Unidirectional Photoisomerization of the Green Fluorescent Protein Chromophore in a Reversibly Photoswitchable Fluorescent Protein rsKiiro: Insights from Quantum Mechanics/Molecular Mechanics Simulations

In this study, a quantum mechanics/molecular mechanics (QM/MM) framework combined with the CASPT2//CASSCF approach was used to investigate the excited-state decay and isomerization of the rsKiiro green fluorescent protein (GFP) from its neutral “OFF” trans state. Upon irradiation at 400 nm, the trans conformation is initially excited to the bright S₁ state. A rapid decay of the excited state then occurs and ultimately leads the molecule to the ground state. Notably, the clockwise and counterclockwise rotations of the C₈C₉C₁₁N₁₂ [or C₅C₈C₉C₁₁] dihedral angle are asymmetric or unidirectional, with only one direction of rotation effectively driving the excited-state relaxation. This process is shaped by hydrogen-bonding networks and steric constraints within the protein. In addition, trans–cis isomerization may not occur directly in the S₁ state because the energy of the S₁ cis minimum is relatively higher than that of the S₁ trans minimum. Instead, the S₁ cis minimum may be generated through the reabsorption of light near 400 nm, as the vertical excitation energy of the S₀ cis minimum is close to that of the S₀ trans minimum. This work provides important insights into the early photodynamics of rsKiiro GFP and aids in the design of novel GFP-like fluorescent proteins.