The photoreaction process of a large-Stokes-shift red fluorescence protein-LSSmCherry1

Large Stokes shift red fluorescent proteins are highly valued in fluorescence imaging due to their considerable spectral separation and minimal self-absorption. However, a notable gap remains in our understanding of these proteins’ excited-state dynamics. Unlocking this knowledge could potentially drive significant advancements in cellular and molecular biology. In this study, we systematically examine the excited-state dynamics of LSSmCherry1, a large Stokes shift red fluorescent protein, under varying isotopic compositions and temperatures. Our aim is to elucidate its distinctive spectral properties. Through steady fluorescence spectral experiments at different temperatures, we demonstrate that the large Stokes shift is significantly reduced by approximately 80 nm at low temperatures compared to room temperature. Using transient fluorescence and absorption spectroscopy, we dissect the excited-state dynamics of LSSmCherry1 in neutral environments. We also investigate the kinetic isotopic effect by comparing spectra in water and heavy water. These analyses allow us to delineate plausible photocyclic pathways. Our results reveal that the excited-state dynamics of LSSmCherry1 follow a model similar to that observed in other fluorescent proteins, such as Green Fluorescent Protein (GFP). Notably, we demonstrate that the excited-state proton transfer (ESPT) process is the primary origin of the large Stokes shift in LSSmCherry1. This ESPT process occurs rapidly, within approximately 365 fs after excitation in the neutral environment. This study provides crucial insights into the mechanisms underlying large Stokes shift fluorescent proteins, potentially paving the way for the development of improved fluorescent probes for biological imaging.