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.

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.

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 (-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 - to -ester transfer reactions (uncatalyzed) to generate a highly fluorescent -carbamoyl peptide. This enables detection of enzyme catalyzed -deacetylation, -demalonylation, -desuccinylation and -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,β-unsaturated ketone (conjugate addition) and α-halomethyl ketone (S2 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 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.

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.