moxMaple3: a Photoswitchable Fluorescent Protein for PALM and Protein Highlighting in Oxidizing Cellular Environments

Area of Science:

• Biochemistry
• Molecular Biology
• Microscopy

Background:

• Fluorescent proteins (FPs) are crucial for biological imaging, relying on robust folding for chromophore formation.
• Understanding the impact of specific amino acid mutations on FP folding is essential for protein engineering.
• Oxidizing cellular environments, like the secretory pathway, pose challenges for FP folding and function.

Purpose of the Study:

• To engineer a monomeric photoswitchable fluorescent protein (FP) suitable for oxidizing cellular environments.
• To investigate the effect of specific mutations on FP folding and chromophore formation.
• To enhance FP performance for super-resolution microscopy techniques.

Main Methods:

• Engineering of a monomeric photoswitchable FP, moxMaple3.
• Introduction of point mutations, including cysteine replacement.
• Assessment of FP folding, chromophore formation, and performance in cellular environments.
• Evaluation using photoactivated localization microscopy (PALM).

Main Results:

• A single point mutation replacing a cysteine residue substantially improved moxMaple3 FP folding and chromophore formation.
• The engineered moxMaple3 demonstrated robust performance in oxidizing environments, including the eukaryotic secretory pathway.
• Improved folding led to a higher fraction of visibly tagged fusion proteins.
• Enhanced FP performance was observed in PALM super-resolution microscopy.

Conclusions:

• The engineered moxMaple3 is a robust monomeric FP with improved folding characteristics.
• This FP is well-suited for applications in oxidizing cellular compartments and super-resolution imaging.
• The findings highlight the non-intuitive impact of single amino acid mutations on FP functionality.