Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead.

Within a cell, multiple copies of the same protein coexist in different pathways and behave differently. Being able to individually analyze the constant actions of proteins in a cell is crucial to know the pathways through which they pass and which physiological functions they are deeply involved in. However, until now, it has been difficult to distinguish protein copies with distinct translocation properties by fluorescence labeling with different colors in living cells. In this study, we have created an unnatural ligand with an unprecedented protein-tag labeling property in living cells and overcome the above-mentioned problem. Of special interest is that some fluorescent probes with the ligand can selectively and efficiently label intracellular proteins without binding to cell-surface proteins, even if the proteins are present on the cell membrane. We also developed a cell-membrane impermeable fluorescent probe that selectively labels cell-surface proteins without labeling of intracellular proteins. These localization-selective properties enabled us to visually discriminate two kinetically distinct glucose transporter 4 (GLUT4) molecules that show different multiple subcellular localization and translocation dynamics in live cells. Taking advantage of the probes, we revealed that N-glycosylation of GLUT4 influences intracellular localization. Furthermore, we were able to visually distinguish active GLUT4 molecules that underwent membrane translocation at least twice within an hour from those that remained intracellularly, discovering previously unrecognized dynamic behaviors of GLUT4. This technology provides not only a valuable tool for study on multiple localization and dynamics of proteins but also important information on diseases caused by protein translocation dysfunction.

An "OFF-ON-OFF" fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems.

The ability to monitor proteolytic pathways that remove unwanted and damaged proteins from cells is essential for understanding the multiple processes used to maintain cellular homeostasis. In this study, we have developed a new protein-labeling probe that employs an 'OFF-ON-OFF' fluorescence switch to enable real-time imaging of the expression (fluorescence ON) and degradation (fluorescence OFF) of PYP-tagged protein constructs in living cells. Fluorescence switching is modulated by intramolecular contact quenching interactions in the unbound probe (fluorescence OFF) being disrupted upon binding to the PYP-tag protein, which turns fluorescence ON. Quenching is then restored when the PYP-tag-probe complex undergoes proteolytic degradation, which results in fluorescence being turned OFF. Optimization of probe structures and PYP-tag mutants has enabled this fast reacting 'OFF-ON-OFF' probe to be used to fluorescently image the expression and degradation of short-lived proteins.

Fluorogenic probes for detecting deacylase and demethylase activity towards post-translationally-modified lysine residues.

Reversible enzymatic post-translational modification of the ε-amino groups of lysine residues (e.g. N-acylation reactions) plays an important role in regulating the cellular activities of numerous proteins. This study describes how enzyme catalyzed N-deprotection of lysine residues of non-fluorescent peptide-coumarin probes can be used to generate N-deprotected peptides that undergo spontaneous O- to N-ester transfer reactions (uncatalyzed) to generate a highly fluorescent N-carbamoyl peptide. This enables detection of enzyme catalyzed N-deacetylation, N-demalonylation, N-desuccinylation and N-demethylation reactions activities towards the N-modified lysine residues of these probes using simple 'turn on' fluorescent assays.

Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies.

Covalent labeling systems that employ protein-tags or chemical probes to convert proteins into fluorescent conjugates are powerful tools for carrying out real time imaging and pulse-chase tracking studies that enable the spatiotemporal role of proteins in complex biological systems to be investigated. In this study, we have covalently modified a specific nucleophilic cysteine residue of the PYP-tag protein with weakly fluorescent α,ß-unsaturated ketone (conjugate addition) and α-halomethyl ketone (SN2 reaction) acceptors to afford highly fluorescent PYP-tag-dimethylaminocoumarin (DMAC) conjugates, whose ligands are covalently bound to the PYP-protein through stable thioether linkers. A chloromethylketone derived DMAC-CMK reagent was found to afford the best kinetic and stability profile for labeling the PYP-tag in cellular systems, with in vitro studies demonstrating that PYP-DMAC-CMK conjugates exhibit excellent photostability and cellular stability profiles which enables them to be used for long-term protein imaging studies in cellular systems. The potential of using this no wash fluorescent labeling PYP-tag-DMAC system to visualise dividing cells undergoing mitosis and for imaging a PYP-tag fused telomere binding protein bound to chromatin in cell nuclei has been demonstrated.

SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for In†Vivo Imaging of Cell Metabolism.

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small-sized multi-colored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.