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首页> 外文期刊>Biochemistry >The vnd/NK-2 homeodomain: Thermodynamics of reversible unfolding and DNAbinding for wild-type and with residue replacements H52R and H52R/T56W inhelix III
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The vnd/NK-2 homeodomain: Thermodynamics of reversible unfolding and DNAbinding for wild-type and with residue replacements H52R and H52R/T56W inhelix III

机译：vnd / NK-2同源结构域：野生型和残基置换H52R和H52R / T56W inhelix III的可逆展开和DNA结合的热力学

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摘要

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix UI of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K-1 mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T-m = 35.5 degreesC to T-m 43-51 degreesC; DeltaH(vH) congruent to 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K-D' = 2.6 +/- 0.3 mM phosphate) is reversed similar to 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degreesC). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K-1 mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA. respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol-1 and DeltaG ' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol-l with a binding stoichiometry of 1.0 +/-. 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.
机译：vnd（腹侧神经系统缺陷）/ NK-2同源域[HD（wt）;已通过差示扫描量热法（DSC）和椭圆率变化研究了包含60个残基同源域的残基1-80和DNA识别位点螺旋UI中具有突变的残基[HD（H52R）和HD（H52R / T56W）]在222 nm pH 7.4下的热解折叠反应是可逆的，并且在存在DeltaC（p）= 0.52 +/- 0.04 kcal K-1 mol（-1）的50-500 mM NaCl的情况下是可重复的。通过50 mM磷酸盐或向50 mM Hepes pH 7.4中添加100-500 mM NaCl缓冲液（从Tm = 35.5摄氏度到Tm 43-51摄氏度; DeltaH（vH））可以基本稳定HD（wt）相当于47 +/- 5 kcal mol（-1））。稳定顺序为HD（H52R / T56W）> HD（H52R）> HD（wt），与存在的阴离子无关。通过两态展开模型很好地拟合了随温度升高而在222 nm处的椭圆率变化的进度曲线，并且突变体同源结构域的二级结构变化的协同作用大于HD（wt），并且通过增加100而增加mM NaCl至Hepes缓冲液。通过磷酸盐结合（K-D'= 2.6 +/- 0.3 mM磷酸盐），HD（wt）的固有色氨酸残基荧光的33％淬灭与DNA结合的60％相似。 vnd / NK-2同源域蛋白结合序列特异性18 bp DNA的热力学参数已通过等温滴定热法（10-30摄氏度）确定。 HD（wt），HD（H52R）和HD（H52R / T56W）结合双链体DNA的DeltaC（p）值为+ 0.25，-0.17和-0.10 +/- 0.04 kcal K-1 mol（-1） 。分别。同源域与DNA的相互作用在298 K和pH 7.4下被焓控制，相应的DeltaH值为-6.6 +/- 0.5，-10.8 +/- 0.1和-9.0 +/- 0.6 kcal mol-1，而DeltaG'值为- 11.0 +/- 0.1，-11.0 +/- 0.1和-11.3 +/- 0.3 kcal mol-1，结合化学计量为1.0 +/-。 0.1。 DNA结合的热力学参数不是根据与DNA复合时发生的同源域结构变化预测的，并且必须反映溶剂以及可能的DNA重排。

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