Riboflavin-binding proteins for singlet oxygen production.

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• miniSOG is a genetically encoded photosensitizer used in cell biology.
• Its efficiency in generating singlet oxygen is limited but can be enhanced by blue light.
• Previous work identified chromophore photolysis as the mechanism for enhancement.

Purpose of the Study:

• To improve miniSOG's singlet oxygen generation yield.
• To investigate the effect of altering the flavin chromophore on photosensitization.
• To engineer flavoprotein specificity for enhanced photophysical properties.

Main Methods:

• Replacement of flavin mononucleotide (FMN) with riboflavin (RF) in miniSOG.
• Site-directed mutagenesis of miniSOG to alter flavin binding specificity.
• Structural determination of miniSOG mutants.
• Measurement of singlet oxygen quantum yield (ΦΔ).

Main Results:

• Replacing FMN with RF significantly increased the singlet oxygen quantum yield (ΦΔ).
• The mutant miniSOG-R57Q selectively binds RF in cells, showing a modest ΦΔ improvement.
• The crystal structure of miniSOG-Q103L revealed a significantly increased ΦΔ and suggested an alternative oxygen access channel.

Conclusions:

• Altering the flavin chromophore, such as using RF instead of FMN, enhances miniSOG's photosensitizing capabilities.
• Engineering flavoprotein specificity is a viable strategy to tune photophysical properties.
• Structural insights provide a basis for further optimization of genetically encoded photosensitizers.