Generation of Pseudocontact Shifts in Protein NMR Spectra with a Genetically Encoded Cobalt(II)-Binding Amino Acid

摘要

There is increasing interest in the paramagnetic labeling of proteins for structural studies by NMR spectroscopy. The resulting paramagnetic effects, particularly pseudocontact shifts (PCSs) and paramagnetic relaxation enhancement (PRE), provide powerful long-range structural information for the rapid structure analysis of proteins, protein-protein complexes, and protein-ligand complexes.Different strategies have been applied for the site-specific labeling of proteins with paramagnetic metal ions. Most rely on single cysteine residues in the protein or peptide fusions.A more widely applicable method would make use of a non-natural metal-binding amino acid that could be incorporated anywhere in the protein without restriction to the N or C terminus of the protein and without consideration of the presence of cysteine residues or disulfide bonds. Herein we show that the site-specific incorporation of the genetically encoded non-natural amino acid bipyridylalanine (BpyAla, Figure 1 a)' endows the target protein with a site-specific binding site for Co~(II) that generates significant long-range PCSs.