Thermodynamic characterization of non-sequence-specific DNA-binding by the Sso7d protein from Sulfolobus solfataricus
Thermodynamic characterization of non-sequence-specific DNA-binding by the Sso7d protein from Sulfolobus solfataricus
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DOI：
10.1006/jmbi.1997.1558
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发表时间：
1998-03-06
影响因子：
5.6
通讯作者：
Härd, T
中科院分区：
文献类型：
--
作者：
Lundbäck, T;Hansson, H;Härd, T
我们采用等温滴定量热法和荧光光谱法研究了古菌硫磺硫化叶菌Sso 7 d蛋白与非序列特异性DNA结合的热力学。我们报告了Sso 7 d-聚（dGdC）结合热力学作为缓冲液组成（Tris-HCl或磷酸盐），温度（15至45 ℃），pH（7.1至8.0），渗透压应力和溶剂（H2O/（H2O）-H-2）的函数，并将其与聚（dAdT）结合进行比较;我们以前也报道了盐浓度依赖性。结合等温线可由McGhee-von Hippel模型表示，用于非合作结合，结合位点大小为4至5个DNA碱基对，结合自由能在Δ G度接近-7至Δ G度接近-10 kcal mol（-1）的范围内，这取决于实验条件。结合的非特异性性质反映在与聚（dAdT）和聚（dGdC）结合的类似热力学中。Sso 7 d的天然赖氨酸甲基化对结合热力学只有很小的影响。Sso 7 d与聚（dGdC）的结合在25 ℃是吸热的，在pH 7.6的磷酸盐和Tris-HCl缓冲液中，结合焓Δ H度接近10 kcal mol（-1），这表明Δ H度不包括在该pH下来自耦合缓冲液电离平衡的大贡献。结合焓是温度依赖性的，具有测量的热容变化Δ C-0度= -0.25（+/-0.01）kcal mol（-1）K-1和热力学数据的外推表明该配合物是热稳定的，在接近S的生长温度（75至80 ℃）时具有放热结合。太阳神添加中性溶质（渗透压）对Δ G度的影响很小，H2O与（H2O）-H-2的交换对Δ H度的影响很小，这与复合物形成不伴随表面水合作用的净变化的推断一致。因此，其他机制的热容量的变化必须被发现。所观察到的热力学讨论的性质，非序列特异性DNA结合蛋白质。(C)出版社：Academic Press Limited。
We used isothermal titration calorimetry and fluorescence spectroscopy to investigate the thermodynamics of non-sequence-specific DNA-binding by the Sso7d protein from the archaeon Sulfolobus solfataricus. We report the Sso7d-poly(dGdC) binding thermodynamics as a function of buffer composition (Tris-HCl or phosphate), temperature (15 to 45 degrees C), pH (7.1 to 8.0), osmotic stress and solvent (H2O/(H2O)-H-2), and compare it to poly(dAdT) binding; and we have previously also reported the salt concentration dependence. Binding isotherms can be represented by the McGhee-von Hippel model for non-cooperative binding, with a binding site size of four to five DNA base-pairs and binding free energies in the range Delta G degrees approximate to -7 to Delta G degrees approximate to -10 kcal mol(-1), depending on experimental conditions. The non-specific nature of the binding is reflected in similar thermodynamics for binding to poly(dAdT) and poly(dGdC). The native lysine methylation of Sso7d has only minor effects on the binding thermodynamics. Sso7d binding to poly(dGdC) is endothermic at 25 degrees C with a binding enthalpy Delta H degrees approximate to 10 kcal mol(-1) in both phosphate and Tris-HCl buffers at pH 7.6, indicating that Delta H degrees does not include large contributions from coupled buffer ionization equilibria at this pH. The binding enthalpy is temperature dependent with a measured heat capacity change Delta C-o degrees = -0.25(+/-0.01) kcal mol(-1) K-1 and extrapolations of thermodynamic data indicate that the complex is heat stable with exothermic binding close to the growth temperature (75 to 80 degrees C) of S. solfataricus. Addition of neutral solutes (osmotic stress) has minor effects on Delta G degrees and the exchange of H2O for (H2O)-H-2 has only a small effect on Delta H degrees, consistent with the inference that complex formation is not accompanied by net changes in surface hydration. Thus, other mechanisms for the heat capacity change must be found. The observed thermodynamics is discussed in relation to the nature of non-sequence-specific DNA-binding by proteins. (C) 1998 Academic Press Limited.