Dark state-mediated photobleaching in mCherry-basedred fluorescent proteins

Developing bright and photostable red fluorescent proteins (RFPs) is one of the holy grails of the protein engineering community. Despite several attempts, finding such fluorescent proteins (FPs) has remained elusive. One bottleneck to engineering next generation RFPs is our lack of understanding of non-fluorescent or dark state properties in such constructs. Here, we develop a theoretical and experimental framework that describes how photobleaching decays in FPs relates 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 non-fluorescent states. Collectively, this work quantifies dark state kinetics and gives insights into engineering dark states in RFPs to develop bright yet photostable molecular probes.