Peters William B, Edmondson Stephen P, Shriver John W
Laboratory for Structural Biology, Graduate Program in Biotechnology Science and Engineering, Department of Chemistry, Materials Science Building, John Wright Drive University of Alabama in Huntsville, 35899, USA.
J Mol Biol. 2004 Oct 15;343(2):339-60. doi: 10.1016/j.jmb.2004.08.042.
Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.
Sac7d是一种嗜热染色质蛋白，它能非特异性地结合双链DNA的小沟，并通过缬氨酸和蛋氨酸侧链的插入诱导66度的急剧弯曲。我们利用Sac7d的热稳定性和缺乏序列特异性来定义在很宽温度范围内DNA结合的热力学。对聚（dGdC）的结合亲和力在25℃时适中（Ka = 3.5（±1.6）×10⁶ M⁻¹），并且从10℃到80℃增加了近一个数量级。结合焓在25℃时是不利的，并且从5℃到60℃线性下降。25℃时的正结合热部分归因于扭曲DNA的能量，并确保最大结合亲和力的温度（75.1±5.6℃）接近嗜酸热硫化叶菌的生长温度。将两个插入残基截短为丙氨酸导致在25℃时弯曲和解开DNA的能力下降，结合亲和力略有降低。从插入获得的能量略大于弯曲双链DNA的自由能损失。令人惊讶的是，双丙氨酸取代导致的扭曲减少并没有导致25℃时结合热的显著降低。此外，双丙氨酸突变蛋白观察到结合的异常正ΔCp，这不能用极性和非极性可及表面积的变化来解释。大于预期的结合焓和正热容都可以通过蛋白质-DNA复合物中温度依赖性的结构转变来解释，其熔解温度为15 - 20℃，ΔH为15千卡/摩尔。讨论的数据表明复合物中的吸热转变与DNA扭曲一致。
