Freyer Matthew W, Buscaglia Robert, Hollingsworth Amy, Ramos Joseph, Blynn Meredith, Pratt Rachael, Wilson W David, Lewis Edwin A
Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, Arizona 86011-5698, USA.
Biophys J. 2007 Apr 1;92(7):2516-22. doi: 10.1529/biophysj.106.098723. Epub 2007 Jan 19.
Studies performed in our laboratory demonstrated the formation of two thermodynamically distinct complexes on binding of netropsin to a number of hairpin-forming DNA sequences containing AATT-binding regions. These two complexes were proposed to differ only by a bridging water molecule between the drug and the DNA in the lower affinity complex. A temperature-dependent isothermal titration calorimetry (ITC)-binding study was performed using one of these constructs (a 20-mer hairpin of sequence 5'-CGAATTCGTCTCCGAATTCG) and netropsin. This study demonstrated a break in the heat capacity change for the formation of the complex containing the bridging water molecule at approximately 303 K. In the plot of the binding enthalpy change versus temperature, the slope (DeltaCp) was -0.67 kcal mol-1 K-1 steeper after the break at 303 K. Because of the relatively low melting temperature of the 20-mer hairpin (341 K (68 degrees C)), the enthalpy change for complex formation might have included some energy of refolding of the partially denatured hairpin, giving the suggestion of a larger DeltaCp. Studies done on the binding of netropsin to similar constructs, a 24-mer and a 28-mer, with added GC basepairs in the hairpin stem to increase thermal stability, exhibit the same nonlinearity in DeltaCp over the temperature range of from 275 to 333 K. The slopes (DeltaCp) were -0.69 and -0.64 kcal mol-1 K-1 steeper after 303 K for the 24-mer and 28-mer, respectively. This observation strengthens the argument regarding the presence of a bridging water molecule in the lower affinity netropsin/DNA complex. The DeltaCp data seem to infer that because the break in the heat capacity change function for the lower affinity binding occurs at the isoequilibrium temperature for water, water may be included or trapped in the complex. The fact that this break does not occur in the heat capacity change function for formation of the higher affinity complex can similarly be taken as evidence that water is not included in the higher affinity complex.
我们实验室进行的研究表明，当纺锤菌素与一些含有AATT结合区域的发夹状DNA序列结合时，会形成两种热力学性质不同的复合物。有人提出，这两种复合物的区别仅在于低亲和力复合物中药物与DNA之间存在一个桥连水分子。使用其中一种构建体（序列为5'-CGAATTCGTCTCCGAATTCG的20聚体发夹）和纺锤菌素进行了温度依赖性等温滴定量热法（ITC）结合研究。这项研究表明，在大约303 K时，含有桥连水分子的复合物形成过程中的热容变化出现了突变。在结合焓变与温度的关系图中，在303 K发生突变后，斜率（ΔCp）变得更陡，为-0.67 kcal mol-1 K-1。由于20聚体发夹的解链温度相对较低（341 K（68℃）），复合物形成的焓变可能包含了部分变性发夹重新折叠的一些能量，这表明ΔCp更大。对纺锤菌素与类似构建体（一个24聚体和一个28聚体）的结合进行的研究，在发夹茎中添加了GC碱基对以提高热稳定性，在275至333 K的温度范围内，ΔCp也表现出相同的非线性。对于24聚体和28聚体，在303 K之后，斜率（ΔCp）分别变得更陡，为-0.69和-0.64 kcal mol-1 K-1。这一观察结果强化了关于低亲和力纺锤菌素/DNA复合物中存在桥连水分子的观点。ΔCp数据似乎推断，由于低亲和力结合的热容变化函数的突变发生在水的等平衡温度，水可能被包含或捕获在复合物中。同样，高亲和力复合物形成的热容变化函数中未出现这种突变这一事实，可以作为水不包含在高亲和力复合物中的证据。
