Protein F-labeling using transglutaminase for the NMR study of intermolecular interactions.

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic F-labeling readily provided NMR detection of protein-drug and protein-protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The F-labeling method was 3.5-fold more sensitive than N-labeling, and could be combined with other chemical modification techniques such as lysine C-methylation. C-dimethylated-F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.