title: Selective two-step labeling of proteins with an off/on fluorescent probe.
abstract: We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis(6)-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His(6)Cys-tagged enhanced blue fluorescent protein (EBFP), but not His(6)-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.
authors: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: Kazuhisa
authors: K
authors: Hirabayashi
authors: Kenjiro
authors: K
authors: Hanaoka
authors: Manabu
authors: M
authors: Shimonishi
authors: Takuya
authors: T
authors: Terai
authors: Toru
authors: T
authors: Komatsu
authors: Tasuku
authors: T
authors: Ueno
authors: Tetsuo
authors: T
authors: Nagano
mesh_terms: D001665:Binding Sites; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D015394:Molecular Structure; D009532:Nickel / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D009942:Organometallic Compounds; D010455:Peptides; D011506:Proteins; D049452:Green Fluorescent Proteins; D009532:Nickel; D009571:Nitrilotriacetic Acid