Beatty E J, Cox M C, Frenkiel T A, Tam B M, Mason A B, MacGillivray R T, Sadler P J, Woodworth R C
Department of Chemistry, Birkbeck College, University of London, U.K.
Biochemistry. 1996 Jun 18;35(24):7635-42. doi: 10.1021/bi960684g.
[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.
报道了[1H, 13C]核磁共振对血清转铁蛋白（80 kDa）中金属诱导的构象变化的研究。这些变化被认为在该蛋白被其细胞受体识别过程中发挥重要作用。基于对重组N端结构域（40 kDa，含五个甲硫氨酸残基）的研究、NOESY中继[1H, 13C] HMQC谱以及结构考虑，给出了重组去糖基化人转铁蛋白所有九个甲硫氨酸13CH3基团的[1H, 13C]核磁共振共振归属。首次给出了转铁蛋白C端结构域共振的具体归属。以甲硫氨酸13CH3共振为探针，结果表明，以草酸盐作为协同阴离子时，Ga3+优先结合到C端结构域，随后结合到N端结构域。位于C端结构域螺旋5中以Trp460为中心的疏水区域且与阴离子和金属结合位点接触的Met464的核磁共振位移表明，Ga3+结合导致该螺旋内的侧链移动，N端结构域情况也是如此。当Ga3+结合到N端结构域时，与N端结构域铰链区接触的C端结构域残基Met382受到扰动，这表明存在结构域间通讯，这一特征可能控制完全金属化的转铁蛋白被其受体识别。这些结果表明，选择性13C标记是探测高分子量蛋白质结构和动力学的有力方法。
