Selective two-step labeling of proteins with an off/on fluorescent probe

Kazuhisa Hirabayashi, Kenjiro Hanaoka, Manabu Shimonishi, Takuya Terai, Toru Komatsu, Tasuku Ueno, Tetsuo Nagano

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

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 6Cys-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.

Original languageEnglish
Pages (from-to)14763-14771
Number of pages9
JournalChemistry - A European Journal
Volume17
Issue number52
DOIs
Publication statusPublished - 2011 Dec 23
Externally publishedYes

Keywords

  • fluorescence
  • fluorescent probes
  • molecular recognition
  • peptides
  • sensors

ASJC Scopus subject areas

  • Catalysis
  • Organic Chemistry

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