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

Abstract

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.

Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures

“…Preliminary calculations (Table S1) performed at the ωB97XD/6-31+G­(d) level on the dark-AppA crystal structure showed that the first excited state (S 1 ) is a bright state corresponding to the first absorption band of the measured spectrum, whereas the next bright state which corresponds to the second observed band is S 3 . At this level of theory, the excitation energies of the two bands are significantly overestimated, as observed before, particularly for the second band. This also leads to overestimating the energy difference between the first two bands.…”

“…Between the TD-DFT methods, B3LYP shows the best agreement with experiments, but closer inspection showed that with this functional both bright states were strongly mixed with dark states. This issue was observed before, and it does not depend on the basis setall that finally encouraged us to employ ωB97X-D/6-31+G­(d) in subsequent calculations.…”

Ultrafast Transient Infrared Spectroscopy of Photoreceptors with Polarizable QM/MM Dynamics

“…The long-range corrected ωB97X-D functional was chosen to avoid artificial mixing of the S 1 state, as found in our previous work on another flavoprotein. 55 This is extremely important in order to avoid an unphysical description of the excited-state potential energy surface.…”

“…The choice of the QM level was dictated by the need of a qualitatively correct description of ground and excited states, while at the same time limiting the computational cost. The long-range corrected ωB97X-D functional was chosen to avoid artificial mixing of the S 1 state, as found in our previous work on another flavoprotein . This is extremely important in order to avoid an unphysical description of the excited-state potential energy surface.…”

All-Atom Quantum Mechanical Calculation of the Second-Harmonic Generation of Fluorescent Proteins

“…Figure displays their chromophore structures. iLOV is an engineered extrinsically fluorescent protein that binds the flavin mononucleotide (FMN). , bR is a light-driven transmembrane protein pump. Its retinal chromophore is covalently linked via a Schiff base to the protein backbone .…”