Abstract
A straightforward protocol for the site-specific incorporation of a 19F label into any protein in vivo is described. This is done using a plasmid containing an orthogonal aminoacyl-tRNA synthetase/tRNACUA that incorporates L-4-trifluoromethylphenylalanine in response to the amber codon UAG. This method improves on other in vivo methods because the 19F label is incorporated into only one location on the protein of interest and that protein can easily be produced in large quantities at low cost. The protocol for producing 19F-labeled protein is similar to expressing protein in Escherichia coli and takes 4 d to obtain pure protein starting from the appropriate vectors.
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References
Danielson, M.A. & Falke, J.J. Use of 19F NMR to probe protein structure and conformational changes. Annu. Rev. Biophys. Biomol. Struct. 25, 163–195 (1996).
Ulrich, A.S. Solid state 19F NMR methods for studying biomembranes. Prog. Nucl. Magn. Reson. Spectrosc. 46, 1–21 (2003).
Gerig, J.T. Fluorine NMR of proteins. Prog. Nucl. Magn. Reson. Spectrosc. 26, 293–370 (1994).
Bai, P., Luo, L. & Peng, Z. Side chain accessibility and dynamics in the molten globule state of alpha-lactalbumin: a (19)F-NMR study. Biochemistry 39, 372–380 (2000).
Bann, J.G. & Frieden, C. Folding and domain–domain interactions of the chaperone PapD measured by 19F NMR. Biochemistry 43, 13775–13786 (2004).
Hoeltzli, S.D. & Frieden, C. Refolding of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase in the presence of ligand: a stopped-flow NMR spectroscopy study. Biochemistry 37, 387–398 (1998).
Furter, R. Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli. Protein Sci. 7, 419–426 (1998).
Vaughan, M.D., Cleve, P., Robinson, V., Duewel, H.S. & Honek, J.F. Difluoromethionine as a novel 19F NMR structural probe for internal amino acid packing in proteins. J. Am. Chem. Soc. 121, 8475–8478 (1999).
Duewel, H., Daub, E., Robinson, V. & Honek, J.F. Incorporation of trifluoromethionine into a phage lysozyme: implications and a new marker for use in protein 19F NMR. Biochemistry 36, 3404–3416 (1997).
Feeney, J.M. et al. 19F nuclear magnetic resonance chemical shifts of fluorine containing aliphatic amino acids in proteins: studies on Lactobacillus casei dihydrofolate reductase containing (2S,4S)-5-fluoroleucine. J. Am. Chem. Soc. 118, 8700–8706 (1996).
Frieden, C., Hoeltzli, S.D. & Bann, J.G. The preparation of 19F-labeled proteins for NMR studies. Methods Enzymol. 380, 400–415 (2004).
Luck, L.A. & Falke, J.J. 19F NMR studies of the D-galactose chemosensory receptor. 2. Ca(II) binding yields a local structural change. Biochemistry 30, 4257–4261 (1991).
Lee, K.H., Lee, H.Y., Slutsky, M.M., Anderson, J.T. & Marsh, E.N. Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine. Biochemistry 43, 16277–16284 (2004).
Higuchi, M. et al. 19F and 1H MRI detection of amyloid beta plaques in vivo. Nat. Neurosci. 8, 527–533 (2005).
Wang, L., Brock, A., Herberich, B. & Schultz, P.G. Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001).
Jackson, J.C., Hammill, J.T. & Mehl, R.A. Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity. J. Am. Chem. Soc. 129, 1160–1166 (2007).
Xie, J. & Schultz, P.G. An expanding genetic code. Methods 36, 227–238 (2005).
Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).
Wang, L. & Schultz, P.G. Expanding the genetic code. Angew. Chem. Int. Ed. Engl. 44, 34–66 (2004).
Acknowledgements
We thank Cinthia Kinsland for her helpful discussions on molecular biology and media. This work was supported by F&M Hackman and Eyler funds and NSF-MCB-0448297, Research Corporation (CC6364) and ACS-PRF (42214-GB4).
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Hammill, J., Miyake-Stoner, S., Hazen, J. et al. Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization. Nat Protoc 2, 2601–2607 (2007). https://doi.org/10.1038/nprot.2007.379
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DOI: https://doi.org/10.1038/nprot.2007.379
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