Trp128Tyr mutation in the N-lobe of recombinant human serum transferrin: 1H- and 15N-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 spectra and energy calculations show that the mutation causes little perturbation of the overall structure of hTF/2N although the chelating agent Tiron removed Fe3+ from the mutant protein about three times faster than from wild-type hTF/2N. 1H-NMR resonances of residues in the Leu122-Trp128-Ile132 hydrophobic patch are assigned both by ring current calculations and with the aid of the mutation. [1H, 15N]-NMR resonances for 11 of the 14 Tyr residues were observed in the spectra of 15N-Tyr-hTF/2N and a resonance for Tyr128 was assignable in spectra of the mutant. The 15N resonance of Y128 was sensitive to oxalate and Ga3+ binding, and Ga3+ binding perturbed 15N 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.