F-19 NMR-STUDIES OF THE RECOMBINANT HUMAN TRANSFERRIN N-LOBE AND 3 SINGLE-POINT MUTANTS

F-19 NMR was utilitized to study human serum transferrin, which is a b ilobal protein with a high-affinity metal-binding site in each lobe. T he 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-fluoro tryptophan (5-F-Trp). The three tryptophan residues gave three well re solved F-19 MMR resonances, which were assigned by site-directed mutag enesis of two of the three Trp residues to Tyr. It was found that conf ormational changes are induced by metal binding to hTF/2N and a site-d irected mutant H207E which has a higher binding affinity for Fe(III). Shifts in the F-19 NMR spectra indicated changes when proteins bound F e(III) or Ga(III) along with a synergistic anion, e.g. oxalate or carb onate. The resonance for 5-F-Trp 264 did not change frequency during t itration with either metal in hTF/2N or the H207E mutant. Two resonanc es corresponding to Trp 128 and Trp 8 showed high-field shifts upon me tal binding. These studies have shown that the fluorine nucleus is sen sitive to local conformational changes in the binding pocket when the synergistic anion is changed from carbonate to oxalate. In addition, t he fluorine nucleus can identify areas of the protein which experience secondary structural change when ligand binds. The solution NMR studi es showing dynamic changes are complementary to the crystal structures of this family of Fe(III) binding proteins. (C) 1997 by John Wiley & Sons, Ltd.