Peek at the Potential Energy Surfaces of the LSSmKate1 and LSSmKate2 Proteins

Abstract

To determine the energetic feasibility of the mechanisms involved in the generation of the fluorescent species in red fluorescent proteins LSSmKate1 and LSSmKate2 developed by Piatkevich et al. (Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5369-5374 and J. Am. Chem. Soc. 2010, 132, 10762-10770), a potential energy scan for the respective reaction coordinates was performed in large cluster models including the surroundings of the chromophores, based on the respective crystallographic structures, using DFT and TDDFT. The predicted absorption wavelengths agree to within 5 nm with experiment, thus confirming the accuracy of the calculational level and modeling done. In both proteins, it was found that the adiabatic electronic state with the largest oscillator strength at the Franck-Condon region was not the one from which fluorescence could occur in the products. A diabatization procedure was used to determine an approximate photoactive state, based on selecting the state with the largest oscillator strength throughout. For LSSmKate1, this led to a rather flat potential energy profile but still did not predict a minimum in the product side. It is suggested that relaxation processes, absent from the model, could bring about such a minimum. LSSmKate2, on the other hand, clearly displays a favorable exoergic process in the photoactive state, and its double-proton transfer can be described as concerted but highly asynchronous, involving a barrier in the transfer of the first proton. In this way, the model provides strong support for the mechanism proposed for LSSmKate2.

Chromophore interactions leading to different absorption spectra in mNeptune1 and mCardinal red fluorescent proteins

“…The experimental values reported are 600 nm (2.07 eV) and 604 nm (2.05 eV), respectively. In this spectral region, this wavelenth difference of about 75 nm is of about +0.3 eV, which agrees with the expected overestimation of excitation energies for the TDDFT methodology used 38 and is in line with results reported by us 21,28,29,34,46,47,51 and other groups using this methodology. [43][44][45] The broad shape of band I in both proteins can be quantified by measuring the full width at half maximum (FWHM), which turns out to be 37 nm for mNeptune1 and 46 nm for mCardinal.…”

Molecular modelling of the pH influence in the geometry and the absorbance spectrum of near-infrared TagRFP675 fluorescent protein

“…The first 15 excited electronic states have been computed for each of the 400 structures. The CAM-B3LYP functional 27 which properly describes long-range charge transfer effects [28][29][30] and the 6-31+G(d,p) basis set complete the set. The second region consists of the rest of the atoms within the considered sphere and it has been treated with molecular mechanics, considering all the atoms as point charges according to the CHARMM-22 force field.…”

New insights into the structure–spectrum relationship in S65T/H148D and E222Q/H148D green fluorescent protein mutants: a theoretical assessment

“…This kind of excitation has been previously described for GFP-like chromophores and seems to be dominant in all of them. 54,55 Looking at the orbitals involved, it is easy to decipher why a negative charge near the tyrosine ring reduces the energy needed for the transition, thus increasing the corresponding wavelength: the excitation implies a loss of electron density on the tyrosine ring and a gain of electron density on the imidazole ring, so it is reasonable to think that a negative charge near the tyrosine ring will favour that electron density loss. In this direction, we predict that the presence of a positive charge placed near the imidazole ring (and sufficiently far from the tyrosine one) would also reduce the energy implied in the transition and produce a red-shift of the absorption band.…”