The role of protonation states of the chromophore and its neighboring amino acid side chains of the reversibly switching fluorescent protein rsEGFP2 upon photoswitching is characterized by molecular modeling methods. Numerous conformations of the chromophore-binding site in computationally derived model systems are obtained using the quantum chemistry and QM/MM approaches. Excitation energies are computed using the extended multiconfigurational quasidegenerate perturbation theory (XMCQDPT2). The obtained structures and absorption spectra allow us to provide interpretation of the observed structural and spectral properties of rsEGFP2 in the active ON- and inactive OFF-states. To identify intermediates along the routes of chromophore transformations between the ON- and OFF-states, molecular dynamics trajectories with the QM/MM potentials are examined. The results demonstrate that in addition to the dominating anionic and neutral forms of the chromophore, the cationic and zwitterionic forms may participate in the photoswitching of rsEGFP2. Conformations and protonation forms of the Glu223 and His149 side chains in the chromophore-binding site play an essential role in stabilizing specific protonation forms of the chromophore.
Atomic coordinates of the computed structures
The files with atomic coordinates of the structures obtained in geometry optimization of molecular systems within the protein and cluster models are available in the general-purpose open-access repository ZENODO and can be accessed via https://doi.org/10.5281/zenodo.5128055.