Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• Smart fluorophores, like reversibly switchable fluorescent proteins, are essential for advanced fluorescence imaging.
• Existing smart fluorophores often have limited availability and reduced biological performance compared to classical variants.

Purpose of the Study:

• To develop robustly photoswitchable variants of enhanced green fluorescent protein (EGFP) with improved biological performance.
• To investigate the mechanisms of photochromism in these new fluorescent proteins.
• To demonstrate their application in advanced fluorescence imaging techniques.

Main Methods:

• Engineering of enhanced green fluorescent protein (EGFP) to create photoswitchable variants (rsGreens).
• Expression and characterization of rsGreens in bacterial (E. coli) and mammalian (HEK293T) cell lines.
• Determination of crystal structures of rsGreens in both "on" and "off" states.
• Application of rsGreens in multimodal, diffraction-unlimited fluorescence imaging techniques (pcSOFI, RESOLFT).

Main Results:

• rsGreens exhibited up to 30-fold higher fluorescence in E. coli and over 4-fold higher fluorescence in HEK293T cells compared to their ancestor rsEGFP.
• Enhanced fluorescence was attributed to increased expression levels, not intrinsic brightness.
• rsGreens with varied photoswitching kinetics were developed and utilized for imaging.
• Achieved a spatial resolution of approximately 70 nm using pcSOFI and RESOLFT.
• Crystal structures confirmed cis-trans isomerization, providing insights into photochromism.

Conclusions:

• Genetically encoded smart fluorophores can be readily optimized for enhanced biological performance.
• rsGreens offer a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.
• These optimized probes advance the capabilities of super-resolution fluorescence microscopy.