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Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy

Nature Protocols volume 4, pages 1641–1648 (2009)Cite this article

Abstract

The utility of nuclear magnetic resonance (NMR) spectroscopy as a tool for the study of biomolecular structure and dynamics has benefited from the development of facile labeling methods that incorporate NMR active probes at key positions in the molecule. Here we describe a protocol for the labeling of proteins that facilitates their study using a technique that is sensitive to millisecond conformational exchange processes. The samples necessary for an analysis of exchange dynamics are discussed, using the Abp1p SH3 domain from Saccharomyces cerevisiae as an example. For this system, the time frame for production of each sample, including in vitro refolding, is about 80 h. The samples so produced facilitate the measurement of accurate chemical shifts of low populated, invisible conformers that are part of the exchange pathway. The accuracy of the methodology has been established experimentally and the chemical shifts that are obtained provide important restraints in structure calculations of the excited state.

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Acknowledgements

This work was supported by funding from the Canadian Institutes of Health Research (CIHR) to L.E.K. P.L. is supported by the Swedish Research Council and D.F.H. holds a CIHR postdoctoral fellowship. L.E.K. is the recipient of a Canada Research Chair in Biochemistry.

P.L. and L.E.K. conceived the labeling strategies. P.L., P.V. and D.F.H. produced the protein samples and validated the methodology. P.L. and L.E.K. wrote the paper. L.E.K. supervised the project.

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Authors and Affiliations

  1. Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden

    Patrik Lundström

  2. Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, Ontario, Canada

    Pramodh Vallurupalli, D Flemming Hansen & Lewis E Kay

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  1. Patrik Lundström
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Correspondence to Lewis E Kay.

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Lundström, P., Vallurupalli, P., Hansen, D. et al. Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy. Nat Protoc 4, 1641–1648 (2009). https://doi.org/10.1038/nprot.2009.118

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