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Quantifying millisecond time-scale exchange in proteins by CPMG relaxation dispersion NMR spectroscopy of side-chain carbonyl groups

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Abstract

A new pulse sequence is presented for the measurement of relaxation dispersion profiles quantifying millisecond time-scale exchange dynamics of side-chain carbonyl groups in uniformly 13C labeled proteins. The methodology has been tested using the 87-residue colicin E7 immunity protein, Im7, which is known to fold via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. Comparison of exchange parameters extracted for this folding ‘reaction’ using the present methodology with those obtained from more ‘traditional’ 15N and backbone carbonyl probes establishes the utility of the approach. The extracted excited state side-chain carbonyl chemical shifts indicate that the Asx/Glx side-chains are predominantly unstructured in the Im7 folding intermediate. However, several crucial salt-bridges that exist in the native structure appear to be already formed in the excited state, either in part or in full. This information, in concert with that obtained from existing backbone and side-chain methyl relaxation dispersion experiments, will ultimately facilitate a detailed description of the structure of the Im7 folding intermediate.

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Acknowledgments

The authors thank Dr. Sara Whittaker and Professors Geoff Moore and Sheena Radford for the gift of Im7 constructs. A.L.H. acknowledges the National Science Foundation for post-doctoral support (OISE-0852964). This work was supported by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada (LEK). L.E.K. holds a Canada Research Chair in Biochemistry.

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Authors and Affiliations

  1. Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON, M5S 1A8, Canada

    Alexandar L. Hansen & Lewis E. Kay

  2. Program in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada

    Lewis E. Kay

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  1. Alexandar L. Hansen
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  2. Lewis E. Kay
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Correspondence to Lewis E. Kay.

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10858_2011_9520_MOESM1_ESM.pdf

Supplementary material 1 (PDF 272 kb). Table of side-chain carbonyl \( |\Updelta \varpi | \) values for the Im7 protein along with pulse sequence code for recording side-chain carbonyl CPMG relaxation dispersion profiles in U-[13C] labeled proteins

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Hansen, A.L., Kay, L.E. Quantifying millisecond time-scale exchange in proteins by CPMG relaxation dispersion NMR spectroscopy of side-chain carbonyl groups. J Biomol NMR 50, 347–355 (2011). https://doi.org/10.1007/s10858-011-9520-6

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  • DOI: https://doi.org/10.1007/s10858-011-9520-6

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