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结合域，其功能是转录调节，在胚胎细胞对特定发育途径的承诺中指定位置和时间信息。与发育相关的一系列事件的早期步骤是转录调节因子(含有同源结构域的蛋白)与特定的DNA序列或特定序列的特定结合。通过差示扫描量热法(DSC)、222 nm处的椭圆度变化和Trp荧光研究了VND(腹侧神经系统缺陷)/NK-2同源结构域[(HD(Wt)；包含60个残基的同源结构域的残基1-80]的构象稳定性和DNA识别位点螺旋III的突变[(HD(H52R)和HD(H52R/T56W)])。在50-500 mM的氯化钠存在下，pH为7.4的热去折叠反应是可逆的和可重复的。通过50 mM磷酸盐或将50-500 mM的氯化钠添加到50 mM的HEPES pH 7.4缓冲液中，可产生对HD(Wt)的实质稳定。稳定性顺序为HD(H52R/T56W)&GT；HD(H52R)&GT；HD(Wt)，与HD(Wt)相比，HD(H52R)在Tm的展开熵显著增加。比较HD(H52R/T56W)和HD(H52R)，差示扫描量热法(DSC)的结果表明，Tm处的熵变最多只有很小的下降。HD(Wt)、HD(H52R)和HD(H52R/T56W)的二态去折叠模型很好地拟合了222 nm处椭圆度随温度升高的变化曲线。此外，HD(Wt)或HD(H52R)的本征色氨酸残基荧光在展开过程中增加了1.6倍，这表明Trp48是通过折叠形式的能量转移猝灭的；磷酸结合产生了额外的Trp48 50%的猝灭，这可通过DNA结合而不是热展开来解除。用等温滴定热法(10-30℃)测定了VND/NK-2同源结构域蛋白结合序列特异性18BP双链DNA的热值，发现其在298K受热控制。与其他特定蛋白质-DNA相互作用观察到的大的负热容变化不同，VND/NK-2同源结构域结合特定DNA的热容变化较小且为正。对于HD(Wt)、HD(H52R)和HD(H52R/T56W)结合序列特异的双链DNA，蛋白质结构的有序性、溶剂重排和可能的DNA调节在形成络合物时发生。

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.