Lundbäck T, Hansson H, Knapp S, Ladenstein R, Härd T
Department of Biosciences, Karolinska Institute, Huddinge, Sweden.
J Mol Biol. 1998 Mar 6;276(4):775-86. doi: 10.1006/jmbi.1997.1558.
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/2H2O), 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 DeltaG degrees approximately -7 to DeltaG degrees approximately -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 DeltaH degrees approximately 10 kcal mol-1 in both phosphate and Tris-HCl buffers at pH 7.6, indicating that DeltaH 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 DeltaCp 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 degreesC) of S. solfataricus. Addition of neutral solutes (osmotic stress) has minor effects on DeltaG degrees and the exchange of H2O for 2H2O has only a small effect on DeltaH 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.
我们采用等温滴定量热法和荧光光谱法，研究了嗜热栖热菌（Sulfolobus solfataricus）的Sso7d蛋白与非序列特异性DNA结合的热力学性质。我们报道了Sso7d与聚（dGdC）的结合热力学性质，作为缓冲液组成（Tris-HCl或磷酸盐）、温度（15至45摄氏度）、pH（7.1至8.0）、渗透压和溶剂（H2O/2H2O）的函数，并将其与聚（dAdT）的结合进行比较；我们之前也报道过盐浓度的依赖性。结合等温线可以用McGhee-von Hippel非协同结合模型来表示，结合位点大小为4至5个DNA碱基对，结合自由能范围为ΔG°约-7至ΔG°约-10 kcal mol-1，具体取决于实验条件。结合的非特异性本质体现在与聚（dAdT）和聚（dGdC）结合的相似热力学性质中。Sso7d的天然赖氨酸甲基化对结合热力学性质只有轻微影响。在25摄氏度时，Sso7d与聚（dGdC）的结合是吸热的，在pH 7.6的磷酸盐和Tris-HCl缓冲液中，结合焓ΔH°约为10 kcal mol-1，这表明在该pH值下，ΔH°不包括来自耦合缓冲液电离平衡的大量贡献。结合焓与温度有关，测得的热容变化ΔCp° = -0.25（±0.01）kcal mol-1 K-1，热力学数据的外推表明，该复合物在接近嗜热栖热菌生长温度（75至80摄氏度）时具有热稳定性，结合为放热。添加中性溶质（渗透压）对ΔG°影响较小，用2H2O替换H2O对ΔH°只有很小的影响，这与复合物形成不伴随表面水化净变化的推断一致。因此，必须找到热容变化的其他机制。我们结合蛋白质与非序列特异性DNA结合的性质，对观察到的热力学性质进行了讨论。
