Dynamic Features Control the Stabilization of the Green and Red Forms of the Chromophore in AzamiGreen Fluorescent Protein Variants

Fluorescent proteins find application as biocompatible, genetically encoded labels for visualization of living organisms tissues. Green fluorescent proteins (GFPs) are the most diverse, but proteins with red fluorescence have advantages, such as lower phototoxicity and better penetration into biological tissues. A promising approach is to obtain red fluorescent proteins (RFPs) from GFPs by introducing mutations that stabilize the oxidized chromophore state with an extended conjugated π-system. However, to date this remains a non-trivial task and experimental developments are carried out mainly by random mutagenesis. Development of descriptors obtained in molecular modeling can rationalize this field. Herein, we rely on experimental data on the AzamiGreen fluorescent protein and its variants that are oxidized to the red form. We perform classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) simulations to determine structural and dynamic features that govern oxidation. We demonstrate that the red state is predominantly stabilized by interactions of polar lysine residues with chromophore oxygen atoms. Dynamic network analysis demonstrates that in red fluorescent proteins the chromophore motions are correlated with the movement of surrounding protein side chains to a higher extent than in green variants. The presence of different resonance forms of the chromophore determines the fluorescence band maximum value: a decrease in the phenolate form population leads to the red shift.