Kesvatera T, Jönsson B, Thulin E, Linse S
Physical Chemistry, University of Lund, Sweden.
J Mol Biol. 1996 Jun 21;259(4):828-39. doi: 10.1006/jmbi.1996.0361.
A pH titration study of calbindin D9k was performed using heteronuclear 1H-13C two-dimensional NMR spectroscopy. The protein was produced with carbon-13 label in the side-chain of lysine residues, next to the titrating group. The site-specific pKa values of these lysine residues, ranging from 10.1 to 12.1, were obtained from the analysis of pH-dependent chemical shifts of 13C and 1H resonances. Ionization constants for both the Ca(2+)-free (apo) and Ca(2+)-loaded forms of the protein were determined. The proton uptake by lysine residues in the apo form was shifted up to 1.7 units towards high pH as compared to that for the model compound. The binding of calcium affected the pKa values of all lysine residues. The largest reduction of one pK unit was observed for Lys55, which is also the closest to the calcium binding sites. A threefold increase in protein concentration, from 0.5 to 1.5 mM, reduced the pKa values by 0.1 to 0.4 pK unit in agreement with the screening concept of ionic interactions. All the observed pKa shifts were site-specific, depending on the local electrostatic environment and were reproduced in Monte Carlo simulations based on the three-dimensional structure of calbindin D9k and a dielectric continuum model for the electrostatic interactions.
使用异核1H-13C二维核磁共振光谱对钙结合蛋白D9k进行了pH滴定研究。该蛋白质在赖氨酸残基侧链上用碳-13标记生成，靠近滴定基团。通过分析13C和1H共振的pH依赖性化学位移，获得了这些赖氨酸残基的位点特异性pKa值，范围为10.1至12.1。测定了蛋白质的无钙（脱辅基）和钙负载形式的电离常数。与模型化合物相比，脱辅基形式中赖氨酸残基的质子摄取向高pH方向移动了1.7个单位。钙的结合影响了所有赖氨酸残基的pKa值。观察到Lys55的一个pK单位下降幅度最大，它也是最接近钙结合位点的。蛋白质浓度从0.5 mM增加到1.5 mM，pKa值降低了0.1至0.4个pK单位，这与离子相互作用的筛选概念一致。所有观察到的pKa位移都是位点特异性的，取决于局部静电环境，并在基于钙结合蛋白D9k的三维结构和静电相互作用的介电连续体模型的蒙特卡罗模拟中得到重现。
