The vnd/NK-2 Homeodomain Stability and DNA Binding

同源结构域是一类蛋白质的高度保守的DNA结合结构域，其作为转录调节因子起作用，在胚胎细胞向特定发育途径的定向中指定位置和时间信息。 与发育相关的级联反应的早期步骤是转录调节因子的序列特异性结合蛋白质（含有同源结构域的蛋白质）与DNA的特定序列或特定序列组的构象稳定性。（腹神经系统缺陷）/NK-2同源域[（HD（wt）;包括60个残基同源结构域的残基1-80）]和在DNA识别位点的螺旋III中具有突变的那些[（HD（H52 R）和HD（H52 R/T56 W）]的差示扫描量热法（DSC）、222 nm处的椭圆率变化和Trp荧光进行了研究。 在50-500 mM NaCl存在下，pH 7.4下的热去折叠反应是可逆的和可重复的。 通过50 mM磷酸盐或通过向50 mM Hepes pH 7.4缓冲液中加入50-500 mM NaCl产生HD（wt）的基本稳定。 与阴离子存在无关，稳定性的顺序为HD（H52 R/T56 W）> HD（H52 R）> HD（wt），与HD（wt）相比，HD（H52 R）在Tm处表现出的去折叠熵显著增加。将HD（H52 R/T56 W）与HD（H52 R）进行比较，DSC结果表明在Tm处的熵变最多有小的降低。在222 nm处的椭圆率变化作为温度升高的函数的进展曲线拟合好由HD（wt），HD（H52 R），和HD（H52 R/T56 W）的两状态展开模型。 此外，HD（wt）或HD（H52 R）的内在双羟基残基荧光在解折叠期间增加1.6倍，这表明Trp 48通过折叠形式的能量转移而猝灭;磷酸盐结合产生Trp 48的另外50%猝灭，其通过DNA结合而不是通过热解折叠而减轻。vnd/NK-2同源结构域蛋白结合序列特异性18 bp双链体DNA的焓值已通过等温滴定量热法（10-30 ℃）测定，并发现其在298 K下受热力学控制。 与其他特异性蛋白质-DNA相互作用观察到的大的负热容量变化相反，vnd/NK-2同源结构域结合特异性DNA的热容量变化小且为正。对于HD（wt）、HD（H52 R）和HD（H52 R/T56 W）结合序列特异性双链体DNA，在复合物形成时发生蛋白质结构的排序、溶剂重排和可能的DNA调节。

The homeodomain is the highly conserved DNA-binding domain of a class of proteins that function as transcriptional regulators, specifying positional and temporal information in the commitment of embryonic cells to specific developmental pathways. An early step in the cascade of events associated with development is the sequence-specific binding of the transcriptional regulator (the homeodomain-containing protein) to a specific sequence or a specific set of sequences of DNA.The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [(HD(wt); residues 1-80 that encompasses the 60 residue homeodomain)] and those harboring mutations in helix III of the DNA recognition site [(HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC), ellipticity changes at 222 nm, and Trp fluorescence. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl. A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 50-500 mM NaCl to 50 mM Hepes pH 7.4 buffer. The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present, with a substantial increase in the unfolding entropy at Tm exhibited by HD(H52R) compared to that of HD(wt). Comparing HD(H52R/T56W) to HD(H52R), DSC results indicate that there is at most a small decrease in the entropy change at Tm. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model for HD(wt), HD(H52R), and HD(H52R/T56W). Also, the intrinsic tryptophanyl residue fluorescence of HD(wt) or HD(H52R) increases 1.6-fold during unfolding, which indicates that Trp48 is quenched by energy transfer in the folded form; phosphate binding produces an additional 50% quench of Trp48 which is relieved by DNA binding but not by thermal unfolding. Enthalpy values for the vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp duplex DNA have been determined by isothermal titration calorimetry (10-30 C) and found to be enthalpically controlled at 298 K. In contrast to the large negative heat capacity change observed for other specific protein-DNA interactions, the heat capacity change for the vnd/NK-2 homeodomain binding specific DNA is small and positive. For HD(wt), HD(H52R), and HD(H52R/T56W) binding sequence-specific duplex DNA, ordering of protein structure, solvent rearrangements, and possible DNA accommodations occur upon complex formation.