Janus-Type AIE Fluorophores: Synthesis and Properties of π-Extended Coumarin-Bearing Triskelions.

Janus-type triskelion-shaped fluorophores comprising coumarins bearing various electron-donating substituents (1aad, 1add, 1ccd, and 1cdd) were successfully synthesized via an intramolecular Ullmann coupling. Density functional theory (DFT) calculations indicated that all the compounds presented two different molecular surfaces, similar to Janus-type molecules. The absorption and fluorescence spectra of asymmetrical derivatives 1aad, 1add, 1ccd, and 1cdd exhibited a bathochromic shift due to their narrow highest occupied molecular orbital (HOMO) -lowest unoccupied molecular orbital (LUMO) gap. Natural transition orbital (NTO) analysis indicated that the excited state orbital overlaps differ among the C3 symmetrical and asymmetrical dyes. These triskelion-shaped fluorophores were found to form molecular nanoaggregates in THF/H2O mixtures and demonstrated aggregation-induced emission (AIE) enhancement characteristics as a result of restricting their molecular inversion. These results indicate that Janus-type AIE fluorophores are potentially applicable as solid-state fluorescent chiral materials, which can be optimized by controlling their molecular rearrangement in the solid state.

Free-Energy Profile Analysis of the Catalytic Reaction of Glycinamide Ribonucleotide Synthetase.

The second step in the de novo biosynthetic pathway of purine is catalyzed by PurD, which consumes an ATP molecule to produce glycinamide ribonucleotide (GAR) from glycine and phosphoribosylamine (PRA). PurD initially reacts with ATP to produce an intermediate, glycyl-phosphate, which then reacts with PRA to produce GAR. The structure of the glycyl-phosphate intermediate bound to PurD has not been determined. Therefore, the detailed reaction mechanism at the molecular level is unclear. Here, we developed a computational protocol to analyze the free-energy profile for the glycine phosphorylation process catalyzed by PurD, which examines the free-energy change along a minimum energy path based on a perturbation method combined with the quantum mechanics and molecular mechanics hybrid model. Further analysis revealed that during the formation of glycyl-phosphate, the partial atomic charge distribution within the substrate molecules was not localized according to the formal charges, but was delocalized overall, which contributed significantly to the interaction with the charged amino acid residues in the ATP-grasp domain of PurD.

Dynamic residue interaction network analysis of the oseltamivir binding site of N1 neuraminidase and its H274Y mutation site conferring drug resistance in influenza A virus.

BACKGROUND: Oseltamivir (OTV)-resistant influenza virus exhibits His-to-Tyr mutation at residue 274 (H274Y) in N1 neuraminidase (NA). However, the molecular mechanisms by which the H274Y mutation in NA reduces its binding affinity to OTV have not been fully elucidated. METHODS: In this study, we used dynamic residue interaction network (dRIN) analysis based on molecular dynamics simulation to investigate the correlation between the OTV binding site of NA and its H274Y mutation site. RESULTS: dRIN analysis revealed that the OTV binding site and H274Y mutation site of NA interact via the three interface residues connecting them. H274Y mutation significantly enhanced the interaction between residue 274 and the three interface residues in NA, thereby significantly decreasing the interaction between OTV and its surrounding loop 150 residues. Thus, we concluded that such changes in residue interactions could reduce the binding affinity of OTV to NA, resulting in drug resistant influenza viruses. Using dRIN analysis, we succeeded in understanding the characteristic changes in residue interactions due to H274Y mutation, which can elucidate the molecular mechanism of reduction in OTV binding affinity to influenza NA. Finally, the dRIN analysis used in this study can be widely applied to various systems such as individual proteins, protein-ligand complexes, and protein-protein complexes, to characterize the dynamic aspects of the interactions.

Path Ensembles for Pin1-Catalyzed Cis-Trans Isomerization of a Substrate Calculated by Weighted Ensemble Simulations.

Pin1 enzyme protein recognizes specifically phosphorylated serine/threonine (pSer/pThr) and catalyzes the slow interconversion of the peptidyl-prolyl bond between cis and trans forms. Structural dynamics between the cis and trans forms are essential to reveal the underlying molecular mechanism of the catalysis. In this study, we apply the weighted ensemble (WE) simulation method to obtain comprehensive path ensembles for the Pin1-catalyzed isomerization process. Associated rate constants for both cis-to-trans and trans-to-cis isomerization are calculated to be submicroseconds time scales, which are in good agreement with the calculated free energy landscape where the cis form is slightly less favorable. The committor-like analysis indicates the shift of the transition state toward trans form (at the isomerization angle ω ∼ 110°) compared to the intrinsic position for the isolated substrate (ω ∼ 90°). The calculated structural ensemble clarifies a role of both the dual-histidine motif, His59/His157, and the basic residues, Lys63/Arg68/Arg69, to anchor both sides of the peptidyl-prolyl bond, the aromatic ring in Pro, and the phosphate in pSer, respectively. The rotation of the torsion angle is found to be facilitated by relaying the hydrogen-bond partner of the main-chain oxygen in pSer from Cys113 in the cis form to Arg68 in the trans form, through Ser154 at the transition state, which is really the cause of the shift in the transition state. The role of Ser154 as a driving force of the isomerization is confirmed by additional WE and free energy calculations for S154A mutant where the isomerization takes place slightly slower and the free energy barrier increases through the mutation. The present study shows the usefulness of the WE simulation for substantial path samplings between the reactant and product states, unraveling the molecular mechanism of the enzyme catalysis.

Free energy profile analysis to identify factors activating the aggregation-induced emission of a cyanostilbene derivative.

An approach to quantitatively analyze the factors contributing to the activation of aggregation-induced emission (AIE) of a molecule is proposed using molecular simulations. A cyanostilbene derivative, 1-cyano-1,2-bis-(4'-methylbiphenyl)ethylene (CN-MBE), has two isomers, E and Z forms. The E-form of CN-MBE exhibits AIE, and is non-emissive in dilute solutions but becomes highly emissive in aggregated states. The Z-form is non-emissive, even in the solid state, that is, the E-form of CN-MBE is AIE-active, while its Z-form is AIE-inactive. In this study, the free energy profiles of the AIE processes of the E and Z forms of CN-MBE are investigated using the free energy perturbation method at the quantum mechanics/molecular mechanics level. The free energy profiles reveal significant differences in the extent to which steric hindrance from surrounding molecules restricts the intramolecular motions of the E and Z forms in the aggregated states. The structural features of the E and Z forms are characterized based on the conformational changes in the excited state relaxation process to reach the conical intersections and the free volume space around the molecules in the aggregated states. This study determines the contributing factors that cause the AIE activity of the molecule by identifying characteristic differences in the free energy profiles of the AIE processes of the AIE-active E-form of CN-MBE and the inactive Z-form. The approach used in this study can be applied to the rational design of highly efficient AIE luminogens utilizing computer modeling.

Free Energy Profile Analysis for the Aggregation-Induced Emission of Diphenyldibenzofulvene.

An improved understanding of the contributions of various factors to aggregation-induced emission (AIE) is required to advance the development and application of highly efficient AIE luminogens. Herein, the AIE of diphenyldibenzofulvene (DPDBF), which is nonemissive in dilute solutions but becomes highly emissive in aggregated states, was investigated using molecular simulations. Electronic structure calculations showed that the ground and first singlet excited states of DPDBF were degenerate after rotation of the ethylenic C═C bond, which results in fluorescence quenching via conical intersections (CIs). In this study, free energy (FE) profiles were used to quantify the extent to which the intramolecular motions of DPDBF required to reach the CIs were restricted in condensed phases. In acetonitrile solution, the FE profile showed that the ethylenic C═C bond of DPDBF could easily rotate to reach the CIs, which enabled nonradiative internal conversion. In contrast, in an aggregated state, the FE profile showed that the rotation around the ethylenic C═C bond of DPDBF was markedly restricted, thus preventing quenching through the CIs. These findings provide quantitative insights into the AIE mechanism of DPDBF.

Crystal structure of adenylate kinase from an extremophilic archaeon Aeropyrum pernix with ATP and AMP.

The crystal structure of an adenylate kinase from an extremophilic archaeon Aeropyrum pernix was determined in complex with full ligands, ATP-Mg2+ and AMP, at a resolution of 2.0 Å. The protein forms a trimer as found for other adenylate kinases from archaea. Interestingly, the reacting three atoms, two phosphorus and one oxygen atoms, were located almost in line, supporting the SN2 nucleophilic substitution reaction mechanism. Based on the crystal structure obtained, the reaction coordinate was estimated by the quantum mechanics calculations combined with molecular dynamics. It was found that the reaction undergoes two energy barriers; the steps for breaking the bond between the oxygen and γ-phosphorus atoms of ATP to produce a phosphoryl fragment and creating the bond between the phosphoryl fragment and the oxygen atom of the ß-phosphate group of ADP. The reaction coordinate analysis also suggested the role of amino-acid residues for the catalysis of adenylate kinase.

Draft Genome Sequence of Rhodococcus aetherivorans JCM 14343T, a Bacterium Capable of Degrading Recalcitrant Noncyclic and Cyclic Ethers.

Here, we report the draft genome sequence of Rhodococcus aetherivorans JCM 14343T, which possesses the versatile ability to degrade recalcitrant noncyclic and cyclic ether compounds. The 4.2-Mbp genome of this bacterium contains alkane hydroxylase and propane monooxygenase genes involved in the degradation of noncyclic and cyclic ethers, respectively.

Stimulatory and inhibitory effects of metals on 1,4-dioxane degradation by four different 1,4-dioxane-degrading bacteria.

This study evaluates the effects of various metals on 1,4-dioxane degradation by the following four bacteria: Pseudonocardia sp. D17; Pseudonocardia sp. N23; Mycobacterium sp. D6; and Rhodococcus aetherivorans JCM 14343. Eight transition metals [Co(II), Cu(II), Fe(II), Fe(III), Mn(II), Mo(VI), Ni(II), and Zn(II)] were used as the test metals. Results revealed, for the first time, that metals had not only inhibitory but also stimulatory effects on 1,4-dioxane biodegradation. Cu(II) had the most severe inhibitory effects on 1,4-dioxane degradation by all of the test strains, with significant inhibition at concentrations as low as 0.01-0.1 mg/L. This inhibition was probably caused by cellular toxicity at higher concentrations, and by inhibition of degradative enzymes at lower concentrations. In contrast, Fe(III) enhanced 1,4-dioxane degradation by Mycobacterium sp. D6 and R. aetherivorans JCM 14343 the most, while degradation by the two Pseudonocardia strains was stimulated most notably in the presence of Mn(II), even at concentrations as low as 0.001 mg/L. Enhanced degradation is likely caused by the stimulation of soluble di-iron monooxygenases (SDIMOs) involved in the initial oxidation of 1,4-dioxane. Differences in the stimulatory effects of the tested metals were likely associated with the particular SDIMO types in the test strains.

1,4-Dioxane degradation characteristics of Rhodococcus aetherivorans JCM 14343.

Rhodococcus aetherivorans JCM 14343 can degrade 1,4-dioxane as a sole carbon and energy source. This study aimed to characterize this 1,4-dioxane degradation ability further, and assess the potential use of the strain for 1,4-dioxane removal in industrial wastewater. Strain JCM 14343 was able to degrade 1,4-dioxane inducibly, and its 1,4-dioxane degradation was also induced by tetrahydrofuran and 1,4-butanediol. The demonstration that 1,4-butanediol not only induced but also enhanced 1,4-dioxane degradation was a novel finding of this study. Although strain JCM 14343 appeared not to be an effective 1,4-dioxane degrader considering the maximum specific 1,4-dioxane degradation rate (0.0073 mg-dioxane/mg-protein/h), half saturation concentration (59.2 mg/L), and cell yield (0.031 mg-protein/mg-1,4-dioxane), the strain could degrade over 1100 mg/L of 1,4-dioxane and maintain its degradation activity at a wide range of temperature (5-40 °C) and pH (4-9) conditions. This suggests the usefulness of strain JCM 14343 in 1,4-dioxane treatment under acidic and cold conditions. In addition, 1,4-dioxane degradation experiments in the presence of ethylene glycol (EG) or other cyclic ethers revealed that 1,4-dioxane degradation by strain JCM 14343 was inhibited in the presence of other cyclic ethers, but not by EG, suggesting certain applicability of strain JCM 14343 for industrial wastewater treatment.

Characterization of newly isolated Pseudonocardia sp. N23 with high 1,4-dioxane-degrading ability.

This study was conducted to elucidate the 1,4-dioxane degradation characteristics of a newly isolated 1,4-dioxane-degrading bacterial strain and evaluate the applicability of the strain to biological 1,4-dioxane removal from wastewater. A bacterial strain (designated strain N23) capable of degrading 1,4-dioxane as the sole carbon and energy source was isolated from an enrichment culture prepared from 1,4-dioxane-contaminated groundwater. Strain N23 was phylogenetically identified as belonging to the genus Pseudonocardia, based on 16S rRNA gene sequencing. 1,4-Dioxane degradation experiments revealed that strain N23 is capable of constitutive 1,4-dioxane degradation. Further, this strain exhibited the highest specific 1,4-dioxane degradation rate of 0.230 mg-1,4-dioxane (mg-protein)-1 h-1 among 1,4-dioxane-degrading bacteria with constitutively expressed degrading enzymes reported to date. In addition, strain N23 was shown to degrade up to 1100 mg L-1 of 1,4-dioxane without significant inhibition, and to maintain a high level of 1,4-dioxane degradation activity under a wide pH (pH 3.8-8.2) and temperature (20-35 °C) range. In particular, the specific 1,4-dioxane degradation rate, even at pH 3.8, was 83% of the highest rate at pH 7.0. In addition, strain N23 was capable of utilizing ethylene glycol and diethylene glycol, which are both considered to be present in 1,4-dioxane-containing industrial wastewater, as the sole carbon source. The present results indicate that strain N23 exhibits the potential for 1,4-dioxane removal from industrial wastewater.

Draft Genome Sequence of Pseudonocardia sp. Strain N23, a 1,4-Dioxane-Degrading Bacterium.

Pseudonocardia sp. strain N23 is a 1,4-dioxane-degrading bacterium that is capable of utilizing 1,4-dioxane as the sole carbon and energy source. Here, we report the draft genome sequence of strain N23, with a size of 6.5 Mbp, to identify the genes associated with 1,4-dioxane degradation.

Excited-state intramolecular proton transfer and conformational relaxation in 4'-N,N-dimethylamino-3-hydroxyflavone doped in acetonitrile crystals.

The effect of intermolecular interactions on excited-state intramolecular proton transfer (ESIPT) in 4'-N,N-dimethylamino-3-hydroxyflavone (DMHF) doped in acetonitrile crystals was investigated by measuring its temperature dependence of steady-state fluorescence excitation and fluorescence spectra and picosecond time-resolved spectra. The relative intensity of emission from the excited state of the normal form (N*) to that from the excited state of the tautomer form (T*) and spectral features changed markedly with temperature. Unusual changes in the spectral shift and spectral features were observed in the fluorescence spectra measured between 200 and 218 K, indicating that a solid-solid phase transition of DMHF-doped acetonitrile crystals occurred. Time-resolved fluorescence spectra suggested conformational relaxation of the N* state competed with ESIPT after photoexcitation and the ESIPT rate increased with temperature in the low-temperature phase of acetonitrile. However, the intermolecular interaction of N* with acetonitrile in the high-temperature phase markedly stabilized the potential minimum of the fluorescent N* state and slowed the ESIPT. This stabilization can be explained by reorganization of acetonitrile originating from the strong electric dipole-dipole interaction between DMHF and acetonitrile molecules.

1,4-Dioxane degradation potential of members of the genera Pseudonocardia and Rhodococcus.

In recent years, several strains capable of degrading 1,4-dioxane have been isolated from the genera Pseudonocardia and Rhodococcus. This study was conducted to evaluate the 1,4-dioxane degradation potential of phylogenetically diverse strains in these genera. The abilities to degrade 1,4-dioxane as a sole carbon and energy source and co-metabolically with tetrahydrofuran (THF) were evaluated for 13 Pseudonocardia and 12 Rhodococcus species. Pseudonocardia dioxanivorans JCM 13855T, which is a 1,4-dioxane degrading bacterium also known as P. dioxanivorans CB1190, and Rhodococcus aetherivorans JCM 14343T could degrade 1,4-dioxane as the sole carbon and energy source. In addition to these two strains, ten Pseudonocardia strains could degrade THF, but no Rhodococcus strains could degrade THF. Of the ten Pseudonocardia strains, Pseudonocardia acacia JCM 16707T and Pseudonocardia asaccharolytica JCM 10410T degraded 1,4-dioxane co-metabolically with THF. These results indicated that 1,4-dioxane degradation potential, including degradation for growth and by co-metabolism with THF, is possessed by selected strains of Pseudonocardia and Rhodococcus, although THF degradation potential appeared to be widely distributed in Pseudonocardia. Analysis of soluble di-iron monooxygenase (SDIMO) α-subunit genes in THF and/or 1,4-dioxane degrading strains revealed that not only THF and 1,4-dioxane monooxygenases but also propane monooxygenase-like SDIMOs can be involved in 1,4-dioxane degradation.

External Electric Field Effects on Excited-State Intramolecular Proton Transfer in 4'-N,N-Dimethylamino-3-hydroxyflavone in Poly(methyl methacrylate) Films.

The external electric field effects on the steady-state electronic spectra and excited-state dynamics were investigated for 4'-N,N-(dimethylamino)-3-hydroxyflavone (DMHF) in a poly(methyl methacrylate) (PMMA) film. In the steady-state spectrum, dual emission was observed from the excited states of the normal (N*) and tautomer (T*) forms. Application of an external electric field of 1.0 MV·cm(-1) enhanced the N* emission and reduced the T* emission, indicating that the external electric field suppressed the excited-state intramolecular proton transfer (ESIPT). The fluorescence decay profiles were measured for the N* and T* forms. The change in the emission intensity ratio N*/T* induced by the external electric field is dominated by ESIPT from the Franck-Condon excited state of the N* form and vibrational cooling in potential wells of the N* and T* forms occurring within tens of picoseconds. Three manifolds of fluorescent states were identified for both the N* and T* forms. The excited-state dynamics of DMHF in PMMA films has been found to be very different from that in solution due to intermolecular interactions in a rigid environment.

Hot spot of structural ambivalence in prion protein revealed by secondary structure principal component analysis.

The conformational conversion of proteins into an aggregation-prone form is a common feature of various neurodegenerative disorders including Alzheimer's, Huntington's, Parkinson's, and prion diseases. In the early stage of prion diseases, secondary structure conversion in prion protein (PrP) causing ß-sheet expansion facilitates the formation of a pathogenic isoform with a high content of ß-sheets and strong aggregation tendency to form amyloid fibrils. Herein, we propose a straightforward method to extract essential information regarding the secondary structure conversion of proteins from molecular simulations, named secondary structure principal component analysis (SSPCA). The definite existence of a PrP isoform with an increased ß-sheet structure was confirmed in a free-energy landscape constructed by mapping protein structural data into a reduced space according to the principal components determined by the SSPCA. We suggest a "spot" of structural ambivalence in PrP-the C-terminal part of helix 2-that lacks a strong intrinsic secondary structure, thus promoting a partial α-helix-to-ß-sheet conversion. This result is important to understand how the pathogenic conformational conversion of PrP is initiated in prion diseases. The SSPCA has great potential to solve various challenges in studying highly flexible molecular systems, such as intrinsically disordered proteins, structurally ambivalent peptides, and chameleon sequences.

Ferryl-oxo species produced from Fenton's reagent via a two-step pathway: minimum free-energy path analysis.

A mixture of ferrous ions and hydrogen peroxide, known as Fenton's reagent, is an effective oxidant and has been widely used in various industrial applications; however, there is still controversy about what the oxidizing agents are and how they are produced. In this study, we have determined minimum free-energy paths (MFEPs) from Fenton's reagent to possible oxidizing agents such as hydroxyl radicals and ferryl-oxo species by combining ab initio molecular dynamics simulations and an MFEP search method. Along the MFEPs, representative free-energy profiles of the Fenton reaction were elucidated. On the basis of the free-energy profiles, we revealed that the reaction producing ferryl-oxo species from Fenton's reagent is more energetically favorable than that yielding a free hydroxyl radical, by 24.4 kcal mol(-1), which indicates that the ferryl-oxo species is the primary oxidizing agent in reactions of Fenton's reagent. Moreover, we clarified that the ferryl-oxo species is favorably formed via a two-step reaction pathway, which reaches the product through a dihydroxyiron(IV) intermediate. The energetics charting the free-energy profiles provided valuable information for a comprehensive understanding of Fenton reactions. We concluded that a ferryl-oxo species produced from Fenton's reagent serves as the primary oxidizing agent in the Fenton reaction.

Excited-state intramolecular proton transfer and charge transfer in 2-(2'-hydroxyphenyl)benzimidazole crystals studied by polymorphs-selected electronic spectroscopy.

Crystal structures of polymorphs of 2-(2'-hydroxyphenyl)benzimidazole (HPBI), Forms α and ß, are analyzed by X-ray crystallography. The fluorescence excitation (FE) and fluorescence spectra of the polymorphs are separately observed at temperatures 77-298 K. It has been found that the electronic spectra of the two crystal forms are significantly different from each other. Photo-excitation of the enol forms in Forms α and ß induces the excited-state intramolecular proton transfer (ESIPT) to produce the S(1) state of the keto forms. In the FE spectra of Forms α and ß, the S(1) ← S(0) (ππ*) transition of the keto form is observed in the 360-420 nm region in addition to that of the enol form in the 250-420 nm region. In the FE spectrum of Form ß a new band peaking at 305 nm is observed, which is assigned to the S(1) ← S(0) transition of a non-planar enol form based on the observation of dual fluorescence in the UV and visible regions and quantum chemical calculation on the transition energy against the twisted angle between the benzimidazole and hydroxyphenyl rings. The fluorescence quantum yield (φ(T)) for the keto form is remarkably dependent on polymorphs at room temperature; φ(T) = 0.53 for Form α is much larger than φ(T) ≤ 0.23 for Form ß. At 77 K the φ(T) values for Forms α and ß increase to 0.67 and ≤0.57, respectively. The changes in the φ(T) values are associated with the intramolecular charge transfer (ICT) state. The potential barrier height between the S(1)-keto and S(1)-ICT states is significantly lower for Form ß than for Form α. At 77 K the S(1)-keto → S(1)-ICT process followed by S(1)-ICT → S(0)-keto internal conversion is significantly suppressed in Form ß. We compare difference in the dynamics between Forms α and ß in the electronic ground and excited states.

Regulating the conformation of prion protein through ligand binding.

Although some antiprion compounds have been shown to interfere with the pathological conversion of prion protein into a misfolded isoform, the actual mechanism has not been elucidated. In this study, we compared different conformations of prion protein with and without ligand binding, based on molecular dynamics simulations, to clarify the role of a typical antiprion compound termed GN8. In our approach, urea-driven unfolding simulations were employed to assay whether or not GN8 prevents denaturation of prion protein. We found that urea mediates partial unfolding at helix B of the prion protein, suggesting a transition into the intermediate states of the pathological conversion. However, GN8 efficiently suppressed local fluctuations by binding to flexible spots on helix B and prevented its urea-induced denaturation. We conclude that GN8 inhibits pathological conversion by suppressing the level of the intermediate. This is the first evidence supporting the chemical chaperone hypothesis, which states that GN8 acts as a chaperone to stabilize the normal form of the prion protein. Our basic principle constitutes a promising strategy for a dynamics-based drug design of therapeutic compounds, particularly for prion diseases and other diseases related to protein misfolding.