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April 10, 2014

New Fluorophores

A review of literature describing the development of fluorogenic probes for live-cell imaging of intracellular proteins.

New Fluorophores

The authors of the article were able to achieve specific intracellular protein labeling with rapid kinetics and fluorogenic response through the design of a new PYP-tag ligand. [Sebastian Kaulitzki - Fotolia.com]

  • Studying intracellular proteins through fluorescence is ideally performed in live cells in a rapid manner. The use of fluorescent proteins such as green fluorescent protein (GFP) has been widely applied using GFP-fusions to a protein of interest as well as split GFP systems to study intracellular protein–protein interactions. This article* describes the use of a small protein (125 amino acids) from a photoactive yellow protein (PYP) as a fluorescent tag to label proteins.

    Previous work has shown that certain environmentally sensitive fluorophores that are derivatives of cinnamic acid or coumarin become strongly fluorescent upon binding to a hydrophobic pocket of PYP and react with Cys-69 of PYP to form a thioester. However, these previous fluorophores were not cell penetrant. Here, new cell penetrant environmentally sensitive fluorophores were synthesized that can be used to label PYP-tagged proteins (TMBDMA and CMBDMA; see Figure).

  • Click Image To Enlarge +
    Figure: (a) Principle of labeling system based on PYP-tag and environment-sensitive fluorogenic probe. (b) Structures of new probes for labeling PYP-tag.

    The half-life of labeling PYP was ~1 min or 13 min for TMBDMA and CMBDMA, respectively. TMBDMA is cationic and the pI of PYP is 4.3, yielding an anionic protein under physiological pH, so it's likely that favorable electrostatic interactions promote the TMBDMA reaction more efficiently than reaction with anionic CMBDMA. Labeling of receptors on the cell surface was shown using an N-terminally PYP-tagged epidermal growth factor receptor (PYP-EGFR). Imaging showed expression of PYP-EGFR at the cell surface of HEK293 cells using a no-wash protocol (enabled since the background fluorescence of the unbound fluorophores employed is extremely low). Labeling of intracellular proteins using no-wash protocols was also demonstrated with PYP tagged with a nuclear localization signal as well as a methyl-CpG-binding domain 1 (MBD1) PYP–tagged construct.

    The PYP-MBD1 tag showed puncta intranuclear staining within 15 min of labeling with either TMBDMA or CMBDMA consistent with labeling of 5-methyl cytosine with CpG sequences of heterochromatin. This puncta staining was blocked with a DNA methylation inhibitor, 5-aza-2-deoxycytidine. The small PYP-tag and fluorophores described here should provide a useful alternative to fluorescent protein fusions. 

  • *Abstract from J Am Chem Soc 2013, Vol. 135: 12360–12365

    Click Image To Enlarge +

    We developed novel fluorogenic probes for no-wash live-cell imaging of proteins fused to PYP-tag, which is a small protein tag recently reported by our group. Through the design of a new PYP-tag ligand, specific intracellular protein labeling with rapid kinetics and fluorogenic response was accomplished. The probes crossed the cell membrane, and cytosolic and nuclear localizations of PYP-tagged proteins without cell washing were visualized within a 6-min reaction time. The fluorogenic response was due to the environmental effect of fluorophore upon binding to PYP-tag. Furthermore, the PYP-tag-based method was applied to the imaging of methyl-CpG-binding domain localization. This rapid protein-labeling system combined with the small protein tag and designed fluorogenic probes offers a powerful method to study the localization, movement, and function of cellular proteins.

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