title: Rapid labeling of intracellular His-tagged proteins in living cells.
abstract: 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.
authors.affiliation: Departments of Chemistry and.
authors.forename: Yau-Tsz
authors.initials: YT
authors.lastname: Lai
authors.affiliation: Departments of Chemistry and.
authors.forename: Yuen-Yan
authors.initials: YY
authors.lastname: Chang
authors.affiliation: Departments of Chemistry and.
authors.forename: Ligang
authors.initials: L
authors.lastname: Hu
authors.affiliation: Departments of Chemistry and.
authors.forename: Ya
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Departments of Chemistry and.
authors.forename: Ailun
authors.initials: A
authors.lastname: Chao
authors.affiliation: School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, People's Republic of China.
authors.forename: Zhi-Yan
authors.initials: ZY
authors.lastname: Du
authors.affiliation: Biochemistry.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Tanner
authors.affiliation: School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, People's Republic of China.
authors.forename: Mee-Len
authors.initials: ML
authors.lastname: Chye
authors.affiliation: Biochemistry.
authors.forename: Chengmin
authors.initials: C
authors.lastname: Qian
authors.affiliation: Departments of Chemistry and.
authors.forename: Kwan-Ming
authors.initials: KM
authors.lastname: Ng
authors.affiliation: Departments of Chemistry and.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Li
authors.affiliation: Departments of Chemistry and hsun@hku.hk.
authors.forename: Hongzhe
authors.initials: H
authors.lastname: Sun
mesh_terms: D005456:Fluorescent Dyes; D006367:HeLa Cells; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D011506:Proteins / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D006639:Histidine
keywords: His-tagged protein; Ni-NTA; fluorescent probe; live cell; photoactivation