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J Biomol NMR. 2003 Mar;25(3):235-42.

A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes.

Author information

1
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave. Boston, MA 02115, USA.

Abstract

A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. (13)C/(1)H-I,L,V-methyl, U-(2)H labeled protein is produced using the biosynthetic precursors [gamma-(13)C]-alpha-ketobutyrate and [gamma,gamma'-(13)C(2)]-alpha-ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D (13)C-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs. A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported. The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle. This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.

PMID:
12652135
DOI:
10.1023/a:1022890112109
[Indexed for MEDLINE]

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