[C-13]methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride

The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each of the five methionine residues was individually mutated have be en produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, C-13 N MR of the [epsilon-C-13]methionine-labelled proteins has been used to monit or conformational changes of the protein during metal binding. All five met hionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, Mac Gillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The ten tative two-dimensional NMR assignment for two of the five methionine residu es, namely Met(26) and Met(109), has been corrected. A series of NMR spectr a for the complexes of C-13-Met-labelled hTF/2N with six different metal io ns, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate tha t the conformational change of the protein upon metal binding can be observ ed by means of the changes in the NMR chemical shifts associated with certa in methionine residues, regardless of whether diamagnetic or paramagnetic m etals are used. Changing any of the methionine residues should have minimal effects on transferrin function, since structural analysis shows that none of these residues contacts functional amino acids or has any obvious role in iron uptake or release. In fact, UV-visible spectra show little perturba tion of the electronic spectra of any of the mutants. Nevertheless, the M10 9L mutant (Met(109) --> Leu) releases iron at half the rate of the wild-typ e N-lobe, and chloride shows a significantly greater retarding effect on th e rate of iron release from all five mutants. All the methionine mutants (e specially in the apo form) show a poor solubility in Hepes buffer lacking a nions such as bicarbonate. These findings imply a more general effect of an ion binding to surface residues than previously realized.