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

J Biomol NMR. 2009 Jan;43(1):31-7. doi: 10.1007/s10858-008-9287-6. Epub 2008 Nov 12.

Abstract

A simple NMR method is presented for the identification and assignment of phosphorylated serine and threonine residues in (13)C- or (13)C/(15)N-labeled proteins. By exploiting modest (~5 Hz) 2- and 3-bond (13)C-(31)P scalar couplings, the aliphatic (1)H-(13)C signals from phosphoserines and phosphothreonines can be detected selectively in a (31)P spin-echo difference constant time (1)H-(13)C HSQC spectrum. Inclusion of the same (31)P spin-echo element within the (13)C frequency editing period of an intraHNCA or HN(CO)CA experiment allows identification of the amide (1)H(N) and (15)N signals of residues (i) for which( 13)C(alpha)(i) or ( 13)C(alpha)(i - 1), respectively, are coupled to a phosphate. Furthermore, (31)P resonance assignments can be obtained by applying selective low power cw (31)P 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Isotopes / chemistry*
  • Escherichia coli / genetics
  • Nitrogen Isotopes / chemistry
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Phosphoproteins / chemistry
  • Phosphorus Isotopes / chemistry*
  • Phosphoserine / analysis*
  • Phosphothreonine / analysis*
  • Proteins / chemistry*
  • Proto-Oncogene Protein c-ets-1 / chemistry
  • Proto-Oncogene Protein c-ets-1 / genetics

Substances

  • Carbon Isotopes
  • Nitrogen Isotopes
  • Phosphoproteins
  • Phosphorus Isotopes
  • Proteins
  • Proto-Oncogene Protein c-ets-1
  • Phosphothreonine
  • Phosphoserine