The Characterization of Evolved mMaple3 Fluorescent Proteins

Fluorescent proteins (FPs) have been a useful tool to highlight cells under the microscope. One class of proteins known as photoconvertible fluorescent proteins (pcFPs) have been developed as biosensors for cell tracking and detecting protein-protein interactions. These proteins undergo a permanent color shift when irradiated with UV light. For example, the pcFP mMaple3 undergoes a color shift from green to red when exposed to 405nm UV radiation. In order to improve the performance of mMaple3 in imaging applications we performed directed evolution to improve its brightness and stability. We characterized three different variants of mMaple3, and expressed and purified them from Escherichia coli. Post-purification, we assessed their biophysical and photophysical properties, comparing them to mMaple3. We found that all the variants show increased brightness and stability, however this was accompanied by an increase in the photoconversion time. We also found that these engineered variants have a shift in pKa, creating an increase of the ionized version of the mMaple3 fluorophore, and supporting the increase in brightness. Collectively, these findings indicate that the mMaple3 variants exhibit superior optical and structural properties compared to the wild-type, making them promising candidates for next-generation live-cell imaging and biosensing applications.