Turning on Fluorescence in Silico: From Radical Cations to 11-cis Locked Rhodopsin Analogues

Over the last decade, a significant progress has been made in the design and development of novel fluorescent probes with the major focus on genetically encoded fluorescent proteins (FP). The conventional route to the FP design is based on tuning and improving the spectral properties of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from other marine organisms. The major challenge of this work was to investigate computationally the possibility of turning a non-fluorescent protein into a fluorescent one. At the quantum-mechanical level, this means to find a way of increasing the excited state lifetime of a molecule by changing the shape of its barierless excited state potential energy surface to a barrier-controlled one. Here, we report the results of the ab initio CASPT2//CASSCF/6-31G*/AMBER hybrid quantum mechanics/molecular mechanics (QM/MM) study indicating that members of the rhodopsin family may be engineered to yield alternative source of FPs, despite the ultrafast photoisomerization reaction characterizing these systems. Indeed, the replacement of the natural chromophore with an artificial (locked) one in the visual pigment rhodopsin leads to a three-order of magnitude increase in excited state lifetime: from ca. 100 fs to 85 ps. To explain the origin of such an increase, we constructed consistent models of the wild-type and artificial rhodopsins and investigated the shapes of their excited state potential energy surfaces in a comparative way. Our results show that observed fluorescence (λfmax= 620 nm) is due to a locally excited intermediate whose lifetime is controlled by a small energy barrier. The analysis of the decay path of such an intermediate provides information useful for engineering rhodopsin variants with augmented fluorescence efficiencies. Preliminarily, to gain more insight into the phenomenon of the barrier-controlled fluorescence lifetime, we also investigated using ab initio multiconfigurational QM and QM/MM protocols the photochemistry and photophysics of N,N,N’,N’-tetramethyl-p-phenylenediamine radical cation, known as Wurster’s Blue (WB). This relatively small and stable organic species, exhibiting a temperature-dependent fluorescence behavior, was used as a training system for mapping of the excited state potential energy surfaces featuring a barrier