title: Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs.
abstract: Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L and V are stereo-specifically labeled. We show that 2-ketobutyrate cannot be combined with Ala and 2-acetolactate (for the stereo-specific labeling of L and V) as this results in co-incorporation incompatibility and isotopic scrambling. Thus, we developed a robust and cost-effective enzymatic synthesis of the isoleucine precursor, 2-hydroxy-2-(1'-[(2)H2], 2'-[(13)C])ethyl-3-keto-4-[(2)H3]butanoic acid, as well as an incorporation protocol that eliminates metabolic leakage. We show that application of this labeling scheme to a large 82 kDa protein permits the detection of long-range (1)H-(1)H NOE cross-peaks between methyl probes separated by up to 10 Å.
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, 71 Avenue des Martyrs, CS 10090, 38044, Grenoble Cedex 9, France.
authors.forename: Rime
authors.initials: R
authors.lastname: Kerfah
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Plevin
authors.forename: Ombeline
authors.initials: O
authors.lastname: Pessey
authors.forename: Olivier
authors.initials: O
authors.lastname: Hamelin
authors.forename: Pierre
authors.initials: P
authors.lastname: Gans
authors.forename: Jerome
authors.initials: J
authors.lastname: Boisbouvier
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D017434:Protein Structure, Tertiary
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D000095:Acetolactate Synthase