Juffer A H, Vogel H J
Biocenter and the Department of Biochemistry, The University of Oulu, Oulun Yliopisto, Oulu, Finland.
Proteins. 2000 Dec 1;41(4):554-67.
Calbindin is a small (75 residues) helix-loop-helix ("EF-hand") calcium-binding protein belonging to the calmodulin superfamily. It binds two Ca(2+) ions. Continuum electrostatics in combination with the boundary element method was employed for the calculation of the acid-dissociation constants K(a) (pK(a) = -log K(a)) values of all titratable residues in the protein. The objectives were to determine quantitatively the effects of divalent ion binding and small ion-induced structural changes on predicted pK(a)'s. Computations were carried out for the apo and holo form of calbindin, for which both X-ray and NMR structures were available. Comparison was made with several sets of experimental pK(a) values determined by NMR spectroscopy. Different choices of the dielectric constant (ranging from 4 to 78.5) for calbindin and variations in ionic strength (from 0 to 0.3 M) were investigated in a systematic fashion. Removal of the two bound Ca(2+) ions increases the pK(a) values of all residues if no conformational changes were allowed. If conformational differences between the apo and holo were accounted for, shifts in either direction were observed. Titrating groups that are directly involved in Ca(2+) binding (Asp and Glu) required a dielectric constant of 78.5 for the holo structure to obtain a reasonable estimate of their pK(a)'s. For the apo structure, passable values for the pK(a)'s of these ligating groups could be determined if the structure was allowed to relax upon ion removal.
钙结合蛋白是一种小的（75个残基）螺旋-环-螺旋（“EF手”）钙结合蛋白，属于钙调蛋白超家族。它结合两个Ca(2+)离子。采用连续介质静电学与边界元方法相结合来计算蛋白质中所有可滴定残基的酸解离常数K(a)（pK(a)=-log K(a)）值。目的是定量确定二价离子结合和小离子诱导的结构变化对预测pK(a)值的影响。对钙结合蛋白的脱辅基形式和全蛋白形式进行了计算，这两种形式都有X射线和核磁共振结构。与通过核磁共振光谱法测定的几组实验pK(a)值进行了比较。系统研究了钙结合蛋白介电常数的不同选择（范围从4到78.5）以及离子强度的变化（从0到0.3 M）。如果不允许构象变化，去除两个结合的Ca(2+)离子会增加所有残基的pK(a)值。如果考虑脱辅基形式和全蛋白形式之间的构象差异，则会观察到向任何一个方向的变化。对于全蛋白结构，直接参与Ca(2+)结合的滴定基团（Asp和Glu）需要介电常数为78.5才能合理估计它们的pK(a)值。对于脱辅基结构，如果在去除离子时允许结构松弛，则可以确定这些连接基团pK(a)的可接受值。
