Dipole Moment Variation Clears Up Electronic Excitations in the π-Stacked Complexes of Fluorescent Protein Chromophores

Abstract

We propose a quantitative structure–property relationship (QSPR) model for prediction of spectral tuning in cyan, green, orange, and red fluorescent proteins, which are engineered by motifs of the green fluorescent protein. Protein variants, in which their chromophores are involved in the π-stacking interaction with amino acid residues tyrosine, phenylalanine, and histidine, are prospective markers useful in bioimaging and super-resolution microscopy. In this work, we constructed training sets of the π-stacked complexes of four fluorescent protein chromophores (of the green, orange, red, and cyan series) with various substituted benzenes and imidazoles and tested the use of dipole moment variation upon excitation (DMV) as a descriptor to evaluate the vertical excitation energies in these systems. To validate this approach, we computed and analyzed electron density distributions of the π-stacked complexes and correlated the QSPR predictions with the reference values of the transition energies obtained using the high-level ab initio quantum chemistry methods. According to our results, the use of the DMV descriptor allows one to predict excitation energies in the π-stacked complexes with errors not exceeding 0.1 eV, which makes this model a practically useful tool in the development of efficient fluorescent markers for in vivo imaging.

Modeling Spectral Tuning in Red Fluorescent Proteins Using the Dipole Moment Variation upon Excitation

“…To assist future efforts, a computational problem of modeling fine spectral tuning in RFPs should be solved. Results of the present work, as well as of the previous studies 26,28,[31][32][33] are encouraging toward this goal.…”

“…The favorable performance of the CAM-B3LYP functional for the GFP-like chromophores is well documented in the literature. 26,28,60 Additionally we utilized 6 more functionals. Two of them, LC-BLYP and B97M-D3BJ, demonstrate poor correlations between the calculated DMV and the observed excitation energy.…”

“…5, i.e. the cRed and cOrange chromophores, 28 in the gas phase. The cRed chromophore has the same conjugated π-system as the DsRed-type chromophore considered in the present work.…”

“…Therefore, the request for spectral tuning in RFPs encourages seeking alternative theoretical approaches to evaluate excitation energies, including the development of quantitative structure -property relationship (QSPR) models. [26][27][28] In this respect, the use of the dipole moment variation upon excitation (DMV) electronic structure descriptor, which reflects redistribution of charge upon electron excitation, 29 is an attractive strategy. As shown by Drobizhev et al, the spectral tuning in fluorescent proteins, including RFPs, is associated with the internal electrostatic field of the protein that affects the excess dipole moment of the chromophore or its DMV.…”

“…34 From theoretical perspective, this correlation has been successfully explored in modeling the yellow-to-cyan tuning in the GFP family, 26 as well as in understanding the origin of the shifts in the electronic excitation energies of different anionic FP chromophores in the π-stacked complexes with aromatic molecules. 27,28 In this work, we examine a quantitative correlation between the computationally derived DMV values and the experimental electronic excitation energies at the excitation band maxima, ΔEex, for a series of RFPs, which operate with the DsRed-type chromophore. 35,36 Their excitation band maxima range between 570 and 605 nm because of the extended conjugated π-system as compared with the GFP-type chromophore.…”