INTERLOBE COMMUNICATION IN C-13-METHIONINE-LABELED HUMAN TRANSFERRIN

[H-1, C-13] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These a re thought to play an important role in the recognition of this protei n by its cellular receptors. [H-1, C-13] NMR resonance assignments are presented for all nine methionine (CH3)-C-13 groups of recombinant de glycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [H-1, C-13] HMQC spe ctra (1) and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine (CH3)-C-13 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subseq uently to the N-lobe. The NMR shifts of Met464, which is in the Trp460 -centered hydrophobic patch of helix 5 in the C-lobe in contact with t he anion and metal binding site, show that Ga3+ binding causes movemen t of side chains within this helix, as is also the case in the N-lobe. The C-lobe residue Met382, which contacts the N-lobe hinge region, is perturbed when Ga3+ binds to the N-lobe, indicative of interlobe comm unication, a feature which may control the recognition of fully-metall ated transferrin by its receptor. These results demonstrate that selec tive C-13 labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins.