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

同源结构域是蛋白质中的小结构基序，其结合到特定的靶DNA序列，从而调节真核生物中的基因转录。同源结构域氨基酸序列决定了对DNA的结合亲和力和特异性。通过差示扫描量热法（DSC）、远紫外圆二色性（CD）和内源色氨酸荧光变化研究了突变型H52 R/NK-2同源结构域蛋白（9713 MW）的热去折叠和温度诱导的二级结构变化。在50 mM磷酸钠缓冲液（pH 7.4）中，H52 R/NK-2蛋白可逆地解折叠，转变温度（Tm）约为100 ℃。56 C，相比之下，Tm值为ca. 39 ℃下在50 mMHepes，pH7.4缓冲液中。磷酸根阴离子对H52 R/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，在Hepes中的解折叠比在磷酸盐缓冲液中的合作性更低，总体解折叠能力约为1000。34和40千卡/摩尔。加入100 mM NaCl缓冲液，在加热和冷却循环过程中，改善了再折叠，如CD测量。在222 nm处由DSC和温度引起的CD变化获得的热力学参数之间获得了良好的协议。然而，DSC数据的热去折叠的H52 R/NK-2蛋白最适合于非两态模型，与热量的比率vant霍夫焓变化小于1。目前，H52 R，H52 R/W56 T突变体和野生型蛋白质与含有特定靶DNA序列的短双链DNA寡聚体的相互作用正在通过等温滴定量热法（ITC），DSC和蛋白质双酰荧光滴定法进行研究。同源结构域蛋白质-DNA相互作用的关联常数以及形成同源结构域-DNA复合物时发生的蛋白质构象变化和稳定性变化正在被测量。- 同源结构域蛋白质，DNA，热去折叠，稳定性，圆二色性，量热法，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