[13C]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 been produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, (13)C NMR of the [epsilon-(13)C]methionine-labelled proteins has been used to monitor conformational changes of the protein during metal binding. All five methionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, MacGillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The tentative two-dimensional NMR assignment for two of the five methionine residues, namely Met(26) and Met(109), has been corrected. A series of NMR spectra for the complexes of (13)C-Met-labelled hTF/2N with six different metal ions, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate that the conformational change of the protein upon metal binding can be observed by means of the changes in the NMR chemical shifts associated with certain methionine residues, regardless of whether diamagnetic or paramagnetic metals 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 perturbation of the electronic spectra of any of the mutants. Nevertheless, the M109L mutant (Met(109)-->Leu) releases iron at half the rate of the wild-type N-lobe, and chloride shows a significantly greater retarding effect on the rate of iron release from all five mutants. All the methionine mutants (especially in the apo form) show a poor solubility in Hepes buffer lacking anions such as bicarbonate. These findings imply a more general effect of anion binding to surface residues than previously realized.
He
Q Y
QY
Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT 05405, USA. qhe@zoo.uvm.edu
Mason
A B
AB
Tam
B M
BM
MacGillivray
R T
RT
Woodworth
R C
RC
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology*; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000494:pharmacology*; D008715:Methionine / Q000235:genetics*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics
D002712:Chlorides; D008670:Metals; D014168:Transferrin; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D008715:Methionine; D007501:Iron