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

TitleDetection and assignment of phosphoserine and phosphothreonine residues by C-13-P-31 spin-echo difference NMR spectroscopy
Publication TypeJournal Article
Year of Publication2009
AuthorsMcIntosh, LP, Kang, HS, Okon, M, Nelson, ML, Graves, BJ, Brutscher, B
JournalJournal of Biomolecular Nmr
Volume43
Pagination31-37
Date PublishedJan
Type of ArticleArticle
ISBN Number0925-2738
KeywordsASSIGNMENT, CENTER-DOT-OP, CONSTANT-TIME, Ets-1, MAP kinase, NUCLEIC-ACIDS, OP HYDROGEN-BONDS, P-31-C-13 scalar coupling, Phosphoprotein, PHOSPHORYLATION, POINTED DOMAIN, PROTEIN, QUANTITATIVE J-CORRELATION, RESONANCE, SCALAR COUPLINGS, TRANSCRIPTION FACTOR
Abstract

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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