Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins

Abstract

iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin’s C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin’s N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4–8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.

Two distinct mechanisms of flavoprotein spectral tuning revealed by low‐temperature and time‐dependent spectroscopy

“…Characteristic time of the transition from the ground state protein conformation to the excited state protein conformation is shorter than the fluorescence lifetime for CagFbFP, and thus the conformational change has a significant impact on the emission spectrum. A similar mechanism was proposed very recently for the Q430E variant of iLOV (residue 430 corresponds to Q89 in CagFbFP): there, the excitation maximum is also relatively unchanged, but emission is red-shifted for 4-8 nm (Kabir et al, 2023).…”

“…Consequently, a common strategy for color tuning of FbFPs was to add or remove charged or polar amino acids around the polar part of the isoalloxazine moiety of flavin. Different research groups used site‐directed mutagenesis guided by QM/MM modeling, including electrostatic spectral tuning maps, or site‐saturation mutagenesis (Davari et al, 2016; Kabir et al, 2023; Nikolaev et al, 2023; Orozco‐Gonzalez et al, 2019; Röllen et al, 2021). While no variants with drastically altered spectra are currently described, a series of moderately color‐shifted variants were gradually developed.…”

“…Some mutations of conserved asparagines near the flavin can also result in 4 nm red shifts (Goncharov et al, 2021; Raffelberg et al, 2011). Mutation of yet another conserved glutamine results in a 4–8 nm red shift (Kabir et al, 2023). Finally, mutation of a position corresponding to the conserved cysteine in LOV domains to a glutamine results in the largest currently reported red shifts of 10–14 nm (Nikolaev et al, 2023).…”

“…The effect of mutations on the emission spectrum is theoretically assessed based on the conformations of the flavin‐proximal amino acids, which are obtained from molecular dynamics simulations (Davari et al, 2016; Khrenova et al, 2015) or x‐ray crystallographic structures where possible (Remeeva et al, 2021; Röllen et al, 2021). However, redistribution of flavin charges after photoexcitation may cause changes in the conformations of surrounding polar amino acids; such a mechanism was recently proposed for the Q430E variant of iLOV (Kabir et al, 2023). This phenomenon becomes especially prominent if the characteristic times of conformational changes are close to the fluorescence lifetime, and can be revealed using low‐temperature and time‐resolved fluorescence.…”

Photoelectron Spectroscopy of Oppositely Charged Molecular Switches in the Aqueous Phase: Theory and Experiment

“…Explicit solvent calculations were performed using the average solvent electrostatic configuration and free energy gradient (ASEC-FEG) quantum mechanical/molecular mechanical (QM/MM) approach. The theoretical details of this method have been described previously. − The details of the model setup and methodology are presented in the Supporting Information. Briefly, the quantum chemical optimization and energy calculations are performed iteratively in the field of a time-averaged environment of the solution obtained from MD snapshots.…”