Proton nuclear magnetic resonance spectroscopy of human transferrin N-terminal half-molecule: titration and hydrogen-deuterium exchange.
The binding of Ga(III) to the proteolytically derived N-terminal half-molecule of human transferrin (HTF/2N) was studied by proton nuclear magnetic resonance spectroscopy. The pH-dependent titration curves of the histidinyl C(2) proton chemical shifts were altered upon formation of the GaIIIHTF/2N(C2O4) ternary complex. Two high-pK'a histidines failed to titrate when the metal and synergistic anion formed a complex with the protein. These results implicated two histidinyl residues as direct ligands to the metal. The rates of hydrogen-deuterium exchange for the C(2) protons of certain histidinyl residues were substantially decreased by metal ion binding. The two ligand histidines were protected from exchange, and a third, low-pK'a, histidinyl residue was protected. We propose that this third histidinyl residue is involved in anion binding and may serve as the base in the putative proton-relay scheme proposed for complex formation.
D001059:Apoproteins / Q000378:metabolism; D003903:Deuterium; D017666:Deuterium Oxide; D005708:Gallium / Q000378:metabolism; D006639:Histidine; D006801:Humans; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D014168:Transferrin / Q000378:metabolism*; D014867:Water
D001059:Apoproteins; D014168:Transferrin; C028284:apotransferrin; D014867:Water; D006639:Histidine; D006859:Hydrogen; D003903:Deuterium; D005708:Gallium; D017666:Deuterium Oxide