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Updated: Jun 28, 2026

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins
Published on: December 27, 2016
Detection and assignment of phosphoserine and phosphothreonine residues by (13)C- (31)P spin-echo difference NMR
Lawrence P McIntosh1, Hyun-Seo Kang, Mark Okon
1Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada. mcintosh@chem.ubc.ca
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.
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