Linse S, Brodin P, Drakenberg T, Thulin E, Sellers P, Elmdén K, Grundström T, Forsén S
Department of Physical Chemistry, Lund University, Sweden.
Biochemistry. 1987 Oct 20;26(21):6723-35. doi: 10.1021/bi00395a023.
Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.
编码牛钙结合蛋白D9k的次要A组分的基因——已知最小的具有一对EF手型钙结合位点的蛋白质——带有氨基酸取代和/或缺失的该基因已在大肠杆菌中合成并表达，并用不同的生物物理技术进行了表征。这些突变局限于N端钙结合位点，包括Pro-20→Gly（M1）、Pro-20→Gly且Asn-21缺失（M2）、Pro-20缺失（M3）以及Tyr-13→Phe（M4）。1H、43Ca和113Cd核磁共振研究表明，诱导产生的结构变化主要局限于修饰区域，对C端钙结合位点几乎没有影响。N端位点的Ca2+交换速率在野生型蛋白（M0）和M4中为3 s-1，在M2和M3中变为5000 s-1，而C端位点的Ca2+交换没有可检测到的变化。宏观Ca2+结合常数是通过在荧光螯合剂2-[[2-[双（羧甲基）-氨基]-甲基苯氧基]甲基]-6-甲氧基-8-[双（羧甲基）氨基]喹啉存在下的平衡或使用Ca2+选择性电极获得的。M4的Ca2+亲和力与M0相似，而在M2和M3的第二个化学计量步骤中发现了最大差异。微量热数据表明，除M2和M3的N端位点（+5 kJ·mol-1）外，所有位点的Ca2+结合焓均为负（-8至-13 kJ·mol-1）。在所有情况下，结合熵均为强正值。通过宏观Ca2+结合常数的值确定了M0和M4中的协同Ca2+结合。通过在Ca2+滴定过程中观察到的1H核磁共振谱变化，我们可以获得M0和M4中位点结合常数之间的值。这些值与宏观结合常数相结合，得出位点间相互作用自由能ΔΔG为-5.1±0.4 kJ·mol-1（M0）且小于-3.9 kJ·mol-1（M4）。有证据（来自113Cd核磁共振）表明M1、M2和M3中也存在位点间相互作用，但由于Ca2+的顺序结合，无法确定ΔΔG的大小。
