Molecular Mechanisms behind Circular Dichroism Spectral Variations between Channelrhodopsin and Heliorhodopsin Dimers.

Channelrhodopsin (ChR) and heliorhodopsin (HeR) are microbial rhodopsins with similar structures but different circular dichroism (CD) spectra: ChR shows biphasic negative and positive bands, whereas HeR shows a single positive band. We explored the physicochemical factors underlying these differences through computational methods. Using the exciton model based on first-principles computations, we obtained the CD spectra of ChR and HeR. The obtained spectra indicate that the protein dimer structures and the quantum mechanical treatment of the retinal chromophore and its interacting amino acids are crucial for accurately reproducing the experimental spectra. Further calculations revealed that the sign of the excitonic coupling was opposite between the ChR and HeR dimers, which was attributed to the contrasting second term of the orientation factor between the two retinal chromophores. These findings demonstrate that slight variations in the intermolecular orientation of the two chromophores can result in significant differences in the CD spectral shape.

In Silico Screening and Experimental Verification of Near-Infrared-Emissive Two-Boron-Doped Polycyclic Aromatic Hydrocarbons.

Embedding two boron atoms into a polycyclic aromatic hydrocarbon (PAH) leads to the formation of a neutral analogue that is isoelectronic to the corresponding dicationic PAH skeleton, which can significantly alter its electronic structure. Based on this concept, we explore herein the identification of near-infrared (NIR)-emissive PAHs with the aid of an in silico screening method. Using perylene as the PAH scaffold, we embedded two boron atoms and fused two thiophene rings to it. Based on this design concept, all possible structures (ca. 2500 entities) were generated using a comprehensive structure generator. Time-dependent DFT calculations were conducted on all these structures, and promising candidates were extracted based on the vertical excitation energy, transition dipole moment, and atomization energy per bond. One of the extracted dithieno-diboraperylene candidates was synthesized and indeed exhibited emission at 724 nm with a quantum yield of 0.40 in toluene, demonstrating the validity of this screening method. This modification was further applied to other PAHs, and a series of thienobora-modified PAHs was synthesized.

Spectral Tuning and Excitation-Energy Transfer by Unique Carotenoids in Diatom Light-Harvesting Antenna.

The light-harvesting antennae of diatoms and spinach are composed of similar chromophores; however, they exhibit different absorption wavelengths. Recent advances in cryoelectron microscopy have revealed that the diatom light-harvesting antenna fucoxanthin chlorophyll a/c-binding protein (FCPII) forms a tetramer and differs from the spinach antenna in terms of the number of protomers; however, the detailed molecular mechanism remains elusive. Herein, we report the physicochemical factors contributing to the characteristic light absorption of the diatom light-harvesting antenna based on spectral calculations using an exciton model. Spectral analysis reveals the significant contribution of unique fucoxanthin molecules (fucoxanthin-S) in FCPII to the diatom-specific spectrum, and further analysis determines their essential role in excitation-energy transfer to chlorophyll. It was revealed that the specificity of these fucoxanthin-S molecules is caused by the proximity between protomers associated with the tetramerization of FCPII. The findings of this study demonstrate that diatoms employ fucoxanthin-S to harvest energy under the ocean in the absence of long-wavelength sunlight and can provide significant information about the survival strategies of photosynthetic organisms to adjust to their living environment.

Extended theoretical modeling of reverse intersystem crossing for thermally activated delayed fluorescence materials.

Thermally activated delayed fluorescence (TADF) materials and multi-resonant (MR) variants are promising organic emitters that can achieve an internal electroluminescence quantum efficiency of ~100%. The reverse intersystem crossing (RISC) is key for harnessing triplet energies for fluorescence. Theoretical modeling is thus crucial to estimate its rate constant (kRISC) for material development. Here, we present a comprehensive assessment of the theory for simulating the RISC of MR-TADF molecules within a perturbative excited-state dynamics framework. Our extended rate formula reveals the importance of the concerted effects of nonadiabatic spin-vibronic coupling and vibrationally induced spin-orbital couplings in reliably determining kRISC of MR-TADF molecules. The excited singlet-triplet energy gap is another factor influencing kRISC. We present a scheme for gap estimation using experimental Arrhenius plots of kRISC. Erroneous behavior caused by approximations in Marcus theory is elucidated by testing 121 MR-TADF molecules. Our extended modeling offers in-depth descriptions of kRISC.

A quantum chemical study on the anti-SARS-CoV-2 activity of TMPRSS2 inhibitors.

Nafamostat and camostat are known to inhibit the spike protein-mediated fusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by forming a covalent bond with the human transmembrane serine protease 2 (TMPRSS2) enzyme. Previous experiments revealed that the TMPRSS2 inhibitory activity of nafamostat surpasses that of camostat, despite their structural similarities; however, the molecular mechanism of TMPRSS2 inhibition remains elusive. Herein, we report the energy profiles of the acylation reactions of nafamostat, camostat, and a nafamostat derivative by quantum chemical calculations using a combined molecular cluster and polarizable continuum model (PCM) approach. We further discuss the physicochemical relevance of their inhibitory activity in terms of thermodynamics and kinetics. Our analysis attributes the strong inhibitory activity of nafamostat to the formation of a stable acyl intermediate and its low activation energy during acylation with TMPRSS2. The proposed approach is also promising for elucidating the molecular mechanisms of other covalent drugs.

Molecular Mechanism of Spectral Tuning by Chloride Binding in Monkey Green Sensitive Visual Pigment.

The visual pigments of the cones perceive red, green, and blue colors. The monkey green (MG) pigment possesses a unique Cl- binding site; however, its relationship to the spectral tuning in green pigments remains elusive. Recently, FTIR spectroscopy revealed the characteristic structural modifications of the retinal binding site by Cl- binding. Herein, we report the computational structural modeling of MG pigments and quantum-chemical simulation to investigate its spectral redshift and physicochemical relevance when Cl- is present. Our protein structures reflect the previously suggested structural changes. AlphaFold2 failed to predict these structural changes. Excited-state calculations successfully reproduced the experimental red-shifted absorption energies, corroborating our protein structures. Electrostatic energy decomposition revealed that the redshift results from the His197 protonation state and conformations of Glu129, Ser202, and Ala308; however, Cl- itself contributes to the blueshift. Site-directed mutagenesis supported our analysis. These modeled structures may provide a valuable foundation for studying cone pigments.

Discovery of 2,6-Dihalopurines as Stomata Opening Inhibitors: Implication of an LRX-Mediated H+-ATPase Phosphorylation Pathway.

Stomata are pores in the leaf epidermis of plants and their opening and closing regulate gas exchange and water transpiration. Stomatal movements play key roles in both plant growth and stress responses. In recent years, small molecules regulating stomatal movements have been used as a powerful tool in mechanistic studies, as well as key players for agricultural applications. Therefore, the development of new molecules regulating stomatal movement and the elucidation of their mechanisms have attracted much attention. We herein describe the discovery of 2,6-dihalopurines, AUs, as a new stomatal opening inhibitor, and their mechanistic study. Based on biological assays, AUs may involve in the pathway related with plasma membrane H+-ATPase phosphorylation. In addition, we identified leucine-rich repeat extensin proteins (LRXs), LRX3, LRX4 and LRX5 as well as RALF, as target protein candidates of AUs by affinity based pull down assay and molecular dynamics simulation. The mechanism of stomatal movement related with the LRXs-RALF is an unexplored pathway, and therefore further studies may lead to the discovery of new signaling pathways and regulatory factors in the stomatal movement.

Impact of Hydrophobic/Hydrophilic Balance on Aggregation Pathways, Morphologies, and Excited-State Dynamics of Amphiphilic Diketopyrrolopyrrole Dyes in Aqueous Media.

We report the thermodynamic and kinetic aqueous self-assembly of a series of amide-functionalized dithienyldiketopyrrolopyrroles (TDPPs) that bear various hydrophilic oligoethylene glycol (OEG) and hydrophobic alkyl chains. Spectroscopic and microscopic studies showed that the TDPP-based amphiphiles with an octyl group form sheet-like aggregates with J-type exciton coupling. The effect of the alkyl chains on the aggregated structure and the internal molecular orientation was examined via computational studies combining MD simulations and TD-DFT calculations. Furthermore, solvent and thermal denaturation experiments provided a state diagram that indicates the formation of unexpected nanoparticles during the self-assembly into nanosheets when longer OEG side chains are introduced. A kinetic analysis revealed that the nanoparticles were obtained selectively as an on-pathway intermediate state toward the formation of thermodynamically controlled nanosheets. The metastable aggregates were used for seed-initiated supramolecular assembly, which allowed establishing control over the assembly kinetics and the aggregate size. The sheet-like aggregates prepared using the seeding method exhibited coherent vibration in the excited state, indicating a well-ordered orientation of the TDPP units. These results underline the significance of fine tuning of the hydrophobic/hydrophilic balance in the molecular design to kinetically control the assembly of amphiphilic π-conjugated molecules into two-dimensional nanostructures in aqueous media.

Determination of FRET orientation factor between artificial fluorophore and photosynthetic light-harvesting 2 complex (LH2).

The orientation factor of fluorescence resonance energy transfer (FRET) between photosynthetic light-harvesting 2 complex (LH2) and artificial fluorophore (Alexa Fluor 647: A647) was theoretically investigated. The orientation factor of 2/3, i.e., the isotropic mean, is widely used to predict the donor-acceptor distance from FRET measurements. However, this approximation seems inappropriate because the movement of A647 is possibly restricted by the bifunctional linker binding to LH2. In this study, we performed molecular dynamics (MD) simulations and electronic coupling calculations on the LH2-A647 conjugate to analyze its orientation factor. The MD results showed that A647 keeps a position approximately 26 Å away from the bacteriochlorophyll (BChl) assembly in LH2. The effective orientation factor was extracted from the electronic coupling calculated using the transition charge from electrostatic potential (TrESP) method. With MD snapshots, an averaged orientation factor was predicted to be 1.55, significantly different from the isotropic mean value. The analysis also suggested that the value of the refractive index employed in the previous studies is not suitable for this system. Furthermore, optimal orientations of A647 with larger orientation factors to improve FRET efficiency were searched using Euler angles. The present approach is useful for extending the applicability of FRET analysis.

Structure-Function Study of a Novel Inhibitor of Cyclin-Dependent Kinase C in Arabidopsis.

The circadian clock, an internal time-keeping system with a period of about 24 h, coordinates many physiological processes with the day-night cycle. We previously demonstrated that BML-259 [N-(5-isopropyl-2-thiazolyl) phenylacetamide], a small molecule with mammal CYCLIN DEPENDENT KINASE 5 (CDK5)/CDK2 inhibition activity, lengthens Arabidopsis thaliana (Arabidopsis) circadian clock periods. BML-259 inhibits Arabidopsis CDKC kinase, which phosphorylates RNA polymerase II in the general transcriptional machinery. To accelerate our understanding of the inhibitory mechanism of BML-259 on CDKC, we performed structure-function studies of BML-259 using circadian period-lengthening activity as an estimation of CDKC inhibitor activity in vivo. The presence of a thiazole ring is essential for period-lengthening activity, whereas acetamide, isopropyl and phenyl groups can be modified without effect. BML-259 analog TT-539, a known mammal CDK5 inhibitor, did not lengthen the period nor did it inhibit Pol II phosphorylation. TT-361, an analog having a thiophenyl ring instead of a phenyl ring, possesses stronger period-lengthening activity and CDKC;2 inhibitory activity than BML-259. In silico ensemble docking calculations using Arabidopsis CDKC;2 obtained by a homology modeling indicated that the different binding conformations between these molecules and CDKC;2 explain the divergent activities of TT539 and TT361.

Machine-Learning- and Knowledge-Based Scoring Functions Incorporating Ligand and Protein Fingerprints.

We propose a novel machine-learning-based scoring function for drug discovery that incorporates ligand and protein structural information into a knowledge-based PMF score. Molecular docking, a simulation method for structure-based drug design (SBDD), is expected to reduce the enormous costs associated with conventional experimental methods in terms of rational drug discovery. Molecular docking has two main purposes: to predict ligand-binding structures for target proteins and to predict protein-ligand binding affinity. Currently available programs of molecular docking offer an accurate prediction of ligand binding structures for many systems. However, the accurate prediction of binding affinity remains challenging. In this study, we developed a new scoring function that incorporates fingerprints representing ligand and protein structures as descriptors in the PMF score. Here, regression analysis of the scoring function was performed using the following machine learning techniques: least absolute shrinkage and selection operator (LASSO) and light gradient boosting machine (LightGBM). The results on a test data set showed that the binding affinity delivered by the newly developed scoring function has a Pearson correlation coefficient of 0.79 with the experimental value, which surpasses that of the conventional scoring functions. Further analysis provided a chemical understanding of the descriptors that contributed significantly to the improvement in prediction accuracy. Our approach and findings are useful for rational drug discovery.

Chemical biology to dissect molecular mechanisms underlying plant circadian clocks.

Circadian clocks regulate the diel rhythmic physiological activities of plants, enabling them to anticipate and adapt to day-night and seasonal changes. Genetic and biochemical approaches have suggested that transcription-translation feedback loops (TTFL) are crucial for Arabidopsis clock function. Recently, the study of chemical chronobiology has emerged as a discipline within the circadian clock field, with important and complementary discoveries from both plant and animal research. In this review, we introduce recent advances in chemical biology using small molecules to perturb plant circadian clock function through TTFL components. Studies using small molecule clock modulators have been instrumental for revealing the role of post-translational modification in the clock, or the metabolite-dependent clock input pathway, as well as for controlling clock-dependent flowering time.

Fluorescent Organic π-Radicals Stabilized with Boron: Featuring a SOMO-LUMO Electronic Transition.

We report on the fluorescence properties of a new class of emissive and stable π-radicals that contain a boron atom at a position distant from the radical center. A fully planarized derivative exhibited an intense red fluorescence with high fluorescence quantum yields (ΦF >0.67) even in polar solvents. To elucidate the origin of this phenomenon, we synthesized another boron-stabilized radical that contains a bulky aryl group on the boron atom. A comparison of these derivatives, as well as with conventional donor-π-acceptor (D-π-A)-type emissive π-radicals, unveiled several characteristic features in their photophysical properties. A theoretical analysis revealed that the SOMO-LUMO electronic transition generates an emissive D1 state. Unlike conventional D-π-A-type π-radicals, this state does not undergo significant structural relaxation. The boron-stabilized π-radicals demonstrated promising potential for organic light-emitting diodes as an emitting material.

In Silico Analysis and Synthesis of Nafamostat Derivatives and Evaluation of Their Anti-SARS-CoV-2 Activity.

Inhibition of transmembrane serine protease 2 (TMPRSS2) is expected to block the spike protein-mediated fusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nafamostat, a potent TMPRSS2 inhibitor as well as a candidate for anti-SARS-CoV-2 drug, possesses the same acyl substructure as camostat, but is known to have a greater antiviral effect. A unique aspect of the molecular binding of nafamostat has been recently reported to be the formation of a covalent bond between its acyl substructure and Ser441 in TMPRSS2. In this study, we investigated crucial elements that cause the difference in anti-SARS-CoV-2 activity of nafamostat and camostat. In silico analysis showed that Asp435 significantly contributes to the binding of nafamostat and camostat to TMPRSS2, while Glu299 interacts strongly only with nafamostat. The estimated binding affinity for each compound with TMPRSS2 was actually consistent with the higher activity of nafamostat; however, the evaluation of the newly synthesized nafamostat derivatives revealed that the predicted binding affinity did not correlate with their anti-SARS-CoV-2 activity measured by the cytopathic effect (CPE) inhibition assay. It was further shown that the substitution of the ester bond with amide bond in nafamostat resulted in significantly weakened anti-SARS-CoV-2 activity. These results strongly indicate that the ease of covalent bond formation with Ser441 in TMPRSS2 possibly plays a major role in the anti-SARS-CoV-2 effect of nafamostat and its derivatives.

Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period.

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional-translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

Electronic Couplings and Electrostatic Interactions Behind the Light Absorption of Retinal Proteins.

The photo-functional chromophore retinal exhibits a wide variety of optical absorption properties depending on its intermolecular interactions with surrounding proteins and other chromophores. By utilizing these properties, microbial and animal rhodopsins express biological functions such as ion-transport and signal transduction. In this review, we present the molecular mechanisms underlying light absorption in rhodopsins, as revealed by quantum chemical calculations. Here, symmetry-adapted cluster-configuration interaction (SAC-CI), combined quantum mechanical and molecular mechanical (QM/MM), and transition-density-fragment interaction (TDFI) methods are used to describe the electronic structure of the retinal, the surrounding protein environment, and the electronic coupling between chromophores, respectively. These computational approaches provide successful reproductions of experimentally observed absorption and circular dichroism (CD) spectra, as well as insights into the mechanisms of unique optical properties in terms of chromophore-protein electrostatic interactions and chromophore-chromophore electronic couplings. On the basis of the molecular mechanisms revealed in these studies, we also discuss strategies for artificial design of the optical absorption properties of rhodopsins.

Spectral Tuning Mechanism of Photosynthetic Light-Harvesting Complex II Revealed by Ab Initio Dimer Exciton Model.

Excited states of two kinds of bacteriochlorophyll (BChl) aggregates, B850 and B800, in photosynthetic light-harvesting complex II (LH2) are theoretically investigated by developing and using an extended exciton model considering efficiently evaluated excitonic coupling. Our exciton model based on dimer fragmentation is shown to reproduce the experimental absorption spectrum of LH2 with good accuracy, entailing their different redshifts originating from aggregations of B850 and B800. The systematic analysis has been performed on the spectra by quantitatively decomposing their spectral shift energies into the contributions of various effects: structural distortion, electrostatic, excitonic coupling, and charge-transfer (CT) effects. Our results show that the spectral redshift of B800 is mainly attributed to its electrostatic interaction with the protein environment, while that of B850 arises from the marked effect of the excitonic coupling between BChl units. The interchromophore CT excitation also plays a key role in the spectral redshift of B850. This CT effect can be effectively described using our dimer model. This suited characterization reveals that the pronounced CT effect originates from the characteristics of B850 that has closely spaced BChls as dimers. We highlight the importance of the refinement of the crystal structure with the use of quantum chemical methods for prediction of the spectrum.

Excitonic coupling effect on the circular dichroism spectrum of sodium-pumping rhodopsin KR2.

We investigate the role of excitonic coupling between retinal chromophores of Krokinobacter eikastus rhodopsin 2 (KR2) in the circular dichroism (CD) spectrum using an exciton model combined with the transition density fragment interaction (TDFI) method. Although the multimer formation of retinal protein commonly induces biphasic negative and positive CD bands, the KR2 pentamer shows only a single positive CD band. The TDFI calculation reveals the dominant contribution of the Coulomb interaction and negligible contributions of exchange and charge-transfer interactions to the excitonic coupling energy. The exciton model with TDFI successfully reproduces the main features of the experimental absorption and CD spectra of KR2, which allow us to investigate the mechanism of the CD spectral shape observed in the KR2 pentamer. The results clearly show that the red shift of the CD band is attributed to the excitonic coupling between retinal chromophores. Further analysis reveals that the weak excitonic coupling plays a crucial role in the shape of the CD spectrum. The present approach provides a basis for understanding the origin of the KR2 CD spectrum and is useful for analyzing the mechanism of chromophore-chromophore interactions in biological systems.

3,4-Dibromo-7-Azaindole Modulates Arabidopsis Circadian Clock by Inhibiting Casein Kinase 1 Activity.

The circadian clock is a timekeeping system for regulation of numerous biological daily rhythms. One characteristic of the circadian clock is that period length remains relatively constant in spite of environmental fluctuations, such as temperature change. Here, using the curated collection of in-house small molecule chemical library (ITbM chemical library), we show that small molecule 3,4-dibromo-7-azaindole (B-AZ) lengthened the circadian period of Arabidopsis thaliana (Arabidopsis). B-AZ has not previously been reported to have any biological and biochemical activities. Target identification can elucidate the mode of action of small molecules, but we were unable to make a molecular probe of B-AZ for target identification. Instead, we performed other analysis, gene expression profiling that potentially reveals mode of action of molecules. Short-term treatment of B-AZ decreased the expression of four dawn- and morning-phased clock-associated genes, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. Consistently, amounts of PRR5 and TIMING OF CAB EXPRESSION 1 (TOC1) proteins, transcriptional repressors of CCA1, LHY, PRR9 and PRR7 were increased upon B-AZ treatment. B-AZ inhibited Casein Kinase 1 family (CK1) that phosphorylates PRR5 and TOC1 for targeted degradation. A docking study and molecular dynamics simulation suggested that B-AZ interacts with the ATP-binding pocket of human CK1 delta, whose amino acid sequences are highly similar to those of Arabidopsis CK1. B-AZ-induced period-lengthening effect was attenuated in prr5 toc1 mutants. Collectively, this study provides a novel and simple structure CK1 inhibitor that modulates circadian clock via accumulation of PRR5 and TOC1.

The anti-human cytomegalovirus drug tricin inhibits cyclin-dependent kinase 9.

4',5,7-trihydroxy-3',5'-dimethoxyflavone (tricin), derived from Sasa albo-marginata, has been reported to suppress significantly human cytomegalovirus (HCMV) replication in human embryonic lung (HEL) fibroblast cells. However, the target protein of tricin remains unclear. This study focused on the anti-HCMV activity of tricin in terms of its binding affinity to cyclin-dependent kinase 9 (CDK9). A molecular docking study predicted that tricin binds well to the ATP-binding site of CDK9. Experimental measurements then revealed that tricin inhibits the kinase activity of CDK9 and affects the phosphorylation of the carboxy-terminal domain of RNA polymerase II. Based on these results, we conclude that CDK9 is one of the target proteins of tricin. We also found that tricin possesses anti-HCMV activity with no cytotoxicity against HEL cells.