Barkley M D, Lewis P A, Sullivan G E
Biochemistry. 1981 Jun 23;20(13):3842-51. doi: 10.1021/bi00516a027.
The effects of ions on the interaction of lac repressor protein and operator DNA have been studied by the membrane filter technique. The equilibrium association constant was determined as a function of monovalent and divalent cation concentrations, anions, and pH. The binding of repressor and operator is extremely sensitive to the ionic environment. The dependence of the observed equilibrium constant on salt concentration is analyzed according to the binding theory of Record et al. [Record, M. T., Jr., Lohman, T. M., & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145]. The number of ionic interactions in repressor--operator complex is deduced from the slopes of the linear log-log plots. About 11 ionic interactions are formed between repressor and DNA phosphates at pH 7.4 and about 9 ionic interactions at pH 8.0, in reasonable agreement with previous estimates. A favorable nonelectrostatic binding free energy of about 9-12 kcal/mol is estimated from the extrapolated equilibrium constants at the 1 M standard state. The values are in good accord with recent results for the salt-independent binding of repressor core and operator DNA. The effects of pH on the repressor--operator interaction are small, and probably result from titration of functional groups in the DNA-binding site of the protein. For monovalent salts, the equilibrium constant is slightly dependent on cation type and highly dependent on anion type. At constant salt concentration, the equilibrium constant decreases about 10000-fold in the order CH3CO2- greater than or equal to F- greater than Cl- greater than Br- greater than NO3- greater than SCN- greater than I-. The wide range of accessible equilibrium constants provides a useful tool for in vitro studies of the repressor--operator interaction.
利用膜过滤技术研究了离子对乳糖阻遏蛋白与操纵基因DNA相互作用的影响。测定了平衡缔合常数与单价和二价阳离子浓度、阴离子及pH的函数关系。阻遏蛋白与操纵基因的结合对离子环境极为敏感。根据Record等人的结合理论[Record, M. T., Jr., Lohman, T. M., & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145]分析了观察到的平衡常数对盐浓度的依赖性。从线性对数-对数图的斜率推导出阻遏蛋白-操纵基因复合物中离子相互作用的数量。在pH 7.4时，阻遏蛋白与DNA磷酸基团之间形成约11个离子相互作用，在pH 8.0时约为9个离子相互作用，这与先前的估计相当一致。根据1M标准状态下外推的平衡常数估计出约9 - 12 kcal/mol的有利非静电结合自由能。这些值与最近关于阻遏蛋白核心与操纵基因DNA的盐无关结合的结果非常吻合。pH对阻遏蛋白-操纵基因相互作用的影响较小，可能是由于蛋白质DNA结合位点中官能团的滴定所致。对于单价盐，平衡常数略微依赖于阳离子类型，高度依赖于阴离子类型。在恒定盐浓度下，平衡常数按CH3CO2-≥F-＞Cl-＞Br-＞NO3-＞SCN-＞I-的顺序降低约10000倍。可获得的平衡常数范围广泛，为阻遏蛋白-操纵基因相互作用的体外研究提供了一个有用的工具。
