Brenowitz M, Pickar A, Jamison E
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Biochemistry. 1991 Jun 18;30(24):5986-98. doi: 10.1021/bi00238a024.
The quantitation of the stability of a protein-mediated "looped complex" of the Lac repressor and DNA containing two protein-binding sites whose centers of symmetry are separated by 11 helical turns (114 bp) was accomplished by footprint and gel mobility-shift titration techniques. Lac repressor binding to this DNA was only moderately cooperative; a cooperative free energy of -1.0 kcal/mol was calculated in a model-independent fashion from the individual-site loading energies obtained from the footprint titration studies. In order to partition the cooperative binding energy into components representing the dimer-tetramer association of Lac repressor and the cyclization probability of the intervening DNA, advantage was taken of the presence of experimental measures that were in proportion to the concentration of the looped complex present in solution. One measure was the DNase I hypersensitivity observed in footprint titrations in bands located between the two binding sites. The second measure resulted from the electrophoretic resolution in the gel mobility-shift titrations of the band representing the doubly liganded "tandem complex" from the band representing the singly liganded complexes, including the looped complex. Analysis of the footprint and mobility-shift titration data utilizing this additional information showed that approximately 65% of the molecules present in solution are looped complexes at pH 7.0, 100 mM KCl, and 20 degrees C when the binding sites on the DNA are saturated with protein. Reconciliation of the observed low binding cooperativity and the high proportion of looped complexes could only be obtained when the titration data were analyzed by a model in which Lac repressor tetramers dissociate into dimers in solution. The proportion of looped complexes present in solution is highly dependent on the dimer-tetramer association constant, delta Gtet. This result is consistent with the determination by high-pressure fluorescence techniques that Lac repressor tetramers dissociate with an association free energy comparable to their DNA-binding free energies [Royer, C. A., Chakerian, A. E., & Matthews, K. S. (1990) Biochemistry 29, 4959-4966]. However, when the value of delta Gtet of -10.6 kcal/mol (at 20 degrees C) reported by Royer et al. (1990) is assumed, the titration data demand that tetramers bind DNA with much greater affinity than dimers: a result inconsistent with the destabilization of tetramers by the operator observed in the dimer-tetramer dissociation studies.(ABSTRACT TRUNCATED AT 400 WORDS)
通过足迹法和凝胶迁移率变动滴定技术，对乳糖阻遏蛋白与含有两个蛋白质结合位点的DNA形成的蛋白质介导的“环状复合物”的稳定性进行了定量分析。这两个结合位点的对称中心相隔11个螺旋圈（114个碱基对）。乳糖阻遏蛋白与该DNA的结合仅有适度的协同性；根据足迹滴定研究获得的单个位点结合能，以一种与模型无关的方式计算出协同自由能为-1.0千卡/摩尔。为了将协同结合能分配到代表乳糖阻遏蛋白二聚体 - 四聚体缔合以及中间DNA环化概率的组分中，利用了与溶液中存在的环状复合物浓度成比例的实验测量值。一种测量方法是在足迹滴定中观察到的位于两个结合位点之间条带处的DNase I超敏感性。第二种测量方法源于凝胶迁移率变动滴定中代表双配体“串联复合物”的条带与代表单配体复合物（包括环状复合物）的条带之间的电泳分辨率。利用这些额外信息对足迹和迁移率变动滴定数据进行分析表明，当DNA上的结合位点被蛋白质饱和时，在pH 7.0、100 mM KCl和20℃条件下，溶液中约65%的分子是环状复合物。只有当通过乳糖阻遏蛋白四聚体在溶液中解离成二聚体的模型来分析滴定数据时，才能使观察到的低结合协同性和高比例的环状复合物相协调。溶液中存在的环状复合物比例高度依赖于二聚体 - 四聚体缔合常数ΔGtet。这一结果与高压荧光技术测定的结果一致，即乳糖阻遏蛋白四聚体以与其DNA结合自由能相当的缔合自由能解离[罗耶，C. A.，查克里安，A. E.，& 马修斯，K. S.（1990年）《生物化学》29，4959 - 4966]。然而，假设罗耶等人（1990年）报道的ΔGtet值为-10.6千卡/摩尔（在20℃），滴定数据要求四聚体比二聚体以更高的亲和力结合DNA：这一结果与在二聚体 - 四聚体解离研究中观察到的操纵基因使四聚体不稳定的情况不一致。（摘要截断于400字）
