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Probing slowly exchanging protein systems via 13Cα-CEST: monitoring folding of the Im7 protein

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Abstract

A 13Cα chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an ‘observable’ ground state and one or more ‘invisible’ excited states. Here a labeling strategy whereby [2-13C]-glucose is the sole carbon source is exploited, producing proteins with 13C at the Cα position, while the majority of residues remain unlabeled at CO or Cβ. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state 13Cα chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.

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Acknowledgments

The authors thank Dr. Sara Whittaker and Professors Geoff Moore and Sheena Radford for the gift of Im7 plasmids. A.L.H. acknowledges the National Science Foundation (OISE-0852964) and the Canadian Institutes of Health Research (CIHR) training grant in protein folding and disease for post-doctoral support. This work was supported by grants from the CIHR 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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Hansen, A.L., Bouvignies, G. & Kay, L.E. Probing slowly exchanging protein systems via 13Cα-CEST: monitoring folding of the Im7 protein. J Biomol NMR 55, 279–289 (2013). https://doi.org/10.1007/s10858-013-9711-4

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