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首页> 外文期刊>Journal of Molecular Biology >Formation of a wrapped DNA-protein interface: Experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H ' DNA
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Formation of a wrapped DNA-protein interface: Experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H ' DNA

机译：包裹的DNA-蛋白质界面的形成：离子和水对IHF与H'DNA结合的热力学的巨大贡献的实验表征和分析

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To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34-base pair (bp) H' DNA sequence to the Escherichia coli DNA-remodeling protein integration host factor (IHF). Isothermal titration calorimetry and fluorescence resonance energy transfer are applied to determine effects of salt concentration [KCl, KF, K glutamate (KGlu)] and of the excluded solute glycine betaine (GB) on the binding thermodynamics at 20 degrees C. Both the binding constant K-obs and enthalpy Delta H degrees(obs) depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., Delta H degrees(obs) = - 20.2 kcal mol(-1) in 0.04 M KCl). Delta H degrees(obs), increases linearly with [salt] with a slope (d Delta H degrees(obs)/d[salt]), which is much larger in KCl (38 +/- 3 kcal mol(-1) M-1) than in KF or KGlu (11 +/- 2 kcal center dot mol- M-1). At 0.33 M [salt], Kobs is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SKobs=dlnK(obs)/dln[salt] is almost twice as large in magnitude in KCl (-8.8 +/- 0.7) as in KF or KGlu (- 4.7 +/- 0.6). A novel analysis of the large effects of anion identity on K-obs, SKobs and on Delta H degrees(obs), dissects coulornbic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K-obs, SKobs, and Delta H degrees(obs) to (i) displacement of a large number of water molecules of ob hydration [estimated to be 1.0(+/- 0.2) x 10(3)] from the 5340 angstrom(2) of IHF and H' DNA surface buried in complex formation, and (ii) significant local exclusion of F- and Glu(-) from this hydration water, relative to the situation with Cl-, which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of GB on K-obs: dlnK(obs)/d[GB] =2.7 +/- 0.4 at constant KCl activity, indicating the net release of ca. 150 H2O molecules from anionic surface. (c) 2007 Elsevier Ltd. All rights reserved.
机译：为了表征类似于核小体的大界面包裹蛋白质-DNA复合物形成过程中的驱动力和驱动过程，我们研究了结合34个碱基对（bp）H'DNA序列与大肠杆菌DNA-的热力学。重塑蛋白整合宿主因子（IHF）。用等温滴定热法和荧光共振能量转移来确定盐浓度[KCl，KF，K谷氨酸盐（KGlu）]和排除的溶质甘氨酸甜菜碱（GB）对20℃下的结合热力学的影响。两个结合常数K-obs和焓Delta H度（obs）强烈取决于[盐]和阴离子的身份。包裹复合物的形成是由焓驱动的，尤其是在低盐度下（例如，在0.04 M KCl中，Delta H度（obs）=-20.2 kcal mol（-1））。 Delta H度（obs），随[salt]的斜率线性增加（d Delta H度（obs）/ d [salt]），在KCl中更大（38 +/- 3 kcal mol（-1）M -1），而不是KF或KGlu（11 +/- 2 kcal中心点mol- M-1）。在0.33 M [盐]时，KGlu或KF中的Kob比KCl大30倍，并且[盐]衍生物SKobs = dlnK（obs）/ dln [盐]的大小几乎是KCl的两倍（- 8.8 +/- 0.7），如KF或KGlu（-4.7 +/- 0.6）。对K-obs，SKobs和Delta H度（obs）上的阴离子同一性的巨大影响进行了新颖的分析，剖析了库仑比，Hofmeister和渗透量对这些量的影响。该分析将K-obs，SKobs和Delta H度（obs）中特定于阴离子的差异归因于（i）大量水合水分子的位移[估计为1.0（+/- 0.2）x 10（ 3）]从5340埃（2）的IHF和H'DNA表面以复杂的形式掩埋，并且（ii）相对于Cl-，这种水化水中F-和Glu（-）的显着局部排斥，我们建议随机分配。为了量化从阴离子表面（埋在复合物中的表面的22％，主要是从DNA磷酸盐中释放出来）的净水释放量，我们确定了GB对K-obs的稳定作用：dlnK（obs）/ d [GB] = 2.7 +/- KCl活性恒定时为0.4，表明约有净释放量。来自阴离子表面的150个H2O分子。 （c）2007 Elsevier Ltd.保留所有权利。

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