Detection and assignment of phosphoserine and phosphothreonine residues by (13)C- (31)P spin-echo difference NMR spectroscopy.
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.
McIntosh
Lawrence P
LP
Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada. mcintosh@chem.ubc.ca
Kang
Hyun-Seo
HS
Okon
Mark
M
Nelson
Mary L
ML
Graves
Barbara J
BJ
Brutscher
Bernhard
B
D002247:Carbon Isotopes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010750:Phosphoproteins / Q000737:chemistry; D010759:Phosphorus Isotopes / Q000737:chemistry*; D010768:Phosphoserine / Q000032:analysis*; D010769:Phosphothreonine / Q000032:analysis*; D011506:Proteins / Q000737:chemistry*; D050784:Proto-Oncogene Protein c-ets-1 / Q000737:chemistry / Q000235:genetics
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010750:Phosphoproteins; D010759:Phosphorus Isotopes; D011506:Proteins; D050784:Proto-Oncogene Protein c-ets-1; D010769:Phosphothreonine; D010768:Phosphoserine