CA-43 NMR-STUDIES OF CALCIUM-BINDING LYSOZYMES AND ALPHA-LACTALBUMINS
CA-43 NMR-STUDIES OF CALCIUM-BINDING LYSOZYMES AND ALPHA-LACTALBUMINS
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DOI：
10.1021/bi00144a016
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发表时间：
1992-07-28
期刊：
影响因子：
2.9
通讯作者：
VOGEL, HJ
中科院分区：
文献类型：
--
作者：
ARAMINI, JM;DRAKENBERG, T;VOGEL, HJ
用Ca-43核磁共振谱研究了马和鸽溶菌酶以及牛和人α-乳清蛋白的钙结合特性。所有的蛋白质被发现含有一个高亲和力的钙结合位点。化学位移，线宽，弛豫时间（T1和T2），和四极耦合常数为各自的Ca-43 NMR信号是非常相似的，这是一个高度的同源性之间的强钙结合位点的这四种蛋白质的指示。测得的化学位移（δ几乎等于-3至-7 ppm）和四极耦合常数（χ几乎等于0.7-0.8 MHz）与典型EF-手型钙结合蛋白的观察结果截然不同，表明钙结合环的几何结构不同。这些蛋白质中结合的钙离子的相关时间为4-8 ns的数量级，表明这些结合位点的灵活性是有限的。高亲和力位点的表观pK（a）值范围为3.4至4.7，证实了含羧酸根的残基参与钙离子的配位。EDTA竞争实验表明，这些蛋白质对钙的亲和力为牛α-乳清蛋白>鸽溶菌酶>马溶菌酶（K（D）= 5 × 10 ~（-8）~ 10 ~（-6）M）。牛α-乳清蛋白中存在第二个弱钙结合位点（K（D）= 3 × 10 ~（-3）M）的证据，而其它蛋白质中没有。从Ca-43 NMR信号的线宽的不寻常的pH依赖性（pK（a）′ = 6.3）推断出在第二个位点处涉及组氨酸残基。本文提出的Ca-43 NMR研究结果提供了直接的光谱证据，支持功能不同的溶菌酶和α-乳白蛋白之间的进化联系。
The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by Ca-43 NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective Ca-43 NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta almost-equal-to -3 to -7 ppm) and quadrupole coupling constants (chi almost-equal-to 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pK(a) values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine a-lactalbumin almost-equal-to human alpha-lactalbumin > pigeon lysozyme > equine lysozyme (K(D) almost-equal-to 5 X 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (K(D) = 3 X 10(-3) M) was obtained for bovine a-lactalbumin, but not for the other proteins studied. The involvement of a histidine residue(s) at this second site was inferred from the unusual pH dependence (pK(a)' = 6.3) of the line width of its Ca-43 NMR signal. The Ca-43 NMR findings presented in this paper provide direct spectroscopic evidence in support of an evolutionary link between the functionally distinct lysozymes and alpha-lactalbumins.