Abstract：
BACKGROUND: Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders.
RESULTS: The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNβ gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNβ). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNβ promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M.
CONCLUSIONS: NTD-IRF6 consists of a mix of α-helix and β-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNβ.
RESULTS: The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNβ gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNβ). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNβ promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M.
CONCLUSIONS: NTD-IRF6 consists of a mix of α-helix and β-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNβ.
摘要：
背景：干扰素调节因子6（IRF6）在胚胎发育过程中的腭融合中起关键作用，IRF6中的突变会导致口面裂开障碍。
结果：进行了IRF6的计算机模拟分析，以获得结构域边界的前导，随后将IRF6的N端结构域亚克隆到pGEX-2TK表达载体中，并成功优化了重组谷胱甘肽S-转移酶融合的NTD-IRF6蛋白在天然条件下的过表达和纯化。切割GST标签后，NTD-IRF6在可变pH下使用圆二色性和固有荧光光谱法进行蛋白质折叠研究，温度,和变性剂。CD研究表明，在pH9.0时，NTD-IRF6的α-螺旋含量和稳定性最高。天然和复性蛋白的比较描述了二级结构含量的损失。内在荧光和猝灭研究已经确定色氨酸残基主要存在于蛋白质的埋藏区域中,并且一小部分在蛋白质表面上或附近。当蛋白质用更高浓度的变性剂尿素展开时，荧光强度的峰值下降和红移，确认色氨酸残基主要存在于更极性的环境中。在病毒感染期间调节IFNβ基因表达的同时，N-末端结构域与病毒应答元件-干扰素β(VRE-IFNβ)的启动子区结合。随着蛋白质折叠分析，这项研究还旨在鉴定DNA结合活性并确定NTD-IRF6与VRE-IFNβ启动子区的结合亲和力。如凝胶延迟测定所证明的，蛋白质-DNA相互作用是特异性的，并且通过生物层干涉法定量的分子相互作用的动力学显示出强亲和力，亲和常数（KD）值为7.96×10-10M。
结论：NTD-IRF6由α-螺旋和β-折叠的混合物组成，在40°C至55°C之间显示温度依赖性的协同展开。尿素诱导的解折叠显示出对尿素的中等耐受性,因为尿素的中间转变浓度(Cm)为3.2M。色氨酸残基主要被掩埋,如通过荧光猝灭研究所描绘的。NTD-IRF6对VRE-IFNβ的启动子区域具有特异性和高亲和力。
结果：进行了IRF6的计算机模拟分析，以获得结构域边界的前导，随后将IRF6的N端结构域亚克隆到pGEX-2TK表达载体中，并成功优化了重组谷胱甘肽S-转移酶融合的NTD-IRF6蛋白在天然条件下的过表达和纯化。切割GST标签后，NTD-IRF6在可变pH下使用圆二色性和固有荧光光谱法进行蛋白质折叠研究，温度,和变性剂。CD研究表明，在pH9.0时，NTD-IRF6的α-螺旋含量和稳定性最高。天然和复性蛋白的比较描述了二级结构含量的损失。内在荧光和猝灭研究已经确定色氨酸残基主要存在于蛋白质的埋藏区域中,并且一小部分在蛋白质表面上或附近。当蛋白质用更高浓度的变性剂尿素展开时，荧光强度的峰值下降和红移，确认色氨酸残基主要存在于更极性的环境中。在病毒感染期间调节IFNβ基因表达的同时，N-末端结构域与病毒应答元件-干扰素β(VRE-IFNβ)的启动子区结合。随着蛋白质折叠分析，这项研究还旨在鉴定DNA结合活性并确定NTD-IRF6与VRE-IFNβ启动子区的结合亲和力。如凝胶延迟测定所证明的，蛋白质-DNA相互作用是特异性的，并且通过生物层干涉法定量的分子相互作用的动力学显示出强亲和力，亲和常数（KD）值为7.96×10-10M。
结论：NTD-IRF6由α-螺旋和β-折叠的混合物组成，在40°C至55°C之间显示温度依赖性的协同展开。尿素诱导的解折叠显示出对尿素的中等耐受性,因为尿素的中间转变浓度(Cm)为3.2M。色氨酸残基主要被掩埋,如通过荧光猝灭研究所描绘的。NTD-IRF6对VRE-IFNβ的启动子区域具有特异性和高亲和力。
