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

John D Gross; Vladimir M Gelev; Gerhard Wagner

doi:10.1023/a:1022890112109

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

John D Gross·Vladimir M Gelev·Gerhard Wagner

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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.

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Butyrates Cholic Acids Eukaryotic Initiation Factor-4E Hemiterpenes Isotopes Keto Acids Ligands Magnetic Resonance Spectroscopy Micelles Models, Chemical Oxalates Proteins Saccharomyces cerevisiae Sensitivity and Specificity Statistics as Topic Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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John D Gross, Vladimir M Gelev, Gerhard Wagner. 2003. A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes.. https://doi.org/10.1023/a%3A1022890112109

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