The vnd/NK-2 Homeodomain Stability and DNA Binding

同源结构域是一类高度保守的dna结合结构域，其功能是转录调控，在胚胎细胞向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（含同源结构域的蛋白质）与特定序列或特定DNA序列集的序列特异性结合。vnd（腹侧神经系统缺陷）/NK-2同源结构域的构象稳定性[(HD(wt)；利用差示扫描量热法（DSC）、222 nm下椭圆度变化和色氨酸荧光研究了DNA识别位点螺旋III突变[(HD（H52R）和HD(H52R/T56W)]和包含60个同源结构域的残基1-80]。在50-500 mM NaCl存在下，pH为7.4的热展开反应是可逆的和可重复的。通过50 mM磷酸盐或在50 mM Hepes pH 7.4缓冲液中加入50-500 mM NaCl，可以实现HD（wt）的稳定。与存在的阴离子无关，其稳定性顺序为HD(H52R/T56W) > HD(H52R) > HD(wt)， HD（H52R）在Tm处的展开熵比HD（wt）显著增加。DSC结果表明，HD（H52R/T56W）与HD（H52R）相比，在Tm处熵变减小幅度不大。利用HD（wt）、HD（H52R）和HD（H52R/T56W）的两态展开模型，可以很好地拟合出222 nm处椭圆率随温度升高的变化曲线。HD（wt）或HD（H52R）的固有色氨酸残基荧光在展开过程中增加1.6倍，表明Trp48在折叠形式下被能量传递猝灭；磷酸盐结合产生额外的50% Trp48猝灭，这是通过DNA结合而不是通过热展开来缓解的。vnd/NK-2同源结构域蛋白结合序列特异性18bp双链DNA的焓值通过等温滴定量热法（10-30℃）测定，发现焓值控制在298 K。与其他特异性蛋白质-DNA相互作用的大的负热容量变化相比，vnd/NK-2同源结构域结合特异性DNA的热容量变化很小，而且是正的。对于HD(wt)， HD（H52R）和HD（H52R/T56W）结合序列特异性双链DNA，蛋白质结构排序，溶剂重排和可能的DNA调节发生在复合物形成过程中。

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.