TRP128TYR MUTATION IN THE N-LOBE OF RECOMBINANT HUMAN SERUM TRANSFERRIN - H-1-NMR AND N-15-NMR AND METAL-BINDING STUDIES

The conserved Trp residue within helix 5 of the N-lobe of human serum transferrin (hTF/2N, 40 kDa) has been mutated to Tyr, NMR and CD spect ra and energy calculations show that the mutation causes little pertur bation of the overall structure of hTF/2N although the chelating agent Tiron removed Fe3+ from the mutant protein about three times faster t han from wild-type hTF/2N, H-1-NMR resonances of residues in the Leu12 2-Trp128-Ile132 hydrophobic patch are assigned both by ring current ca lculations and with the aid of the mutation, [H-1, N-15]-NMR resonance s for 11 of the 14 Tyr residues were observed in the spectra of N-15-T yr-hTF/2N and a resonance for Tyr128 was assignable in spectra of the mutant, The N-15 resonance of Y128 was sensitive to oxalate and Ga3+ b inding, and Ga3+ binding perturbed N-15 resonances for most of the Tyr residues, Since these are well distributed over the N-lobe, it can be concluded that metal-induced structural changes are not merely local to the binding site.