Abstract
A 2D 13C Chemical Exchange Saturation Transfer (CEST) experiment is presented for studying slowly exchanging protein systems using methyl groups as probes. The utility of the method is first established through studies of protein L, a small protein, for which chemical exchange on the millisecond time-scale is not observed. Subsequently the approach is applied to a folding exchange reaction of a G48M mutant Fyn SH3 domain, for which only cross-peaks derived from the folded (‘ground’) state are present in spectra. Fits of 15N and methyl 13C CEST profiles of the Fyn SH3 domain establish that the exchange reaction involves an interchange between folded and unfolded conformers, although elevated methyl 13C transverse relaxation rates for some of the residues of the unfolded (‘invisible, excited’) state indicate that it likely exchanges with a third conformation as well. In addition to the kinetics of the exchange reaction, methyl carbon chemical shifts of the excited state are also obtained from analysis of the 13C CEST data.
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Acknowledgments
G.B. acknowledges the Canadian Institutes of Health Research (CHIR) for a post-doctoral fellowship. This work was supported by grants from the CIHR and the Natural Sciences and Engineering Research Council of Canada. L. E. K. holds a Canada Research Chair in Biochemistry.
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Bouvignies, G., Kay, L.E. A 2D 13C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding. J Biomol NMR 53, 303–310 (2012). https://doi.org/10.1007/s10858-012-9640-7
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DOI: https://doi.org/10.1007/s10858-012-9640-7