Design of Large Stokes Shift Fluorescent Proteins Based on Excited State Proton Transfer of an Engineered Photobase

• Published in: Journal of the American Chemical Society Vol. 143; no. 37; pp. 15091 - 15102
• Main Authors: Santos, Elizabeth M, Sheng, Wei, Esmatpour Salmani, Rahele, Tahmasebi Nick, Setare, Ghanbarpour, Alireza, Gholami, Hadi, Vasileiou, Chrysoula, Geiger, James H, Borhan, Babak
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 22.09.2021; Amer Chemical Soc
• Subjects: Chemistry; Chemistry, Multidisciplinary; Glutamic Acid - chemistry; HeLa Cells; Humans; Luminescent Proteins - chemistry; Photochemical Processes; Physical Sciences; Protein Engineering; Science & Technology; RED; BODIPY; DYES; TRANSFER ESIPT; IN-VIVO; DYNAMICS; FLUOROGENIC PROBES; CYSTEINE; MOLECULAR PROBES; LIVING CELLS
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.1c05039

The incredible potential for fluorescent proteins to revolutionize biology has inspired the development of a variety of design strategies to address an equally broad range of photophysical characteristics, depending on potential applications. Of these, fluorescent proteins that simultaneously exhibit high quantum yield, red-shifted emission, and wide separation between excitation and emission wavelengths (Large Stokes Shift, LSS) are rare. The pursuit of LSS systems has led to the formation of a complex, obtained from the marriage of a rationally engineered protein (human cellular retinol binding protein II, hCRBPII) and different fluorogenic molecules, capable of supporting photobase activity. The large increase in basicity upon photoexcitation leads to protonation of the fluorophore in the excited state, dramatically red-shifting its emission, leading to an LSS protein/fluorophore complex. Essential for selective photobase activity is the intimate involvement of the target protein structure and sequence that enables Excited State Proton Transfer (ESPT). The potential power and usefulness of the strategy was demonstrated in live cell imaging of human cell lines.