Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

Area of Science:

• Biophysics
• Molecular Biology
• Fluorescence Imaging

Background:

• Reversibly switchable fluorescent proteins (RSFPs) are crucial for advanced fluorescence imaging.
• The performance of RSFP-based imaging depends heavily on the specific properties of the protein labels.

Purpose of the Study:

• To investigate the effect of a small binding peptide, Enhancer, on the spectroscopic properties of rsGreen series RSFPs.
• To explore how Enhancer binding influences RSFP brightness, pH stability, switching speed, and fatigue resistance.

Main Methods:

• Fusion constructs of Enhancer with rsGreen1 and rsGreenF were created.
• Spectroscopic properties and cellular expression (in E. coli and HeLa cells) were analyzed.
• Photoswitching kinetics and fatigue resistance were investigated.

Main Results:

• Enhancer binding increased molecular brightness and pH stability of rsGreen RSFPs.
• Cellular expression led to decreased overall emission.
• Enhancer binding accelerated off-switching speed and enhanced resistance to switching fatigue.
• Enhancer preferentially stabilized the fast-switching emissive state of RSFPs.

Conclusions:

• Small protein binding can effectively modulate the photo-physical properties of RSFPs.
• Enhancer binding provides insights into RSFP photoswitching kinetics and fatigue mechanisms.
• This study opens new avenues for engineering RSFPs with tailored properties for diverse applications.