Water-modulated low-wavelength fluorescence emission of PSmOrange

Abstract

The fluorescent protein (FP) family has been a prominent focus for several decades due to its extensive applications in bioimaging. However, the fluorescence mechanisms of fluorescent proteins have not been fully elucidated. Recently, there have been reports suggesting the involvement of water in the fluorescent behaviors of FPs. Although numerous theoretical calculations and a few experimental studies based on protein solutions have been conducted, direct experimental evidence to unravel the role of water within fluorescent proteins remains elusive. Herein, we reported the impact of water on the low-wavelength fluorescence emission of PSmOrange, a model FP known for its excellent photostability and high-water content. We found that the presence of water induces the emergence of this low-wavelength fluorescence emission. This effect can be attributed to a hydrogen bond network involving the fluorophore and water molecules. Manipulating this hydrogen bond network by altering temperature or water types proved to be an effective strategy for modulating the fluorescent behaviors of PSmOrange. This study sheds light on the role of water in modulating the low-wavelength fluorescence emission of FPs and pave the way for the design and development of the next generation biomolecule-based fluorescence probes in real-time temperature monitoring in cold chain storage.