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

Nature Methods volume 3, pages 923929 (2006) | Download Citation

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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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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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Affiliations

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

    • Daisuke Kajihara
    • , Issei Iijima
    •  & Takahiro Hohsaka
  2. Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.

    • Daisuke Kajihara
    • , Ryoji Abe
    • , Chie Komiyama
    •  & Masahiko Sisido
  3. PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.

    • Takahiro Hohsaka

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Takahiro Hohsaka.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    CBB-stained SDS-PAGE of labeled calmodulins.

  2. 2.

    Supplementary Fig. 2

    Fluorescence spectra of double-labeled calmodulins.

  3. 3.

    Supplementary Fig. 3

    Fluorescence spectra of single-labeled calmodulins.

  4. 4.

    Supplementary Fig. 4

    Calcium dependence of double-labeled calmodulins.

  5. 5.

    Supplementary Fig. 5

    Calcium dependence of double-labeled calmodulin-M13 fusion.

  6. 6.

    Supplementary Table 1

    Fluorescent properties of BODIPYFL and BODIPY558.

  7. 7.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth945

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