19F NMR Studies of the Recombinant Human TransferrinN-Lobe and Three Single Point Mutants

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19F NMR was utilitized to study human serum transferrin, which is a bilobal protein with a high-affinity metal-binding site in each lobe. The N-terminal lobe of the recombinant protein (residues 1–337; hTF/2N) and three single point mutants, H207E, W8Y and W128Y, were expressed in baby hamster kidney cells grown in media supplemented with 5-fluorotryptophan (5-F-Trp). The three tryptophan residues gave three well resolved 19F NMR resonances, which were assigned by site-directed mutagenesis of two of the three Trp residues to Tyr. It was found that conformational changes are induced by metal binding to hTF/2N and a site-directed mutant H207E which has a higher binding affinity for Fe(III). Shifts in the 19F NMR spectra indicated changes when proteins bound Fe(III) or Ga(III) along with a synergistic anion, e.g. oxalate or carbonate. The resonance for 5-F-Trp 264 did not change frequency during titration with either metal in hTF/2N or the H207E mutant. Two resonances corresponding to Trp 128 and Trp 8 showed high-field shifts upon metal binding. These studies have shown that the fluorine nucleus is sensitive to local conformational changes in the binding pocket when the synergistic anion is changed from carbonate to oxalate. In addition, the fluorine nucleus can identify areas of the protein which experience secondary structural change when ligand binds. The solution NMR studies showing dynamic changes are complementary to the crystal structures of this family of Fe(III) binding proteins. © 1997 John Wiley & Sons, Ltd.