Methods (PDF 124 kb)

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Kajihara, D., Abe, R., Iijima, I. et al. FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nat Methods 3, 923–929 (2006). https://doi.org/10.1038/nmeth945

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