Interlobe communication in 13C-methionine-labeled human transferrin.
[1H, 13C] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These are thought to play an important role in the recognition of this protein by its cellular receptors. [1H, 13C] NMR resonance assignments are presented for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [1H, 13C] HMQC spectra, and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine 13CH3 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subsequently 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 the anion and metal binding site, show that Ga3+ binding causes movement 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 communication, a feature which may control the recognition of fully-metallated transferrin by its receptor. These results demonstrate that selective 13C labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins.
Beatty
E J
EJ
Department of Chemistry, Birkbeck College, University of London, U.K.
Cox
M C
MC
Frenkiel
T A
TA
Tam
B M
BM
Mason
A B
AB
MacGillivray
R T
RT
Sadler
P J
PJ
Woodworth
R C
RC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002247:Carbon Isotopes; D002460:Cell Line; D006224:Cricetinae; D018360:Crystallography, X-Ray; D006801:Humans; D007668:Kidney; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010070:Oxalates; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D002247:Carbon Isotopes; D009838:Oligodeoxyribonucleotides; D010070:Oxalates; D011994:Recombinant Proteins; D014168:Transferrin; D008715:Methionine