Temperature Controlled Decay and Pendulum Dynamics of Green Fluorescent Protein (GFP) Chromophore

Abstract

The excited-state dynamics of the GFP chromophore, HBDI^– (anionic p-hydroxybenzylidene-2,3-dimethylimidazolinone), were investigated through a combination of theoretical nonadiabatic molecular dynamics (NAMD) simulations and femtosecond transient absorption spectroscopy (fs-TA). The NAMD simulations revealed that the primary dynamics in excited states involve the formation of a P-twisted intermediate (S_1min,P), which undergoes pendulum-like oscillations with respect to ϕ = 90°. This motion serves as a reservoir for the excited-state population and the primary source of fluorescence. Rather than a direct channel from the major S_1min,P, a coordinated pathway of S_1min,P → S_1min → S_1min,I → S₀ is responsible for the decay to the ground state, emphasizing the importance of planar intermediate (S_1min) formation. The experimental fs-TA spectra confirmed these dynamics, revealing three distinct time scales (340–470 fs, 1.4 ps, and 8.3 ps), corresponding to the formation of S_1min,P and its decay governed by the coordinated pathway. At low temperatures, the coordinated decay pathway is suppressed, leading to prolonged fluorescence lifetimes, consistent with low-temperature experimental results. This study presents a new model for the excited-state dynamics of GFP chromophore, suggesting that pendulum motion and the coordinated decay pathway play a crucial role in regulating fluorescence intensity.