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

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 phosphoserinesand phosphothreoninescan be detected selectively in a 31P spin-echodifference constant time 1H-13C HSQC spectrum. Inclusion of the same 31P spin-echoelement 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-echoperiod. 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.