Recombinant Phototagging Enables Fluorescent Labeling of Biomolecules.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Current fluorescent labeling methods for biomolecules often require extra steps or can impair biomolecule function.
• There is a need for genetically encodable and minimally perturbative labeling techniques.

Purpose of the Study:

• To develop a novel, minimally invasive, genetically encodable strategy for fluorescent labeling of biomolecules.
• To leverage the red fluorescence of a photocleavable peptide fragment for post-expression labeling.

Main Methods:

• Genetically fusing the photoconvertible protein PhoCl1 to target peptides and proteins.
• Utilizing the photocleavage of PhoCl1 to release a red-fluorescent C-terminal peptide fragment (CTPF).
• Applying the strategy to label diverse biomolecules and visualize liquid-liquid phase separation.

Main Results:

• Demonstrated successful post-expression fluorescent labeling of peptides, proteins, and enzymes.
• Validated the strategy's applicability in visualizing liquid-liquid phase separation events.
• Achieved red fluorescence induction in nonfluorescent biomolecules via the photocleavable protein system.

Conclusions:

• The developed strategy offers a minimally perturbative, genetically encodable approach for red fluorescent labeling.
• This method provides a versatile tool for studying biomolecules and their interactions, including phase separation.
• The photocleavable protein-based labeling strategy opens new possibilities in bioimaging and molecular analysis.