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Measuring 13Cβ chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

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Abstract

A labeling scheme is introduced that facilitates the measurement of accurate 13Cβ chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of 13C enrichment (30–40%) at Cβ side-chain carbon positions for 15 of the amino acids with little 13C label at positions one bond removed (≈5%). A pair of samples are produced using [1-13C]-glucose/NaH12CO3 or [2-13C]-glucose as carbon sources with isolated and enriched (>30%) 13Cβ positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of 13Cβ chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein–ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.

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Acknowledgments

We thank Dr. Jack Greenblatt, University of Toronto, for the gift of plasmids used for the knockout of succinate dehydrogenase and for valuable advice regarding this procedure. P. Vallurupalli and D. F. Hansen are thanked for useful discussion. This research was supported by a grant from The Canadian Institutes of Health Research. PL is supported by The Swedish Research Council. LEK holds a Canadian Research Chair in Biochemistry.

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Authors and Affiliations

  1. Molecular Biotechnology/IFM, Linköping University, 581 83, Linköping, Sweden

    Patrik Lundström

  2. Molecular Structure & Function, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada

    Hong Lin

  3. Department of Medical Genetics, The University of Toronto, One King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Lewis E. Kay

  4. Department of Biochemistry, The University of Toronto, One King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Lewis E. Kay

  5. Department of Chemistry, The University of Toronto, One King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Lewis E. Kay

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Correspondence to Lewis E. Kay.

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Lundström, P., Lin, H. & Kay, L.E. Measuring 13Cβ chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy. J Biomol NMR 44, 139–155 (2009). https://doi.org/10.1007/s10858-009-9321-3

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