title: Site-selective screening by NMR spectroscopy with labeled amino acid pairs.
abstract: A new method for site-selective screening by NMR is presented. The core of the new method is the dual amino acid sequence specific labeling technique. Amino acid X is labeled with (13)C and amino acid Y is labeled with (15)N. Provided only one XY pair occurs in the amino acid sequence, only one signal in the 1D carbonyl (13)C spectrum will display a splitting due to the (1)J(C'N) coupling. Using this labeling strategy it is possible to screen selectively for binding to a selected epitope without the need for sequence specific assignments. An HNCO spectrum (1D or 2D) can be used either directly as a screening experiment or indirectly to identify what signals to monitor in a 2D (1)H-(15)N correlation spectrum. Chemical shift perturbations upon addition of a potential ligand are easily detected even for large proteins due to the reduced spectral complexity resulting from the use of a selectively labeled sample. The new technique is demonstrated on the human adipocyte fatty acid binding protein FABP-4. Due to the reduced spectral complexity, the method should be applicable to larger proteins than are conventional methods.
authors.affiliation: Department of Structural Chemistry, Biovitrum, S-112 76 Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Weigelt
authors.forename: Maria
authors.initials: M
authors.lastname: van Dongen
authors.forename: Jonas
authors.initials: J
authors.lastname: Uppenberg
authors.forename: Johan
authors.initials: J
authors.lastname: Schultz
authors.forename: Mats
authors.initials: M
authors.lastname: Wikström
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D001665:Binding Sites; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes