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A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins

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Abstract

A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) 13C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ 13C–1H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964–73, 2004) supplemented with a CPMG train of refocusing 1H pulses applied with constant frequency and synchronized with the 13C CPMG pulse train. The optimal 1H ‘decoupling’ scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite 1H pulses. For small-to-medium sized proteins, the MQ 13C CPMG experiment has the advantage over its single quantum (SQ) 13C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ 13C CPMG experiment eliminates complications in the interpretation of MQ 13C–1H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl 1H chemical shifts between ground and excited states. The MQ 13C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale.

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Acknowledgements

We thank Prof. D.F. Hansen (UCL, London, UK) for useful suggestions, Drs. F. Torricella (NIDDK, NIH) and A. Ceccon (University of Bolzano, Italy) for preparation of the NMR samples of Fyn SH3VPL and MSG, respectively, and Drs. Jinfa Ying and Dan Garrett (NIDDK, NIH) for technical support. This work was supported by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK029023 to G.M.C.).

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  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA

    Vitali Tugarinov, James L. Baber & G. Marius Clore

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  1. Vitali Tugarinov
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  2. James L. Baber
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  3. G. Marius Clore
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Contributions

V.T. and G.M.C. designed research; V.T. and J.B. performed research; V.T. analyzed the data; V.T. and G.M.C. wrote the paper. All authors reviewed the manuscript

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Correspondence to Vitali Tugarinov or G. Marius Clore.

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Supplementary Information

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10858_2023_413_MOESM1_ESM.pdf

Supplementary file1 (PDF 1222 KB)— Figures S1-S3 comparing methyl relaxation dispersion profiles obtained with different experimental schemes for Fyn SH3VPL and MSG. Table S1 listing exchange parameters of individual Ile δ1 methyl sites of MSG. ‘Materials and Methods’ describing NMR sample conditions and acquisition parameters of NMR experiments. Bruker pulse code for the experiment in Figure 1 and the associated 1H ‘decoupling’ (composite pulse decoupling) schemes. A Matlab® script for calculation of artifact locations in XY-4-cycled 1H ‘decoupling’ schemes (Figure 2, Right panels)

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Tugarinov, V., Baber, J.L. & Clore, G.M. A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins. J Biomol NMR 77, 83–91 (2023). https://doi.org/10.1007/s10858-023-00413-8

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  • DOI: https://doi.org/10.1007/s10858-023-00413-8

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