THERMODYNAMIC EVALUATION OF BINDING INTERACTIONS IN THE METHIONINE REPRESSOR SYSTEM OF ESCHERICHIA-COLI USING ISOTHERMAL TITRATION CALORIMETRY
THERMODYNAMIC EVALUATION OF BINDING INTERACTIONS IN THE METHIONINE REPRESSOR SYSTEM OF ESCHERICHIA-COLI USING ISOTHERMAL TITRATION CALORIMETRY
复制标题
DOI：
10.1021/bi00010a010
复制
发表时间：
1995-03-14
期刊：
影响因子：
2.9
通讯作者：
SPICER, LD
中科院分区：
文献类型：
--
作者：
HYRE, DE;SPICER, LD
采用量热法研究了大肠杆菌中甲硫氨酸阻遏蛋白MetJ与其同源物metbox DNA序列和辅阻遏物S-腺苷甲硫氨酸的相互作用。该系统的详细热力学表征显示了最近报道的（β α α）（2）结合基序，提供了阻遏物结合循环中每个步骤的Δ G、Δ H和Δ S值。这些研究表明，在辅阻遏物存在的情况下，MetJ与单个metbox操纵基因位点结合，Delta G = -7.7 kcal。mol（-1），而在没有辅阻遏物的情况下，与单个位点相互作用的自由能为-5.8kcal。mol（-1）。与两个相邻位点结合的两个阻遏物分子之间的协同相互作用贡献了-1.3千卡的额外自由能。mol（-1）与第二位点结合。在缺乏辅阻遏物的情况下，结合在遗传学上是不利的，Δ H = +2.6千卡。摩尔（-1），但变得放热，Delta H = -4.6 kcal。mol（-1）。系统的热容显著降低Δ C-p = -290 cal。mol（-1）。当蛋白质结合到DNA时，每个位点的K-1，以及结合到相邻位点的阻遏物分子之间的相互作用产生Δ C-p = -800 cal。mol（-1）。K-1，表明溶剂排斥在该系统中的结合中起着重要作用。辅阻遏物与未结合的阻遏蛋白结合的自由能为Δ G = -6.0 kcal。mol（-1）和MetJ-操作符复合物，Δ G = -6.95 kcal·mol（-1）。阻遏物与随机序列DNA结合的自由能估计为-5.7千卡。mol（-1）。这些数据清楚地表明，MetJ阻遏物二聚体特异性结合其8bp同源metbox操纵子的中心区域，但识别短至6 bp的部分操纵子序列。相邻的MetJ二聚体的双metbox序列的结合中的协同性被证明是重要的，在确定相互作用的能量。最后，辅阻遏物S-腺苷甲硫氨酸将MetJ对其识别位点DNA的亲和力提高了25倍，并显著提高了阻遏物结合的净亲和力。
The binding interactions of the methionine repressor protein, MetJ, from Escherichia coli with its cognate, metbox DNA sequence and corepressor S-adenosylmethionine were examined using calorimetric methods. A detailed thermodynamic characterization of this system which exhibits the recently reported (beta alpha alpha)(2) binding motif provides values for Delta G, Delta H, and Delta S for each step in the repressor binding cycle. These studies show that, in the presence of corepressor, MetJ binds to a single metbox operator site with Delta G = -7.7 kcal . mol(-1), whereas in the absence of corepressor, the free energy of interaction with a single site is -5.8 kcal . mol(-1). Cooperative interactions between two repressor molecules bound to two adjacent sites contribute an additional free energy of -1.3 kcal . mol(-1) to binding at the second site. Binding is enthalpically unfavorable in the absence of the corepressor with Delta H = +2.6 kcal . mol(-1) but becomes exothermic with Delta H = -4.6 kcal . mol(-1) when corepressor is present. The heat capacity for the system decreases significantly by Delta C-p = -290 cal . mol(-1). K-1 on a per site basis when the protein binds to DNA, and interactions between repressor molecules bound to adjacent sites contribute a Delta C-p = -800 cal . mol(-1). K-1 indicating that solvent exclusion plays a significant role in binding in this system. The corepressor binds to the unbound repressor protein with a free energy of Delta G = -6.0 kcal . mol(-1) and to the MetJ-operator complex with Delta G = -6.95 kcal.mol(-1). Repressor binding to random-sequence DNA was estimated to occur with a free energy of -5.7 kcal . mol(-1) in the presence of corepressor. These data clearly indicate that MetJ repressor dimer binds specifically to the central region of its 8 bp cognate metbox operator but recognizes partial operator sequences as short as 6 bp. Cooperativity in binding of adjacent MetJ dimers to a double metbox sequence is demonstrated to be important in determining the energetics of the interaction. Finally, the corepressor S-adenosylmethionine enhances the affinity of MetJ for its recognition site DNA by a factor of 25 and contributes significantly to the net exothermicity of repressor binding.