CALORIMETRIC ANALYSIS OF LAMBDA-CI REPRESSOR BINDING TO DNA OPERATOR SITES
CALORIMETRIC ANALYSIS OF LAMBDA-CI REPRESSOR BINDING TO DNA OPERATOR SITES
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DOI：
10.1021/bi00027a005
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发表时间：
1995-07-11
期刊：
影响因子：
2.9
通讯作者：
ACKERS, GK
中科院分区：
文献类型：
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作者：
MERABET, E;ACKERS, GK
通过等温滴定量热法测定噬菌体 lambda cI 阻遏物与包含三个操纵位点 O(R)1、O(R)2 和 O(R)3 的各种组合（每个包含一个共有半位点和 3 个特定非共有半位点）的 DNA 结合的焓和热容。采用差示扫描量热法来评估特异性 DNA 结合对 N 端和 C 端阻遏物结构域热熔解的影响，本研究的主要发现如下：（1）阻遏物与每个 DNA 操纵子的结合由大的负焓主导，与早期定量足迹滴定数据的 van't Hoff 分析一致 [Koblan 和 Ackers (1992) Biochemistry 31, 57-65]，量热数据还揭示了 cl 结合的负热容，其大小与许多其他系统相当[Spolar & Record (1994) Science 263, 777-784，然而，这一特征与结合焓的大负值相结合导致了整个生理温度范围内的焓优势，由此产生的热力学曲线与在许多系统中观察到的熵主导的结合相反，包括 lambda、cro 阻遏物，它与 cI 结合到相同的位点，也采用螺旋-转角-螺旋结合结构域[Takeda er al, (1992) Proc, Natl, Acad. Sci, U,S.A, 89, 8180-8184]，表明这些热力学差异可能是由于 cI 阻遏物的 N 末端“臂”与 DNA 的相互作用引起的。 (3) 阻遏物单体不会与含有共有半位点或非共有半位点的 DNA 显着结合。对双一致操作符的结合亲和力比对任何天然全位点操作符的结合亲和力弱得多，对于其他半位点组合也发现了同样的情况。一种二聚化严重缺陷的突变阻遏蛋白 (PT158) [Burz et al. (1994) Biochemistry 33, 8399-8405] 还发现仅结合全位点操纵子并显示二聚体化学计量，(3) 发现在不存在 DNA 的情况下 N 末端结构域的热熔解单位在形成野生型蛋白质的高级寡聚物的浓度下达到 6-8 (单体单位) 的值 [Senear 等人，(1993) Biochemistry 32， 6179-6189]，然而，在 DNA 操纵位点存在的情况下，热解折叠的协同单位精确地减少为两个单体，表明 N 端结构域严格作为二聚体结合。 (4) 用全位点操纵子的多种组合测定的阻遏物结合焓和热容量观察到显着的非加和性。这种效应用野生型 cI 和 GD147 观察到，GD147 是一种 C 端突变型阻遏物，它缺乏协同性，但对每个位点具有正常的结合亲和力 [Burz 和 Ackers (1994) Biochemistry 33, 8406-8416]。野生型和 GD147 结果的比较表明，阻遏蛋白与特定 OK 位点的结合可能会诱导邻近位点的结构变化，从而影响它们与阻遏蛋白二聚体的相互作用。考虑了本研究中发现的热力学效应的可能结构起源。
Enthalpies and heat capacities were determined by isothermal titration calorimetry for bacteriophage lambda cI repressor binding to DNA containing various combinations of the three operator sites O(R)1, O(R)2, and O(R)3 (each comprising a consensus half-site and 3 specific nonconsensus half-site). Differential scanning calorimetry was employed to evaluate the effects of specific DNA binding on thermal melting of the N-terminal and C-terminal repressor domains, Principal findings of this study are as follows: (1) Binding of repressor to each of the DNA operators is dominated by a large negative enthalpy, in agreement with earlier van't Hoff analyses of quantitative footprint titration data [Koblan and Ackers (1992) Biochemistry 31, 57-65], The calorimetric data also revealed negative heat capacities for cl binding that are of comparable magnitude with many other systems [Spolar & Record (1994) Science 263, 777-784, However, this feature in combination with the large negative values of binding enthalpies leads to an enthalpic dominance throughout the physiological temperature range, The resulting thermodynamic profile is opposite to the entropically dominated binding observed for many systems, including lambda, cro repressor which binds to the same sites as cI and also employs a helix-turn-helix binding domain [Takeda er al, (1992) Proc, Natl, Acad. Sci, U,S.A, 89, 8180-8184], It is suggested that these thermodynamic differences may arise from interactions of the cI repressor's N-terminal ''arm'' with the DNA. (3) Repressor monomers do not bind significantly to DNA containing either a consensus half-site or a nonconsensus half-site. Binding affinity to the double-consensus operator is much weaker than to any of the natural full-site operators, The same was found with other combinations of half-sites. A mutant repressor (PT158) which is severely defective in dimerization [Burz et al. (1994) Biochemistry 33, 8399-8405] was also found to bind only full-site operators and showed dimeric stoichiometry, (3) The thermal melting unit for N-terminal domains in the absence of DNA was found to reach values of 6-8 (monomer units) at concentrations where high-order oligomers of wild-type protein are formed [Senear ct al, (1993) Biochemistry 32, 6179-6189], However, in the presence of DNA operator sites, the cooperative unit for thermal unfolding was reduced to precisely two monomers, indicating that the N-terminal domain binds strictly as a dimer. (4) Significant nonadditivity was observed for the repressor binding enthalpies and heat capacities determined with multiple combinations of full-site operators, This effect was observed with wild-type cI and also with GD147, a C-terminal mutant repressor which is devoid of cooperativity but has normal binding affinity to each site [Burz and Ackers (1994) Biochemistry 33, 8406-8416]. Comparison of the results from wild type and GD147 suggests that repressor binding to the specific OK sites may induce structural changes at neighboring sites which affect their interactions with repressor dimers. Possible structural origins of the thermodynamic effects found in this study are considered.