Computational Investigation of Structural and Spectroscopic Properties of LOV-Based Proteins with Improved Fluorescence

[Image: see text] Flavin-based fluorescent proteins are a class of fluorescent reporters derived from light, oxygen, and voltage (LOV) sensing proteins. Through mutagenesis, natural LOV proteins have been engineered to obtain improved fluorescence properties. In this study, we combined extended classical Molecular Dynamics simulations and multiscale Quantum Mechanics/Molecular Mechanics methods to clarify the relationship between structural and dynamic changes induced by specific mutations and the spectroscopic response. To reach this goal we compared two LOV variants, one obtained by the single mutation needed to photochemically inactivate the natural system, and the other (iLOV) obtained through additional mutations and characterized by a significantly improved fluorescence. Our simulations confirmed the “flipping and crowding” effect induced in iLOV by the additional mutations and revealed its mechanism of action. We also showed that these mutations, and the resulting differences in the composition and flexibility of the binding pockets, are not reflected in significant shifts of the excitation and emission energies, in agreement with the similarity of the spectra measured for the two systems. However, a small but consistent reduction was found in the Stokes shift of iLOV, suggesting a reduction of the intermolecular reorganization experienced by the chromophore after excitation, which could slow down its internal conversion to the ground state and improve the fluorescence.