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Biosynthetically directed 2H labelling for stereospecific resonance assignments of glycine methylene groups

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Abstract

Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific resonance assignments can readily be obtained by cell-free protein synthesis in D2O, as the serine hydroxymethyltransferase, that is naturally present in E. coli cell extracts, selectively replaces the pro-2S proton of glycine by a deuterium. To encourage the conversion by serine hydroxymethyltransferase, we performed the cell-free reaction without the addition of any glycine, exploiting the capability of the enzyme to convert serine to glycine with the help of tetrahydrofolate. 13C-HSQC spectra of ubiquitin produced with 13C/15N-serine showed that about a quarter of the glycine residues derived from serine were stereospecifically deuterated. Pulse sequences are presented that select the signals from the stereospecifically deuterated glycine residues.

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Acknowledgments

We thank Professor Masatsune Kainosho for stimulating discussions and a sample of stereospecifically deuterated glycine. Financial support by the Australian Research Council is gratefully acknowledged.

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  1. Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia

    Karin V. Loscha & Gottfried Otting

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  1. Karin V. Loscha
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  2. Gottfried Otting
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Correspondence to Gottfried Otting.

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Loscha, K.V., Otting, G. Biosynthetically directed 2H labelling for stereospecific resonance assignments of glycine methylene groups. J Biomol NMR 55, 97–104 (2013). https://doi.org/10.1007/s10858-012-9690-x

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  • DOI: https://doi.org/10.1007/s10858-012-9690-x

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