Ramsden J J, Dreier J
Department of Biophysical Chemistry, Biozentrum, Basel, Switzerland.
Biochemistry. 1996 Mar 26;35(12):3746-53. doi: 10.1021/bi952158f.
Optical waveguide mode spectroscopy was used to determine the binding constants characterizing the interaction of EcoR124II, a type IC restriction modification enzyme from Salmonella typhimurium, with DNA. The DNA is immobilized on the surface of an optical waveguide, and the enzyme is introduced in bulk solution flowing over the DNA under controlled hydrodynamic conditions. The binding kinetics of the protein to the DNA can be directly observed and the number of bound protein molecules per base pair determined to a high accuracy. Dissociation of the protein was measured by switching flowing protein to protein-free buffer. Binding to two different kinds of DNA, with and without the specific sequence recognized by EcoR124II, was investigated. Protein binding and dissociation ("nonspecific" binding), quantified by association and dissociation rate coefficients ka and kd, were the same for both types, but the DNA carrying the recognition site showed an additional process, "irreversible" association (i.e. dissociation was not observed on the time scale of the experiments) of the protein, quantified by a rate coefficient ks. Some inferences regarding the mechanism of base pair searching are made from the measured ka, kd, and ks values.
采用光波导模式光谱法测定了来自鼠伤寒沙门氏菌的I型C类限制修饰酶EcoR124II与DNA相互作用的结合常数。将DNA固定在光波导表面，在可控的流体动力学条件下，将酶引入流过DNA的本体溶液中。可以直接观察蛋白质与DNA的结合动力学，并高精度地确定每碱基对结合的蛋白质分子数。通过将流动的蛋白质切换为无蛋白质缓冲液来测量蛋白质的解离。研究了与两种不同DNA的结合，一种有EcoR124II识别的特定序列，另一种没有。通过缔合和解离速率系数ka和kd定量的蛋白质结合和解离（“非特异性”结合）在两种类型中是相同的，但携带识别位点的DNA显示出蛋白质的额外过程，即“不可逆”缔合（即在实验时间尺度上未观察到解离），通过速率系数ks定量。从测量的ka、kd和ks值对碱基对搜索机制进行了一些推断。
