Multiple Photoisomerization Pathways of the Green Fluorescent Protein Chromophore in a Reversibly Photoswitchable Fluorescent Protein: Insights from Quantum Mechanics/Molecular Mechanics Simulations.

Herein, we have employed a combined CASPT2//CASSCF approach within the quantum mechanics/molecular mechanics (QM/MM) framework to explore the early time photoisomerization of rsEGFP2 starting from its two OFF trans states, i.e., Trans1 and Trans2. The results show similar vertical excitation energies to the S 1 state in their Franck-Condon regions. Considering the clockwise and counterclockwise rotations of the C11-C9 bond, four pairs of the S 1 excited-state minima and low-lying S 1 /S 0 conical intersections were optimized, based on which we determined four S 1 photoisomerization paths that are essentially barrierless to the relevant S 1 /S 0 conical intersections leading to efficient excited-state deactivation to the S 0 state. Most importantly, our work first identified multiple photoisomerization and excited-state decay paths, which must be seriously considered in the future. This work not only sheds significant light on the primary trans-cis photoisomerization of rsEGFP2 but also aids in the understanding of the microscopic mechanism of GFP-like RSFPs and the design of novel GFP-like fluorescent proteins.