Protonation States of Molecular Groups in the Chromophore-Binding Site Modulate Properties of the Reversibly Switchable Fluorescent Protein rsEGFP2

Abstract

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 chromophorebinding site play an essential role in stabilizing specific protonation forms of the chromophore.

Modeling Light-Induced Chromophore Hydration in the Reversibly Photoswitchable Fluorescent Protein Dreiklang

“…The engineered protein Dreiklang [1], a member of the green fluorescent proteins (GFP) family with a chromophore formed from the Gly65-Tyr66-Gly67 tripeptide is a notable moiety, because its optical properties are modified in the chemical reaction of its chromophore. Mostly, the photoswitching in fluorescent proteins is due to the conformational changes of the chromophore, or to the tuning of the hydrogen-bond network near the chromophore [2][3][4][5][6][7][8][9][10][11]. As proposed in the pioneering work of Brakemann et al [1], a structural water molecule in Dreiklang attaches to its chromophore at the imidazolinone ring upon the light illumination at 405 nm (3.06 eV).…”