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

A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. C-13/H-1-I,L,V-methyl, U-H-2 labeled protein is produced using the biosynthetic precursors [gamma-C-13]- alpha-ketobutyrate and [gamma,gamma'-C-13(2)]-alpha-ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D C-13-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.