Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications

Area of Science:

• Biochemistry and Molecular Biology
• Cell Biology
• Biotechnology

Background:

• Fluorescent proteins (FPs) are widely used for visualizing biomolecules but have limitations like slow maturation and photobleaching.
• Existing protein-labeling technologies face challenges including background fluorescence and probe washout.
• Development of advanced protein-labeling systems is crucial for improved cellular imaging and functional studies.

Purpose of the Study:

• To review novel fluorogenic protein-labeling systems developed by the authors.
• To address limitations of current protein-labeling technologies, particularly background fluorescence and imaging speed.
• To present innovative probes for enhanced protein visualization in living systems.

Main Methods:

• Development of a fluorogenic labeling system using a noncatalytic mutant of β-lactamase and specific β-lactam antibiotics.
• Design of fluorogenic probes based on fluorescence resonance energy transfer (FRET) and other principles.
• Creation of a photoactive yellow protein (PYP)-tag system with a turn-on switch utilizing a quenching mechanism.

Main Results:

• Demonstrated a β-lactamase-based system for specific covalent protein labeling with fluorogenic probes.
• Engineered hydrophobic prodrugs for effective intracellular protein labeling.
• Developed PYP-tag probes that enable rapid, washout-resistant protein imaging via a turn-on fluorescence mechanism.

Conclusions:

• The developed fluorogenic protein-labeling systems offer significant advantages over traditional FP-based methods.
• These novel systems provide tools for sensitive and dynamic imaging of proteins in living cells.
• Future protein-labeling technologies with tailored probes will enable advanced biomolecular imaging and functional perturbation.