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An Isotope Labeling Strategy for Methyl TROSY Spectroscopy

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Abstract

Recently we have shown that HMQC spectra of protonated methyl groups in high molecular weight, highly deuterated proteins have large enhancements in sensitivity and resolution relative to HSQC-generated data sets. These enhancements derive from a TROSY effect in which complete cancellation of intra-methyl 1H-1H and 1H-13C dipolar interactions occurs for 50% of the signal in the case of HMQC, so long as the methyl is attached to a molecule tumbling in the macromolecular limit (Tugarinov, V., Hwang, P.M., Ollerenshaw, J.E., Kay, L.E. J. Am. Chem. Soc. (2003) 125, 10420–10428; Ollerenshaw, J.E., Tugarinov, V. and Kay, L.E. Magn. Reson. Chem. (2003) 41, 843–852. The first demonstration of this effect was made for isoleucine δ1 methyl groups in a highly deuterated 82 kDa protein, malate synthase G. As with 1H-15N TROSY spectroscopy high levels of deuteration are critical for maximizing the TROSY effect. Here we show that excellent quality methyl TROSY spectra can be recorded on U-[2H] Ileδ1-[13CH3] Leu,Val-[13CH3/12CD3] protein samples, significantly extending the number of probes available for structural and dynamic studies of high molecular weight systems.

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Authors and Affiliations

  1. Protein Engineering Network Centres of Excellence and the Departments of Medical Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8

    Vitali Tugarinov & Lewis E. Kay

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  1. Vitali Tugarinov
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  2. Lewis E. Kay
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Tugarinov, V., Kay, L.E. An Isotope Labeling Strategy for Methyl TROSY Spectroscopy. J Biomol NMR 28, 165–172 (2004). https://doi.org/10.1023/B:JNMR.0000013824.93994.1f

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  • DOI: https://doi.org/10.1023/B:JNMR.0000013824.93994.1f

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