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Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy

Nature Protocols volume 1, pages 749–754 (2006)Cite this article

Abstract

The development of isotope labeling methodology has had a significant impact on NMR studies of high-molecular-weight proteins and macromolecular complexes. Here we review some of this methodology that has been developed and used in our laboratory. In particular, experimental protocols are described for the production of highly deuterated, uniformly 15N- and 13C-labeled samples of large proteins, with optional incorporation of selective isotope labels into methyl groups of isoleucine, leucine and valine residues. Various types of methyl labeling schemes are assessed, and the utility of different methyl labeling strategies is highlighted for studies ranging from protein structure determination to the investigation of side-chain dynamics. In the case of malate synthase G (MSG), the time frame of the whole preparation, including the protein refolding step, is about 70 h.

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Figure 1: Isotopically labeled α-keto acids that are commercially available and that can be used as biosynthetic precursors in E.coli–based growth of selectively methyl-labeled proteins.
Figure 2: 1H-15N HSQC-TROSY spectrum (37 °C; 800 MHz; 10% D2O/90% H2O) of [U-2H,15N]MSG refolded in H2O after the D2O-based growth as described in the text.
Figure 3: Two-dimensional 1H-13C HMQC spectra (D2O; 800 MHz; 37 °C) acquired with variously labeled MSG samples.
Figure 4: An overlay of two-dimensional 1H-13C correlation maps acquired on {[U-2H,15N]; Ileδ1-[13CH3]}MSG (peaks shown with red contours), {[U-2H,15N]; Ileδ1-[13CH2D]}MSG (blue contours) and {[U-2H,15N]; Ileδ1-[13CHD2]}MSG (green contours; D2O, 800 MHz, 37 °C).

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Acknowledgements

The authors gratefully acknowledge many of the former members of the Kay laboratory who have contributed to the development of the selective methyl protonation approaches described here. V.T. is a recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship. L.E.K. is the recipient of a Canada Research Chair in Biochemistry.

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Authors and Affiliations

  1. Departments of Medical Genetics and Microbiology, Biochemistry and Chemistry, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, M5S 1A8, Ontario, Canada

    Vitali Tugarinov & Lewis E Kay

  2. Program in Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, M5G 1X8, Ontario, Canada

    Voula Kanelis

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  1. Vitali Tugarinov
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Correspondence to Lewis E Kay.

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Tugarinov, V., Kanelis, V. & Kay, L. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc 1, 749–754 (2006). https://doi.org/10.1038/nprot.2006.101

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