QM/MM-Based Calculations of Absorption and Emission Spectra of LSSmOrange Variants Maike Bergeler,* ,† Hideaki Mizuno, ‡ Eduard Fron, § and Jeremy N. Harvey* ,† † Department of Chemistry, Quantum Chemistry and Physical Chemistry Section ‡ Department of Chemistry, Biochemistry, Molecular and Structural Biology Section, § Department of Chemistry, Molecular Imaging and Photonics, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium *S Supporting Information ABSTRACT: The goal of this computational work is to gain new insight into the photochemistry of the fluorescent protein (FP) LSSmOrange. This FP is of interest because besides exhibiting the eponymous large spectral shift (LSS) between the absorption and emission energies, it has been exper- imentally observed that it can also undergo a photoconversion process, which leads to a change in the absorption wavelength of the chromophore (from 437 to 553 nm). There is strong experimental evidence that this photoconversion is caused by decarboxylation of a glutamate located in the close vicinity of the chromophore. Still, the exact chemical mechanism of the decarboxylation process as well as the precise understanding of structure-property relations in the measured absorption and emission spectra is not yet fully understood. Therefore, hybrid quantum mechanics/molecular mechanics (QM/MM) calculations are performed to model the absorption and emission spectra of the original and photoconverted forms of LSSmOrange. The necessary force-field parameters of the chromophore are optimized with CGenFF and the FFToolkit. A thorough analysis of QM methods to study the excitation energies of this specific FP chromophore has been carried out. Furthermore, the influence of the size of the QM region has been investigated. We found that QM/MM calculations performed with time-dependent density functional theory (CAM-B3LYP/D3/6-31G*) and QM calculations performed with the semiempirical ZIndo/S method including a polarizable continuum model can describe the excitation energies reasonably well. Moreover, already a small QM region size seems to be sufficient for the study of the photochemistry in LSSmOrange. Especially, the calculated ZIndo spectra are in very good agreement with the experimental ones. On the basis of the spectra obtained, we could verify the experimentally assigned structures. ■ INTRODUCTION Fluorescent proteins (FPs) are widely used in noninvasive (protein) labeling experiments. 1-3 One of the most famous FPs is the jellyfish green fluorescent protein (GFP), 4 but a broad range of other FPs exist. All FPs have in common that a certain part of the protein serves as a chromophore, which emits light in the visible range of light. Different FPs can fluoresce at different wavelengths, leading to many different colors. The absorption and emission wavelengths can be tuned by, for example, adjusting the length of the mesomeric system, the chemical composition, and the environment of the chromo- phore. One FP with emission in the orange range of light is LSSmOrange. It is special because of the large shift between the absorption and emission bands, which led to its abbreviation “LSS”, meaning “large Stokes shift” (typically for gaps between absorption and emission above 100 nm). Although this synonym is commonly used within the community, it is actually better to refer to LSS in LSSmOrange as “light-induced spectral shift” (suggested by Fron et al. 5 ) because the difference between the absorption and emission wavelengths is not only due to vibrational and environmental relaxation and is hence not a Stokes shift. Because of the large spectral shift, LSSmOrange has many applications in multicolor labeling. 6-9 The chromophore of LSSmOrange is a 2-[(5-)-2-hydroxy- dihydro-oxazole]-4-(p-hydroxybenzylidene)-5-imidazolinone moiety, which is embedded in a β-barrel protein structure (see Figure 1). It is covalently bound to two amino acids of the protein structure. The phenol group of the chromophore can be protonated or deprotonated depending on the protein environment and the electronic state of the system. It has been found that LSSmOrange undergoes (like many other FPs) an excited-state proton transfer (ESPT). 5,10 Interestingly, Fron et al. have observed that besides the ESPT also a Kolbe-like decarboxylation of a glutamate close to the chromophore (Glu215) 5,11,12 can occur (in the first excited state (ES)). This decarboxylation process leads to a change in the absorption wavelength of the chromophore (from 437 to 553 nm) and is thus also referred to as a photoconversion process (Phot. conv.). It is noteworthy that the photoconversion leads to a Received: September 30, 2016 Revised: November 18, 2016 Published: November 22, 2016 Article pubs.acs.org/JPCB © 2016 American Chemical Society 12454 DOI: 10.1021/acs.jpcb.6b09815 J. Phys. Chem. B 2016, 120, 12454-12465