Bertini I, Viezzoli M S, Luchinat C, Stafford E, Cardin A D, Behnke W D, Bhattacharyya L, Brewer C F
Department of Chemistry, University of Florence, Italy.
J Biol Chem. 1987 Dec 15;262(35):16985-94.
Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.
已记录了小扁豆凝集素（CCoLcH）和豌豆凝集素（CCoPSA）的Ca2+-Co2+衍生物（S1位点为Co2+，S2位点为Ca2+）的可见吸收光谱、圆二色性（CD）光谱和磁圆二色性光谱，结果表明这两种蛋白质的这些光谱非常相似。可见吸收光谱和磁圆二色性光谱表明，两种蛋白质中S1位点的高自旋Co2+具有相似的八面体几何结构，就像伴刀豆球蛋白A（CCoPL）的Ca2+-Co2+复合物中那样（Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441 - 451）。然而，可见CD数据表明，与CCoPL相比，CCoLcH和CCoPSA的S1周围环境存在差异。凝集素的Co2+和Ni2+衍生物在90 MHz下的1H NMR光谱显示出一些各向同性位移信号，这些信号来自S1位点紧邻区域的质子。对H2O和D2O中Co2+衍生物光谱的分析使得能够对凝集素中S1位点配位组氨酸的侧链环质子进行共振归属。与小扁豆和豌豆凝集素相比，观察到伴刀豆球蛋白A中S1位点组氨酸NH质子的H-D交换速率存在差异。Ni2+取代蛋白的NMR数据以及Co2+衍生物的光谱还表明，S1位点的羧酸根配体和组氨酸残基的侧链在伴刀豆球蛋白A中的位置与其他两种凝集素不同。这些结果似乎部分解释了在Co2+取代蛋白的可见CD光谱中观察到的差异。此外，单糖的结合不会显著干扰凝集素的光谱。所有三种Co2+取代凝集素的1H NMR光谱中的一个不寻常特征是存在两个可交换的低场位移共振，这似乎与与S2位点的Ca2+离子配位的缓慢交换水分子的两个质子有关。CCoLcH的T1测量提供了Co2+离子到这两个质子的距离估计值，分别为3.7 Å和4.0 Å。
