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13C-detected HN(CA)C and HMCMC experiments using a single methyl-reprotonated sample for unambiguous methyl resonance assignment

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Abstract

Methyl groups provide an important source of structural and dynamic information in NMR studies of proteins and their complexes. For this purpose sequence-specific assignments of methyl 1H and 13C resonances are required. In this paper we propose the use of 13C-detected 3D HN(CA)C and HMCMC experiments for assignment of methyl 1H and 13C resonances using a single selectively methyl protonated, perdeuterated and 13C/15N-labeled sample. The high resolution afforded in the 13C directly-detected dimension allows one to rapidly and unambiguously establish correlations between backbone HN strips from the 3D HN(CA)C spectrum and methyl group HmCm strips from the HMCMC spectrum by aligning all possible side-chain carbon chemical shifts and their multiplet splitting patterns. The applicability of these experiments for the assignment of methyl 1H and 13C resonances is demonstrated using the 18.6 kDa B domain of the Escherichia coli mannose transporter (IIBMannose).

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Acknowledgements

This work was supported by the intramural program of NIDDK, NIH (to G.M.C.). B.V. acknowledges a Fellowship from the Swiss National Science Foundation. We thank Dr. Dusty Baber for technical assistance at the spectrometer.

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Authors and Affiliations

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

    Kaifeng Hu, Beat Vögeli & G. Marius Clore

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  1. Kaifeng Hu
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  2. Beat Vögeli
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  3. G. Marius Clore
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Correspondence to G. Marius Clore.

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Kaifeng Hu, Beat Vögeli: These two authors contributed equally to this work.

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Hu, K., Vögeli, B. & Clore, G.M. 13C-detected HN(CA)C and HMCMC experiments using a single methyl-reprotonated sample for unambiguous methyl resonance assignment. J Biomol NMR 36, 259–266 (2006). https://doi.org/10.1007/s10858-006-9090-1

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  • DOI: https://doi.org/10.1007/s10858-006-9090-1

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