mScarlet3: a brilliant and fast-maturing red fluorescent protein

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Fluorescent proteins are essential tools in biological research.
• Existing red fluorescent proteins often have limitations in maturation speed, brightness, or stability.
• Development of improved fluorescent proteins is crucial for advancing imaging techniques.

Purpose of the Study:

• To engineer and characterize mScarlet3, a novel monomeric red fluorescent protein.
• To evaluate mScarlet3's performance as a fusion tag and reporter in biological systems.
• To compare mScarlet3 with existing red fluorescent proteins.

Main Methods:

• Protein engineering and directed evolution.
• Spectroscopic analysis (brightness, quantum yield, fluorescence lifetime).
• X-ray crystallography to determine protein structure.
• Functional assays in transient expression systems and as fusion tags.

Main Results:

• mScarlet3 exhibits fast and complete maturation.
• Achieved record brightness, 75% quantum yield, and 4.0 ns fluorescence lifetime.
• Crystal structure reveals a rigidified protein core.
• Demonstrated excellent performance as a fusion tag with no apparent cytotoxicity.
• Outperformed existing red fluorescent proteins as a Förster resonance energy transfer acceptor and reporter.

Conclusions:

• mScarlet3 represents a significant advancement in red fluorescent protein technology.
• Its superior properties make it a versatile tool for diverse biological imaging applications.
• mScarlet3 offers enhanced capabilities for FRET-based studies and reporter assays.