Dark-State-Mediated Photobleaching in mCherry-Based Red Fluorescent Proteins

Area of Science:

• Biochemistry
• Biophysics
• Protein Engineering

Background:

• Developing bright and photostable red fluorescent proteins (RFPs) remains a significant challenge in protein engineering.
• Understanding the properties of nonfluorescent or dark states is crucial for advancing RFP development.

Purpose of the Study:

• To establish a theoretical and experimental framework linking photobleaching decay to dark-state conversion and ground-state recovery in fluorescent proteins (FPs).
• To investigate the dark-state behavior of mCherry and a modified version, mCherry-d, to understand factors influencing RFP photostability.

Main Methods:

• Photophysical investigations of mCherry and mCherry-d.
• Theoretical modeling of photobleaching decay kinetics.
• Molecular dynamics simulations of chromophore dynamics.

Main Results:

• Photodestructive dark states were identified in mCherry and mCherry-d.
• A framework was developed to quantify dark-state kinetics and ground-state recovery.
• Molecular dynamics simulations indicated increased chromophore fluctuations in mCherry-d, potentially leading to nonfluorescent states.

Conclusions:

• Dark-state properties significantly influence RFP photostability.
• Insights into dark-state kinetics can guide the engineering of brighter and more photostable RFPs.
• This work provides a foundation for developing next-generation molecular probes with enhanced performance.