Lai Yau-Tsz, Chang Yuen-Yan, Hu Ligang, Yang Ya, Chao Ailun, Du Zhi-Yan, Tanner Julian A, Chye Mee-Len, Qian Chengmin, Ng Kwan-Ming, Li Hongyan, Sun Hongzhe
Departments of Chemistry and.
School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, People's Republic of China.
Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):2948-53. doi: 10.1073/pnas.1419598112. Epub 2015 Feb 23.
Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.
基于小分子的荧光探针已被用于活细胞的实时可视化以及各种细胞事件的追踪，对被研究细胞的扰动最小。鉴于（组氨酸）6-镍（2+）-次氮基三乙酸（Ni-NTA）系统在蛋白质纯化中的广泛应用，基于荧光Ni（2+）-NTA的探针备受关注。不幸的是，先前基于Ni-NTA的探针膜通透性较差，无法标记细胞内蛋白质。在此，据我们所知，我们报告了首个膜通透性荧光探针Ni-NTA-AC的设计与合成，该探针通过将NTA与荧光团和芳基叠氮化物共轭，随后与Ni（2+）离子配位而成。该探针在Ni（2+）-NTA的驱动下，特异性结合与蛋白质基因融合的His标签，随后在芳基叠氮化物光激活后形成共价键，导致荧光增强13倍。芳基叠氮化物不仅对荧光增强不可或缺，而且对加强探针与蛋白质之间的结合也很重要。值得注意的是，Ni-NTA-AC探针能够快速进入不同类型的细胞，甚至植物组织，以靶向His标签蛋白。使用该探针，我们可视化了一种DNA修复蛋白——A型着色性干皮病蛋白（XPA122）的亚细胞定位，已知该蛋白主要富集于细胞核中。我们还证明了该探针能够对植物组织中的基因工程His标签蛋白进行成像。因此，本研究为原位可视化各种原核和真核细胞中大量His标签蛋白文库提供了新机会。
