THERMAL UNFOLDING OF VND/NK-2 HOMEODOMAIN PROTEINS AND MUTANTS

同源结构域是蛋白质中的一个小结构基序，它与特定的靶DNA序列结合，从而调节真核生物的基因转录。同源结构域氨基酸序列决定了与DNA的结合亲和力和特异性。利用差示扫描量热法（DSC）、远紫外圆二色性（CD）和固有色氨酸荧光变化研究了突变体H52R/NK-2同源结构域蛋白（9713 MW）的热展开和温度诱导的二级结构变化。在pH为7.4的50 mM Na-磷酸盐缓冲液中，H52R/NK-2蛋白可逆展开，转变温度（Tm）约为56℃，而在pH为7.4的50 mM Hepes缓冲液中，Tm值约为39℃。磷酸阴离子对H52R/NK-2同源结构域突变蛋白的稳定作用可能与DNA结合特性有关。此外，Hepes中的展开比磷酸盐缓冲液中的展开更不协调，总展开焓分别为0.34和40 kcal/mol。在缓冲液中加入100 mM NaCl，可以改善加热和冷却循环中的再折叠，通过CD测量。DSC得到的热力学参数与温度引起的CD变化在222 nm处吻合良好。然而，H52R/NK-2蛋白热展开的DSC数据最适合非二态模型，其量热与万霍夫焓变的比值小于1。目前，H52R、H52R/W56T突变体和野生型蛋白质与含有特定目标DNA序列的短双链DNA寡聚物的相互作用正在通过等温滴定量热法（ITC）、DSC和蛋白质色氨酸荧光滴定法进行研究。测量了同位结构域蛋白- dna相互作用的结合常数，以及在形成同位结构域- dna复合物时发生的蛋白质构象变化和稳定性变化。-同源结构域蛋白，DNA，热展开，稳定性，圆二色性，量热，荧光

The homeodomain is a small structural motif in proteins that binds to specific target DNA sequences and thereby regulates gene transcription in eukaryotes. The homeodomain amino acid sequence determines the binding affinity and specificity for DNA. Thermal unfolding and temperature-induced changes in the secondary structure of the mutant H52R/NK-2 homeodomain protein (9713 MW) were investigated by differential scanning calorimetry (DSC), far-UV circular dichroism (CD), and intrinsic tryptophan fluorescence changes. In 50 mM Na- phosphate buffer, pH 7.4, the H52R/NK-2 protein reversibly unfolded with a transition temperature (Tm) of ca. 56 C as compared to a Tm value of ca. 39 C in 50 mM Hepes, pH 7.4 buffer. The stabilizing effect of the phosphate anion on the H52R/NK-2 homeodomain mutant protein may relate to DNA binding properties. Moreover, the unfolding was less cooperative in Hepes than in phosphate buffer, with overall unfolding enthalpies of ca. 34 and 40 kcal/mol, respectively. Adding 100 mM NaCl to buffers, improved refolding during heating and cooling cycles, as measured by CD. Good agreement was obtained between thermodynamic parameters obtained by DSC and temperature-induced CD changes at 222 nm. However, DSC data for the thermal unfolding of the H52R/NK-2 protein were fitted best to a non-two-state model, with ratios of calorimetric to vant Hoff enthalpy changes less than unity. Currently, H52R, H52R/W56T mutants and wild type protein interactions with short double-stranded DNA oligomers containing the specific target DNA sequence are being studied by isothermal titration calorimetry (ITC), DSC, and protein tryptophanyl fluorescence titrations. Association constants for the homeodomain protein-DNA interaction as well as protein conformational changes and stability changes that occur upon forming the homeodomain-DNA complex are being measured. - homeodomain proteins, DNA, thermal unfolding, stability, circular dichroism, calorimetry, fluorescence