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Reversible labeling of native and fusion-protein motifs

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Abstract

The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins. Here we demonstrate the full reversibility of post-translational custom pantetheine modification of Escherichia coli acyl carrier protein for visualization and functional studies. We use this iterative enzymatic methodology in vitro to reversibly label acyl carrier protein variants and apply these tools to NMR structural studies of protein-substrate interactions.

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Figure 1: Reversible labeling of E. coli ACP.
Figure 2: Gel detection of reversible ACP labeling.
Figure 3: HSQC spectra of recycled [15N]ACP in various acyl states overlaid with apo-[15N]ACP.

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Acknowledgements

We wish to thank (all from UCSD) T.L. Foley for preparing rhodamine-CoA, coumarin-CoA and coumarin-ACP standard; S. Mayfield for providing the luciferase plasmid template; S. Duan for laboratory support; M. Rothmann for assisting with AcpH method development; R. Tsien for use of GFP-related gel-imaging equipment; the UCSD Chemistry & Biochemistry Mass Spectrometry Facility; and J. La Clair for manuscript design input. This research was funded by US National Institutes of Health grants R21AI090213, R01GM094924 and R01GM095970.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, San Diego (UCSD), La Jolla, California, USA

    Nicolas M Kosa, Robert W Haushalter, Andrew R Smith & Michael D Burkart

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  1. Nicolas M Kosa
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  2. Robert W Haushalter
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  3. Andrew R Smith
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  4. Michael D Burkart
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Contributions

N.M.K. performed all cloning, subcloning, enzymatic reactions, imaging and protein purifications (unless otherwise stated). R.W.H. conducted all protein NMR experiments and provided resulting NMR data. R.W.H. prepared the native Sfp, MBP-CoaA,D,E enzyme stocks used for 'one-pot' chemoenzymatic CoA analog synthesis. A.R.S. synthesized, purified and characterized all oxopantetheine probes in this work. N.M.K., R.W.H., A.R.S. and M.D.B. wrote the manuscript.

Corresponding author

Correspondence to Michael D Burkart.

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The authors declare no competing financial interests.

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Supplementary Figures 1–42, Supplementary Tables 1 and 2, Supplementary Methods and Supplementary Note (PDF 5310 kb)

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Kosa, N., Haushalter, R., Smith, A. et al. Reversible labeling of native and fusion-protein motifs. Nat Methods 9, 981–984 (2012). https://doi.org/10.1038/nmeth.2175

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