ИСТИНА

Fluorescent proteins (FPs) are widely used in biology as tags to trace other proteins in living cells. Photoswitchable fluorescent proteins constitute a unique subclass as they can change their photophysical properties upon laser illumination. Purified from the sea anemone Anemonia sulcata the asCP is one of the most studied FP of this type. Its ability to be reversibly switched between the nonfluorescent “off”-state (λabs = 565 nm) and the fluorescent “on”-state (λabs = 576 nm, λem = 610 nm) upon the intensive irradiation with green light has been attributed to the photochemical E → Z isomerization of its chromophore. The reverse isomerization proceeds either thermally or photochemically by the 445 nm irradiation (“quenching”). The current work is aimed to study the details of asCP photocycle by computational chemistry methods. Structures, spectra and properties of the asCP have been modeled by using ab initio based QM/MM and molecular cluster approaches. The results of these simulations favor a mechanism describing majority of photoinduced asCP transformations solely relying on protein structures with the anionic form of the chromophore. The structures with the zwitterionic chromophore are energetically unfavorable in both ground S0 and excited S1 electronic states. The computed absorption S0 → S1 (561 nm) and emission S1 → S0 (605 nm) band maxima for the model system as well as calculated vibronic structure of the absorption band with the anionic chormophore correlate well with the available experimental data. In particular, we show that a shoulder at 530 nm in the absorption band corresponds to a vibronic transition in the anionic chromophore, but does not originate from contributions of other protonation states. This spectral shoulder primarily arises from the two most active Franck-Condon modes, which are traced to in-plane stretching vibrations with frequencies of 830 and 1285 cm-1. The routes of the radiationless relaxation of the anionic chromophore inside the protein via the twisted S0/S1 conical intersections are also described. Both conical intersections are related to the rotations of cromophore’s bridge bonds. One of them corresponds to the photoinduced E → Z isomerization, and the other – to relaxation back to the “off”-state. Small quantum yield of asCP photoactivation originates from high energy and a sloped nature of the corresponding photodeactivation channel. Photochemical quenching of the asCP “on”-state is explained by neutral chromophore excited state Z → E isomerization. The estimated value of absorption spectra maximum (437 nm) is in a good agreement with an experimental maximum of quenching effectiveness (445 nm). Also, neutral chromophore concentration increases upon “off”-“on” photoswitching that was shown by QM/MM-based MD study. Ground state Z → E isomerization proceeds exclusively through the anionic form of the asCP chromophore.