García-Moreno B, Dwyer J J, Gittis A G, Lattman E E, Spencer D S, Stites W E
Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Biophys Chem. 1997 Feb 28;64(1-3):211-24. doi: 10.1016/s0301-4622(96)02238-7.
The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.
蛋白质内部的介电常数是蛋白质中静电相互作用强度的关键物理决定因素。我们从现象学角度测量了这种介电常数，即确定需要与玻恩方程一起使用的介电常数，以便重现因可电离基团埋藏于蛋白质疏水核心中而观察到的pKa位移。为此构建了在第66位带有埋藏赖氨酸残基的葡萄球菌核酸酶突变体。通过差分电位滴定法测量埋藏赖氨酸的pKa值。通过测量比野生型稳定3.3或6.5千卡/摩尔的超稳定核酸酶形式中的pKa值，评估了pKa与蛋白质整体稳定性之间的耦合程度。测定了一个突变体的晶体结构以描述埋藏赖氨酸的环境。所测量的介电常数范围为10至12。以类似方式分析了其他蛋白质中埋藏可电离残基的已发表pKa值，从这些值获得的介电常数与在核酸酶中测量的结果一致。这些结果强烈反对在基于结构的静电能连续介质模型计算中普遍使用4或更低的介电常数来描述蛋白质内部的介电效应。
