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

Developing bright and photostable red fluorescent proteins (RFPs) is one of the "holy grails" of the protein engineering community. Despite several attempts, such fluorescent proteins (FPs) have remained elusive. One bottleneck to engineering next-generation RFPs is our lack of understanding of nonfluorescent or dark-state properties in such constructs. Here, we develop a theoretical and experimental framework that describes how photobleaching decays in FPs relate to dark-state conversion and ground-state recovery. Our systematic photophysical investigation of mCherry and mCherry-d, an RFP with enhanced dark-state behavior, showed the presence of photodestructive dark states in such FPs. Molecular dynamics simulations reveal enhanced fluctuation around the imidazolinone end of the chromophore in mCherry-d, potentially facilitating conversion to nonfluorescent states. Collectively, this work quantifies dark-state kinetics and provides insights into engineering dark states in RFPs to develop bright, yet photostable, molecular probes.