Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 43, Issue 1, Pages 31-37Publisher
SPRINGER
DOI: 10.1007/s10858-008-9287-6
Keywords
P-31-C-13 scalar coupling; Phosphoprotein; MAP kinase; Ets-1 transcription factor
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Funding
- Scientific Services of the French Embassy in Canada
- CIHR-CNRS International Exchange
- National Cancer Institute of Canada
- Canadian Cancer Society
- National Institutes of Health [GM38663, CA42014-I]
- Huntsman Cancer Institute/Huntsman Cancer Foundation
- Commissariat a l'Energie Atomique (CEA)
- Centre National de la Recherche Scientifique (CNRS)
- University Grenoble
- French Research Agency (ANR)
- NATIONAL CANCER INSTITUTE [P30CA042014] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R29GM038663, R01GM038663] Funding Source: NIH RePORTER
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A simple NMR method is presented for the identification and assignment of phosphorylated serine and threonine residues in C-13- or C-13/N-15-labeled proteins. By exploiting modest (similar to 5 Hz) 2- and 3-bond C-13-P-31 scalar couplings, the aliphatic H-1-C-13 signals from phosphoserines and phosphothreonines can be detected selectively in a P-31 spin-echo difference constant time H-1-C-13 HSQC spectrum. Inclusion of the same P-31 spin-echo element within the C-13 frequency editing period of an intraHNCA or HN(CO)CA experiment allows identification of the amide H-1(N) and N-15 signals of residues (i) for which C-13(alpha)(i) or C-13(alpha)(i - 1), respectively, are coupled to a phosphate. Furthermore, P-31 resonance assignments can be obtained by applying selective low power cw P-31 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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