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

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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 13C and amino acid Y is labeled with 15N. Provided only one XY pair occurs in the amino acid sequence, only one signal in the 1D carbonyl 13C spectrum will display a splitting due to the 1JC‘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 1H−15N 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.