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Towards unambiguous assignment of methyl-containing residues by double and triple sensitivity-enhanced HCCmHm-TOCSY experiments

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Abstract

Chemical shift assignment of methyl-containing residues is essential in protein NMR spectroscopy, as these residues are abundant in protein interiors and provide the vast majority of long-range NOE connectivities for structure determination. These residues also constitute an integral part of hydrophobic cavities, the surroundings for many enzymatic reactions. Here we present a powerful strategy for the assignment of methyl-containing residues in a uniformly 13C/15N double labeled protein sample. The approach is based on novel four-dimensional HCCmHm-TOCSY experiments, two of them utilizing gradient selection and sensitivity enhancement in all three indirectly detected dimensions. Regardless of the number of dimensions, the proposed experiments can be executed using only one transient per FID, providing outstanding resolution and sensitivity. A complete assignment of the 51 methyl-containing residues in the 16 kDa Mus musculus coactosin was accomplished using a four-dimensional HCCmHm-TOCSY spectrum recorded in 16 hours.

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Acknowledgments

This work was financially supported by the grant 106852 (P. P.) from the Academy of Finland.

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Authors and Affiliations

  1. Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, P.O. Box 65, FI-00014, Helsinki, Finland

    Peter Würtz, Maarit Hellman, Helena Tossavainen & Perttu Permi

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  1. Peter Würtz
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  2. Maarit Hellman
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  3. Helena Tossavainen
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  4. Perttu Permi
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Correspondence to Perttu Permi.

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Würtz, P., Hellman, M., Tossavainen, H. et al. Towards unambiguous assignment of methyl-containing residues by double and triple sensitivity-enhanced HCCmHm-TOCSY experiments. J Biomol NMR 36, 13–26 (2006). https://doi.org/10.1007/s10858-006-9056-3

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