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Detection and assignment of phosphoserine and phosphothreonine residues by 13C–31P spin-echo difference NMR spectroscopy

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Abstract

A simple NMR method is presented for the identification and assignment of phosphorylated serine and threonine residues in 13C- or 13C/15N-labeled proteins. By exploiting modest (~5 Hz) 2- and 3-bond 13C–31P scalar couplings, the aliphatic 1H–13C signals from phosphoserines and phosphothreonines can be detected selectively in a 31P spin-echo difference constant time 1H-13C HSQC spectrum. Inclusion of the same 31P spin-echo element within the 13C frequency editing period of an intraHNCA or HN(CO)CA experiment allows identification of the amide 1HN and 15N signals of residues (i) for which 13Cα(i) or 13Cα(i − 1), respectively, are coupled to a phosphate. Furthermore, 31P resonance assignments can be obtained by applying selective low power cw 31P decoupling during the spin-echo period. The approach is demonstrated using a PNT domain containing fragment of the transcription factor Ets-1, phosphorylated in vitro at Thr38 and Ser41 with the MAP kinase ERK2.

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Abbreviations

CT:

Constant time

CW:

Continuous wave

DSS:

2,2-Dimethyl-2-silapentance-5-sulfonic acid

ERK2:

Extracellular regulated kinase 2

HSQC:

Heteronuclear single quantum correlation

MAPK:

Mitogen activated protein kinase

pSer:

Phosphoserine

pThr:

Phosphothreonine

TMP:

Trimethylphosphate

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Acknowledgements

We thank Robert Konrat and Adrien Favier for helpful discussions and technical help. L.P.M. gratefully acknowledges the sabbatical support of a Killam Faculty Research Fellowship, a Séjour Scientifique de Haut Niveau Award from the Scientific Services of the French Embassy in Canada, and a CIHR-CNRS International Exchange Grant. This research was supported by grants from National Cancer Institute of Canada with funds from the Canadian Cancer Society (to L.P.M.) and the National Institutes of Health grants GM38663 (to B.J.G.) and CA42014-I (to the Huntsman Cancer Institute for support of core facilities). B.J.G. also acknowledges funding from the Huntsman Cancer Institute/Huntsman Cancer Foundation. Instrument support was provided by the Canadian Institutes for Health Research (CIHR), the Canadian Foundation for Innovation (CFI), the British Columbia Knowledge Development Fund (BCKDF), the UBC Blusson Fund, and the Michael Smith Foundation for Health Research (MSFHR). B.B. acknowledges financial support from the Commissariat à l’Energie Atomique (CEA), the Centre National de la Recherche Scientifique (CNRS), the University Grenoble, and the French Research Agency (ANR).

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Correspondence to Lawrence P. McIntosh or Bernhard Brutscher.

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McIntosh, L.P., Kang, HS., Okon, M. et al. Detection and assignment of phosphoserine and phosphothreonine residues by 13C–31P spin-echo difference NMR spectroscopy. J Biomol NMR 43, 31–37 (2009). https://doi.org/10.1007/s10858-008-9287-6

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