Ultrafast Interchromophore Energy Transfer in Oligomeric Red Fluorescent Proteins

Red fluorescent proteins (RFPs) are widely used as genetically encoded reporters and biosensors, whose photophysics is often interpreted within a framework that treats the chromophore as an isolated fluorophore. Such descriptions may underrepresent interactions among chromophores and with the surrounding protein matrix that influence ultrafast excited-state dynamics. Here, we investigate these dynamics in a homologous series of RFPs, mCherry, tdTomato, and DsRed2, spanning monomeric to tetrameric quaternary structures, using femtosecond transient absorption spectroscopy and anisotropy measurements. We find that oligomerization promotes interchromophore energy transfer, giving rise to rapid depolarization dynamics absent in the monomer. In the tetramer DsRed2, we further resolve an ultrafast energy transfer pathway between spectrally distinct chromophore forms, revealing coupling between chemically heterogeneous states within a single protein assembly. These results suggest that collective effects arising from quaternary structure and chromophore heterogeneity can play an important role in the excited-state dynamics of oligomeric RFPs and should be considered when interpreting fluorescent protein photophysics.