利用非canonical 氨基酸在 NF-ĸB 中进行生物物理探针的位点特异性整合。
Site-specific incorporation of biophysical probes into NF-ĸB with non-canonical amino acids.
机构信息
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA.
出版信息
Methods. 2023 May;213:18-25. doi: 10.1016/j.ymeth.2023.03.004. Epub 2023 Mar 20.
The transcription factor NF-ĸB is a central mediator of immune and inflammatory responses. To understand the regulation of NF-ĸB, it is important to probe the underlying thermodynamics, kinetics, and conformational dynamics of the NF-ĸB/IĸBα/DNA interaction network. The development of genetic incorporation of non-canonical amino acids (ncAA) has enabled the installation of biophysical probes into proteins with site specificity. Recent single-molecule FRET (smFRET) studies of NF-ĸB with site-specific labeling via ncAA incorporation revealed the conformational dynamics for kinetic control of DNA-binding mediated by IĸBα. Here we report the design and protocols for incorporating the ncAA p-azidophenylalanine (pAzF) into NF-ĸB and site-specific fluorophore labeling with copper-free click chemistry for smFRET. We also expanded the ncAA toolbox of NF-ĸB to include p-benzoylphenylalanine (pBpa) for UV crosslinking mass spectrometry (XL-MS) and incorporated both pAzF and pBpa into the full-length NF-ĸB RelA subunit which includes the intrinsically disordered transactivation domain.
转录因子 NF-κB 是免疫和炎症反应的核心介质。为了理解 NF-κB 的调控机制，探究 NF-κB/IκBα/DNA 相互作用网络的热力学、动力学和构象动力学非常重要。非天然氨基酸（ncAA）的遗传掺入的发展使得能够将生物物理探针以位点特异性方式安装到蛋白质中。最近通过 ncAA 掺入进行的 NF-κB 的单分子荧光共振能量转移（smFRET）研究揭示了 IκBα介导的 DNA 结合的动力学控制的构象动力学。在这里，我们报告了设计和方案，用于将 ncAA p-叠氮苯丙氨酸（pAzF）掺入 NF-κB 中，并通过无铜点击化学进行位点特异性荧光标记，用于 smFRET。我们还将 NF-κB 的 ncAA 工具包扩展到包括 p-苯甲酰基苯丙氨酸（pBpa），用于紫外线交联质谱（XL-MS），并将 pAzF 和 pBpa 都掺入全长 NF-κB RelA 亚基中，其中包括固有无序的转录激活结构域。
Zotero 插件