QM/MM-Based Calculations of Absorption and Emission Spectra of LSSmOrange Variants

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 experimentally 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.

Ultrafast action chemistry in slow motion: atomistic description of the excitation and fluorescence processes in an archetypal fluorescent protein

“…where N(Dt) is the number of snapshots with valid excitation energies that are still ''alive'' after Dt has elapsed since photoexcitation, and L i is the ''spectlet'' (the contribution to the spectrum from snapshot i). The line shape form was chosen to be a simple Lorentzian function 28 centered at the corresponding excitation energy, with the peak intensity given by eqn (4):…”

“…Some recent studies have employed classical MD simulations for the computation of full fluorescence spectra, using a specifically re-parameterized force field that reproduces the charges of the chromophore in the excited state. 28 A similar approach is not feasible in GFP S65T/ H148D, for reasons similar to those that limit the use of force fields in ground-state studies: 26 the extremely short O-O distance between chromophore and Asp148 cannot be reproduced with ordinary force fields as the van der Waals term between these two atoms would keep them much further apart, and the crucial proton transfer between them involves bond making and bond breaking.…”

Unravelling the Turn‐On Fluorescence Mechanism of a Fluorescein‐Based Probe in GABAA Receptors

“…However, there are several reasons why this is still a major endeavor. Fluorescence computations of few other‐purpose biological probes exist, [8, 9] but mechanistic studies of molecular probes bound to macromolecules are scarce [10–14] . In this system, one difficulty is that the subunit composition of GABA A Rs varies depending on their body localization, resulting in hundreds of possible structures.…”

Multistate QM/QM Extrapolation of UV/Vis Absorption Spectra with Point Charge Embedding

“…The accurate simulation of the UV/vis absorption spectra of large chromophores in complex environments is still an open problem for quantum chemistry. The computational scaling is too steep to treat large sections of the system at an accurate quantum mechanical (QM) level, and hybrid methods are necessary to reduce the computational cost and the burden of storage/memory requirements. − In hybrid methods, the system is partitioned in fragments or layers, so that only a small part is treated at a high level at any given point. The region surrounding the main chromophoric moiety can be treated with classical methods, using molecular mechanics (MM) force fields, or even implicit solvation models. − However, there are cases when a classical environment is not sufficient, as quantum effects due to the tail of the electron density may be important for the electron excitation process.…”