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Site-Selective Screening by NMR Spectroscopy with Labeled Amino Acid Pairs

2002; American Chemical Society; Volume: 124; Issue: 11 Linguagem: Inglês

10.1021/ja0178261

1943-2984

Johan Weigelt, Maria van Dongen, J. Uppenberg, Johan Schultz, Mats Wikström,

Analytical Chemistry and Chromatography

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.