Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites
Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites
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DOI：
10.1016/j.jmb.2004.08.101
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发表时间：
2004-10-29
影响因子：
5.6
通讯作者：
Privalov, PL
中科院分区：
文献类型：
--
作者：
Dragan, AI;Frank, L;Privalov, PL
用光学方法、扫描量热法和等温滴定量热法研究了GCN 4-bZIP配合物中AP-1和ATF/CREB位的能量基础。发现bZIP二聚体的解离常数显著高于其分离的亮氨酸拉链结构域的解离常数：在20 ℃时为1.45 μ M，并随温度升高而增加。为了避免这种二聚体解离的并发症，使用bZIP的SS交联版本进行DNA结合实验。在不同的温度和盐浓度下测得的bZIP/DNA协会的热力学特性进行了校正的基础段结合后，从扫描量热实验确定的重折叠的贡献。荧光各向异性滴定实验表明，20 ℃时bZIP与AP-1和ATF/CREB结合位点的缔合常数相差不大，分别为1.5 nM和6.4 nM，对应的吉布斯自由能分别为-49 kJ mol（-1）和-46 kJ mol（-1）。几乎一半的吉布斯能是由于静电分量，导致从熵效应的counterweight释放后，DNA与bZIP协会，是相同的两个网站。与吉布斯自由能相反，完全折叠的bZIP与AM和ATF/CREB位点的缔合熵以及相应的缔合熵是非常不同的。bZIP与AP-1位点结合的特征在于比与ATF/CREB位点结合的负焓和非静电熵大得多，这意味着AM复合物比ATF/CREB复合物结合了更多的水分子。(C)2004爱思唯尔有限公司保留所有权利。
The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry. The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 degreesC it is 1.45 muM and increases with temperature. To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP. The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments. Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 degreesC with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5 nM and 6.4 nM, corresponding to Gibbs energies of -49 kJ mol(-1) and -46 kJ mol(-1), respectively. Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites. In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AM and ATF/CREB sites, and correspondingly the entropies of association, are very different. bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AM complex incorporates significantly more water molecules than the ATF/CREB complex. (C) 2004 Elsevier Ltd. All rights reserved.