Heuristics-Guided Exploration of Reaction Mechanisms

For the investigation of chemical reaction networks, the efficient and accurate determination of all relevant intermediates and elementary reactions is mandatory. The complexity of such a network may grow rapidly, in particular if reactive species are involved that might cause a myriad of side reactions. Without automation, a complete investigation of complex reaction mechanisms is tedious and possibly unfeasible. Therefore, only the expected dominant reaction paths of a chemical reaction network (e.g., a catalytic cycle or an enzymatic cascade) are usually explored in practice. Here, we present a computational protocol that constructs such networks in a parallelized and automated manner. Molecular structures of reactive complexes are generated based on heuristic rules derived from conceptual electronic-structure theory and subsequently optimized by quantum chemical methods to produce stable intermediates of an emerging reaction network. Pairs of intermediates in this network that might be related by an elementary reaction according to some structural similarity measure are then automatically detected and subjected to an automated search for the connecting transition state. The results are visualized as an automatically generated network graph, from which a comprehensive picture of the mechanism of a complex chemical process can be obtained that greatly facilitates the analysis of the whole network. We apply our protocol to the Schrock dinitrogen-fixation catalyst to study alternative pathways of catalytic ammonia production.Comment: 27 pages, 9 figure

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

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.status: publishe

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

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

Efficient switching of mCherry fluorescence using chemical caging

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.status: publishe

Switching of mCherry fluorescence using a novel caging mechanism

status: publishe

Efficient switching of mCherry fluorescence using chemical caging

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores