Stickle D F, Liu G, Fried M G
Department of Biochemistry, Pennsylvania State University College of Medicine, Hershey 17033.
Eur J Biochem. 1994 Dec 15;226(3):869-76. doi: 10.1111/j.1432-1033.1994.00869.x.
The salt concentration dependences of the observed association constants (Kobs) for the binding of wild-type lac repressor tetramer and the dimeric lacI-18 mutant repressor to lactose operator DNA were compared. For both proteins, the data are consistent with a class of linkage models in which ion binding by the protein is driven by differences in the ionic concentrations in bulk solution and in the volume near the DNA surface. The models that best agree with the data are those in which ion-binding reactions are cooperative. In spite of close agreement between these models and the data, the determination of ion stoichiometries and apparent ion-binding affinities requires additional mechanistic or structural information. The simplest ion-binding mechanism consistent with the data is compatible with a current structural model of the repressor-operator complex. At salt concentrations in excess of 50 mM, at which cation displacement from the DNA and anion displacement from the protein are expected to dominate, similar ion stoichiometries are found for the DNA binding of dimeric and tetrameric repressors. This supports the notion that the DNA contacts of these proteins are homologous. At lower salt concentrations, in which cation binding by the proteins is expected to be significant, the net ion stoichiometry of wild-type repressor binding is slightly greater than that of the lacI-18 mutant. This difference may reflect the availability of ion-binding sites in the distal subunits of tetramer that are not present in the dimer, or may be a consequence of the involvement of ion binding in the dimer/monomer equilibrium.
比较了野生型乳糖阻遏物四聚体和二聚体lacI - 18突变阻遏物与乳糖操纵基因DNA结合时观察到的缔合常数（Kobs）对盐浓度的依赖性。对于这两种蛋白质，数据与一类连锁模型一致，在这类模型中，蛋白质的离子结合是由本体溶液和DNA表面附近区域离子浓度的差异驱动的。与数据最吻合的模型是离子结合反应具有协同性的模型。尽管这些模型与数据吻合得很好，但确定离子化学计量和表观离子结合亲和力还需要额外的机制或结构信息。与数据一致的最简单离子结合机制与阻遏物 - 操纵基因复合物的当前结构模型相符。在盐浓度超过50 mM时，预计DNA上的阳离子置换和蛋白质上的阴离子置换占主导，二聚体和四聚体阻遏物的DNA结合具有相似的离子化学计量。这支持了这些蛋白质与DNA的接触是同源的观点。在较低盐浓度下，预计蛋白质的阳离子结合会很显著，野生型阻遏物结合的净离子化学计量略大于lacI - 18突变体。这种差异可能反映了四聚体远端亚基中存在而二聚体中不存在的离子结合位点的可用性，或者可能是离子结合参与二聚体/单体平衡的结果。
