Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 25, Issue 3, Pages 235-242Publisher
SPRINGER
DOI: 10.1023/A:1022890112109
Keywords
biosynthetic precursors; C-13-methyl; intermolecular NOEs; isotope labeling of proteins; protein complex
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Funding
- NCI NIH HHS [CA68262] Funding Source: Medline
- NCRR NIH HHS [RR-00995] Funding Source: Medline
- NIGMS NIH HHS [GM47467, P01 GM047467, GM38608] Funding Source: Medline
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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.
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