Sidorova N Y, Rau D C
Laboratory of Physical and Structural Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
J Mol Biol. 2001 Jul 20;310(4):801-16. doi: 10.1006/jmbi.2001.4781.
We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.
我们测量了特异性结合的EcoRI核酸内切酶的解离速率以及非特异性与特异性结合常数之比对水活度、盐浓度和pH的依赖性，以区分这些溶液成分对特异性和非特异性结合的贡献。对于像EcoRI这样通过沿着非特异性DNA扩散来有效定位其特异性识别位点的蛋白质，特异性位点解离速率可分为两个步骤：酶与DNA的非特异性和特异性结合之间的平衡，以及非特异性结合蛋白的解离。我们之前证明了解离速率的渗透压依赖性主要由特异性和非特异性结合之间的平衡决定，该平衡与渗透溶质的性质无关。与非特异性结合蛋白解离相关的剩余渗透压敏感性显著取决于所使用的特定渗透溶质，这表明溶质可及表面积发生了变化。相比之下，非特异性结合酶的解离几乎占了所有的pH和盐依赖性。我们通过竞争测定法观察到特异性和非特异性结合之间的平衡几乎没有pH依赖性。观察到的特异性和非特异性结合常数之比的弱盐敏感性与渗透压作用而非静电作用一致。对粘度似乎缺乏依赖性表明非特异性结合蛋白解离的限速步骤是离散的构象变化，而不是蛋白质从DNA上的一般扩散。
