T-shaped Interactions Research Articles

Neurotransmitter metabolites in milk ferments of Leuconostoc mesenteroides regulate temperature-sensitive heartbeats in an ex ovo model

Accumulated evidence has supported the probiotic activity of Leuconostoc mesenteroides (L. mesenteroides) which can yield beneficial metabolites via fermentation. Here, bovine milk rich in phenylalanine（PHE) was used as a source for fermentation of L. mesenteroides. The complexes of PHE with bacterial phenylalanine hydroxylase (PheH) at two temperatures were revealed via molecular dynamics simulation. Two carbon hydrogen bonds and a Pi-Alkyl T-shaped interaction were newly formed at an active site of the PheH-PHE complex. The PheH interacted with two different hydrogen atoms in an amine of PHE via conventional hydrogen bonds at 37 °C, a temperature that accelerated the milk fermentation of L. mesenteroides. Twenty-eight metabolites including various neurotransmitters in fermented milk were identified and quantified by liquid chromatography coupled to quadrupole ion trap (Q-Trap) tandem mass spectrometry. Ex ovo injection of milk ferments into the yolk sac of chicken embryos enhanced a rising temperature-induced increase in heartbeats towards the normal resting level. The neurotransmitter-rich milk ferments hold potential for using to adjust energy metabolism, referred from heart rates, during fluctuating temperature conditions.

Integration of High-Resolution LC-Q-TOF Mass Spectrometry and Multidimensional Chemical-Biological Analysis to Detect Nanomolar-Level Acetylcholinesterase Inhibitors from Different Parts of Zanthoxylum nitidum.

Zanthoxyli radix is a popular tea among the elderly, and it is believed to have a positive effect on Alzheimer's disease. In this study, a highly effective three-step strategy was proposed for comprehensive analysis of the active components and biological functions of Zanthoxylum nitidum (ZN), including high-resolution LC-Q-TOF mass spectrometry (HRMS), multivariate statistical analysis for heterogeneity (MSAH), and experimental and virtual screening for bioactivity analysis (EVBA). A total of 117 compounds were identified from the root, stem, and leaf of ZN through HRMS. Bioactivity assays showed that the order of acetylcholinesterase (AChE) inhibitory activity from strong to weak was root > stem > leaf. Nitidine, chelerythrine, and sanguinarine were found to be the main differential components of root, stem, and leaf by OPLS-DA. The IC50 values of the three compounds are 0.81 ± 0.02, 0.14 ± 0.01, and 0.48 ± 0.01 μM respectively, indicating that they are potent and high-quality AChE inhibitors. Molecular docking showed that pi-pi T-shaped interactions and pi-lone pairs played important roles in AChE inhibition. This study not only explains the biological function of Zanthoxyli radix in alleviating Alzheimer's disease to some extent, but also lays the foundation for the development of stem and leaf of ZN.

Functional analysis of three odorant receptors in Plutella xylostella response to repellent activity of 2,3-dimethyl-6-(1-hydroxy)-pyrazine

Plutella xylostella is an important pest showing resistance to various chemical pesticides, development of botanical pesticides is an effective strategy to resolve above problem and decrease utilization of chemical pesticides. Previous study showed that 2,3-dimethyl-6-(1-hydroxy)-pyrazine has significant repellent activity to P. xylostella adult which mainly effect to the olfactory system, however the molecular targets and mechanism are still unclear. Based on the RNA-Seq and RT-qPCR data, eight ORs (Odorant receptor) in P. xylostella were selected as candidate targets response to repellent activity of 2,3-dimethyl-6-(1-hydroxy)-pyrazine. Here, most of the ORs in P. xylostella were clustered into three branches, which showed similar functions such as recognition, feeding, and oviposition. PxylOR29, PxylOR31, and PxylOR46 were identified as the potential molecular targets based on the results of repellent activity and EAG response tests to the adults which have been injected with dsRNA, respectively. Additionally, the three ORs were higher expressed in antenna of P. xylostella, followed by those in the head segment. Furthermore, it was found that the bindings between these three ORs and 2,3-dimethyl-6-(1-hydroxy)-pyrazine mainly depend on the hydrophobic effect of active cavities, and the binding to PxylOR31 was more stabler and easier with an energy of −16.34 kcal/mol, together with the π-π T-shaped interaction at PHE195 site. These findings pave the way for the complete understanding of pyrazine repellent mechanisms.

Ultrasound-assisted extraction of emodin from Rheum officinale Baill and its antibacterial mechanism against Streptococcus suis based on CcpA

Emodin was extracted from Rheum officinale Baill by ultrasound-assisted extraction (UAE), and ethanol was chosen as the suitable solvent through SEM and molecular dynamic simulation. Under the optimum conditions (power 541 W, time 23 min, liquid to material ratio 13:1 mL/g, ethanol concentration 83 %) predicted by RSM, the yield of emodin was 2.18 ± 0.11 mg/g. Moreover, ultrasound power and time displayed the significant effects on the extraction process. Extracting dynamics analysis indicated that the extraction process of emodin by UAE conformed to Fick's second diffusion law. The results of antibacterial experiments suggested that emodin can damage cell membrane and inhibit the expression of cps2A, sao, mrp, epf, neu and the hemolytic activity of S. suis. Biolayer interferometry and FT-IR multi-peak fitting assays demonstrated that emodin induced a secondary conformational shift in CcpA. Molecular docking and molecular dynamics confirmed that emodin bound to CcpA through hydrogen bonding (ALA248, GLU249, GLY129 and ASN196) and π-π T-shaped interaction (TYR225 and TYR130), and the mutation of amino acid residues affected the affinity of CcpA to emodin. Therefore, emodin inhibited the sugar utilization of S. suis through binding to CcpA, and CcpA may be a potential target to inhibit the growth of S. suis.

Investigation of the removal kinetics, thermodynamics and adsorption mechanism of anionic textile dye, Remazol Red RB, with powder pumice, a sustainable adsorbent from waste water.

Excessive growth and abnormal use of dyes and water in the textile industry cause serious environmental problems, especially with excessive pollution of water bodies. Adsorption is an attractive, feasible, low-cost, highly efficient and sustainable technique in terms of green chemistry for the removal of pollutants from water. This study aims to investigate the removal kinetics, thermodynamics and adsorption mechanism of Remazol Red RB, which was chosen as a representative anionic reactive dye, from synthetic wastewater using powdered pumice, taking into account various experimental parameters such as initial dye concentration, adsorption time, temperature and pH. Moreover, to support the proposed adsorption mechanism, before and after adsorption of the samples, the Fourier transform infrared spectrophotometer (FTIR) spectra, X-ray powder diffraction (XRD) diffractograms and High resolution transmission electron microscopy (HRTEM) images were also taken and used. The results show that powder pumice can be an efficient adsorbent for anionic dye removal with a relatively high adsorption capacity of 38.90mg/g, and it is very effective in 30-60min in mild conditions. The experimental data showed a high agreement with the pseudo-second-order kinetic model and the Freundlich adsorption isotherm equation. In addition, thermodynamically, the process exhibited exothermic nature and standard isosteric enthalpy and entropy changes of -4.93kJ/mol and 16.11J/mol. K were calculated. It was determined that the adsorption mechanism was predominantly based on T-shaped pi-pi interactions and had physical characteristics.

Occurrence and classification of T-shaped interactions between nucleobases in RNA structures.

Understanding the frequency and structural context of discrete noncovalent interactions between nucleotides is of pivotal significance in establishing the rules that govern RNA structure and dynamics. Although T-shaped contacts (i.e., perpendicular stacking contacts) between aromatic amino acids and nucleobases at the nucleic acid-protein interface have recently garnered attention, the analogous contacts within the nucleic acid structures have not been discussed. In this work, we have developed an automated method for identifying and unambiguously classifying T-shaped interactions between nucleobases. Using this method, we identified a total of 3261 instances of T-shaped (perpendicular stacking) contacts between two nucleobases in an array of RNA structures from an up-to-date data set of ≤3.5 Å resolution crystal structures deposited in the Protein Data Bank.

Demethyleneberberine alleviated the inflammatory response by targeting MD-2 to inhibit the TLR4 signaling.

The colitis induced by trinitrobenzenesulfonic acid (TNBS) is a chronic and systemic inflammatory disease that leads to intestinal barrier dysfunction and autoimmunedisorders. However, the existing treatments of colitis are associated with poor outcomes, and the current strategies remain deep and long-time remission and the prevention of complications. Recently, demethyleneberberine (DMB) has been reported to be a potential candidate for the treatment of inflammatory response that relied on multiple pharmacological activities, including anti-oxidation and antiinflammation. However, the target and potential mechanism of DMB in inflammatory response have not been fully elucidated. This study employed a TNBS-induced colitis model and acute sepsis mice to screen and identify the potential targets and molecular mechanisms of DMB in vitro and in vivo. The purity and structure of DMB were quantitatively analyzed by high-performance liquid chromatography (HPLC), mass spectrometry (MS), Hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), and infrared spectroscopy (IR), respectively. The rats were induced by a rubber hose inserted approximately 8 cm through their anus to be injected with TNBS. Acute sepsis was induced by injection with LPS via the tail vein for 60 h. These animals with inflammation were orally administrated with DMB, berberine (BBR), or curcumin (Curc), respectively. The eukaryotic and prokaryotic expression system of myeloid differentiation protein-2 (MD-2) and its mutants were used to evaluate the target of DMB in inflammatory response. DMB had two free phenolic hydroxyl groups, and the purity exceeded 99% in HPLC. DMB alleviated colitis and suppressed the activation of TLR4 signaling in TNBS-induced colitis rats and LPS-induced RAW264.7 cells. DMB significantly blocked TLR4 signaling in both an MyD88-dependent and an MyD88-independent manner by embedding into the hydrophobic pocket of the MD-2 protein with non-covalent bonding to phenylalanine at position 76 in a pi-pi T-shaped interaction. DMB rescued mice from sepsis shock induced by LPS through targeting the TLR4-MD-2 complex. Taken together, DMB is a promising inhibitor of the MD-2 protein to suppress the hyperactivated TLR4 signaling in inflammatory response.

Molecular docking and ADMET studies of the interaction of 4-carboxyl-2,6-dinitrophenylazohydroxynaphthalenes with bovine serum albumin

Previous spectrophotometric investigations revealed strong binding affinities between four potential monoazo colourants (code-named AZ-01 to 04) and bovine serum albumin (BSA) which could dictate the tissue distribution and toxicity of the additives. The molecular docking interactions of the dyes with BSA were analyzed using AutoDock vina and PatchDock in order to elucidate the functional groups involved in complex stabilization. Docking conformations confirmed the ligands preferentially inserted into the hydrophobic cavities of BSA site I. Structure-BSA binding relationships revealed the binding of AZ-02 was driven by hydrogen bond donation from its free phydroxynaphthalene substituent to Ser-479 while the predominantly hydrazone form of its positional isomer, AZ-01, increased its lipophilicity and tendency for hydrophobic interactions. The relatively higher C/H ratio of AZ-03 and - 04, which contain additional C-7 substituents, was responsible for their stronger binding and the extensive involvement of their aromatic rings in ligand-site I complex stabilization via Pi-Pi T-shaped, Pi-alkyl and alkyl-alkyl interactions. Moreso, AZ-01, -03 and -04 exist predominantly as hydrazone tautomers with an overall positive charge which provided complementary modes for interaction with negatively charged aspartic and glutamic acids. The structure-BSA binding relationships of the molecules, which can be employed in synthesis of safer congeners, have been elucidated.

A high-resolution α-glucosidase inhibition profiling for targeted identification of natural antidiabetic products from Lycopodiella cernua (L.) Pic. Serm and their inhibitory mechanism study

The targeted identification of α-glucosidase inhibitors from the crude ethyl acetate of Lycopodiella cernua (L.) Pic. Serm (L.cernua) was guided by high-resolution inhibition profiling. The α-glucosidase inhibition profiling and HPLC-QTOF-MS showed tannins and serratenes were the corresponding antidiabetic constituents. Two new serratenes named 3β, 21β-dihydroxyserra-14-en-24-oic acid-3β-(4'-methoxy-5'-hydroxybenzoate) (4), 3β, 21α-dihydroxyserra-14-en-24-oic acid-3β-(4'-methoxy-5'-hydroxybenzoate) (7), together with two known compounds (5 and 6) were isolated. Their structures were elucidated by HR-ESI-MS and NMR. Compounds 5–7 inhibited the α-glucosidase activity in a non-competitive manner with Ki values ranging from 1.29 to 12.9 µM. The molecular docking result unveiled that 4–7 bound to the residues at the channel site, which enabled to block the substrate access. In addition, the molecular dynamics (MD) simulation of the most active compound 7 and α-glucosidase indicated the 4′-methoxy-5′-hydroxybenzoate group formed the stable hydrogen bonds and pi-pi T-shaped interactions with Arg312, Gln350 and Phe300 residues, while the rings D and E were stabilized by hydrophobic interaction.

Biosynthetic incorporation of fluorinated amino acids into the nonribosomal peptide gramicidin S.

Fluorine is a key element in medicinal chemistry, as it can significantly enhance the pharmacological properties of drugs. In this study, we aimed to biosynthetically produce fluorinated analogues of the antimicrobial cyclic decapeptide gramicidin S (GS). However, our results show that the A-domain of the NRPS module GrsA rejects 4-fluorinated analogues of its native substrate Phe due to an interrupted T-shaped aromatic interaction in the binding pocket. We demonstrate that GrsA mutant W239S improves the incorporation of 4-fluorinated Phe into GS both in vitro and in vivo. Our findings provide new insights into the behavior of NRPSs towards fluorinated amino acids and strategies for the engineered biosynthesis of fluorinated peptides.

Apigenin attenuates depressive-like behavior via modulating monoamine oxidase A enzyme activity in chronically stressed mice

Chronic stress is a risk factor for depression and is characterized by elevated levels of brain monoamine oxidase A (MAOA). Mounting evidence has shown that MAOA is a biochemical link between stress and depression. Apigenin (API), a natural flavonoid, as demonstrated in vitro inhibitory effect on MAOA, is suggestive of antidepressant-like activity. However, the in vivo inhibitory effect of API on MAOA and how it affects depression still remain unclear. Here, we report the probable mechanisms of action of API in chronic unpredictable mild stress (CUMS)-induced depression in mice. Treatment with API reversed anhedonia, and reduced anxiety and immobility time in behavioral studies. API reduced brain corticosterone and malondialdehyde (MDA) levels but increased brain levels of glutathione and superoxide dismutase. Furthermore, interleukin-6 and tumor necrosis factor-α were attenuated by API. It also restored cell loss and inhibited the activity of MAOA in the hippocampal brain regions and prefrontal cortex. Comparative binding affinity of API for MAOA (-7.7 kcal/mol) through molecular docking studies was greater than that of reference compound, clorgyline (-6.8 kcal/mol). Favorable hydrophobic interactions important to API binding at MAOA binding cavity was revealed to include conventional hydrogen bond (Cys323 and Tyr444), π-Sulfur (Cys323), π-π Stacked (Tyr407), π-π T-shaped (Phe208), π-lone pair and π-alkyl (Ile335, Ile180) interactions. These results suggest that API is a potent, selective, reversible inhibitor of MAOA with capability of attenuating CUMS-induced depression via inhibiting MAOA enzyme activity and altering other pathomechanisms.

Insighting the inhibitory potential of novel modafinil drug derivatives against estrogen alpha (ERα) of breast cancer through a triple hybrid computational methodology

Breast cancer (Bca) is the prominent, most commonly detected, and the leading cause of mortality among women. Estrogen receptor alpha (ERα) is considered an important receptor for the proliferation of this disease and its blockage is necessary for the treatment of Bca. The purpose of the current study is to design novel potential inhibitors against ERα of Bca. We designed modafinil drug derivatives using quantum chemical methods. These newly designed derivatives were put under an in-silico investigation followed by molecular docking simulation, molecular dynamics simulation, and MMPBSA analysis to find novel inhibitors of ERα. Moreover, three reference anticancer drugs; tamoxifen, raloxifene, and toremifene are also studied against ERα of Bca. The spectroscopic and structural features of sulfoxide-based designed derivatives of modafinil drug M1((R)-2-(banzhydrylsulfinyl-N,N diethylacetamide) have been evaluated using different quantum chemical analyses. The findings of the current investigation demonstrate that all studied ligands exhibit the binding energy ranges (-5.3 to −5.8 Kcal/mol). The designed compounds showed effective hydrophobic (alkyl, π-alkyl, π-sigma, π-amide stacked, π-π T-shaped) interactions and hydrogen bond formation and are anticipated to be potential inhibitors against ERα. Additionally, designed derivatives have a good ADMET (absorption, distribution, metabolism, excretion, toxicity) profile and drug-likeness properties obeying RO5 without any toxicity. The stability profile of designed derivatives (M1-M6) was further validated by molecular dynamics (MD) simulation and calculate binding free energy by the MM-PBSA approach. All ligand–protein complexes showed structural stability over the 120 ns MD simulation time. The MD simulation of the complex system was carried out by RMSD (root-mean-square deviation) of C α atoms of ERα, RMSF (root mean square fluctuations), Rg (radius of gyration), SASA (solvent accessible surface area), and dynamic behavior of hydrogen bonds. The MD simulation results illustrate that RMSD for trajectories of designed derivative complexes over 120 ns are within the acceptable deviation range of ∼ 3 Å. The calculations of net binding free energy (ΔGbind) between the designed derivatives and their complexes are found to be −8.50 Kcal/mol (M1) at maximum and −5.197 Kcal/mol (M3) at a minimum among all derivatives. The outcomes of our current in-silico investigation will evoke the scientific community to carry out further in vivo and in vitro studies on designed modafinil derivatives that can be potential therapeutic drug candidates against ERα.

Antioxidant and α-Glucosidase Inhibitory Activities Guided Isolation and Identification of Components from Mango Seed Kernel.

In the present study, petroleum ether, dichloromethane, ethyl acetate, and n-butanol fractions of mango seed kernel exhibited different degrees of antioxidant and α-glucosidase inhibitory activity. Thus, quantitative and qualitative analysis of the petroleum ether fraction was conducted by GC-MS. Among identified components, four unsaturated fatty acids had never been reported in natural products before, together with 19 known components. In addition, 17 compounds were isolated and elucidated from other active fractions. Compounds 2, 9, 15, and 17 were isolated for the first time from Mangifera genus. Compounds 1 and 2 exhibited prominent DPPH radical scavenging and α-glucosidase inhibitory effects. In order to further explore their mechanism of α-glucosidase inhibition, their enzyme kinetics and in silico modeling experiments were performed. The results indicated that 1 inhibited α-glucosidase in a noncompetitive manner, whereas 2 acted in a competitive manner. In molecular docking, the stability of binding was enhanced by π-π T-shaped, π-alkyl, π-π stacked, hydrogen bond, and electrostatic interactions. Thus, compounds 1 and 2 were determined to be new potent antioxidant and α-glucosidase inhibitors for preventing food oxidation and enhancing hypoglycemic activity.

Engineering ω-transaminase by random mutagenesis and semi-rational design for the synthesis of (R)-(+)-1-(1-naphthyl)ethylamine

(R)-(+)-1-(1-naphthyl)ethylamine is a key chiral intermediate for the synthesis of calcimimetic drug cinacalcet hydrochloride. ω-Transaminase has been considered to be potential for producing (R)-(+)-1-(1-naphthyl)ethylamine by asymmetric reduction of 1-acetonaphthone. Here, ω-transaminase from Arthrobacter sp. was engineered by combinatorial strategies of random mutagenesis and semi-rational design. Variants F225M, C281I, F225M/C281I with improved catalytic efficiency and thermostability were obtained. Compared with WT, variant F225M/C281I showed 85% increased kcat, 56% decreased Km and 3.42-fold kcat/Km. Furthermore, 22% higher conversion rate was achieved by F225M/C281I at 10 mmol/L 1-acetonaphthone after 24 h. Based on molecular docking and molecular dynamics simulation, improved catalytic efficiency of F225M/C281I could be attributed to its increased Pi-Pi T-shaped interaction with substrate 1-acetonaphthone. Additionally, a slightly higher half-life of F225M/C281I was validated by its lower root-mean-square fluctuation (RMSF) value of loop 134-139 compared with WT.

Molecular dynamics and spectral analysis for the binding of methoxylated polybrominated diphenyl ethers to lysozyme

The interaction between three methoxylated polybrominated diphenyl ethers (MeO-PBDEs) and lysozyme was investigated using two complementary techniques: computational simulations and multi-spectroscopic methods. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method was used to determine the relative binding energies. Molecular modeling analysis showed van der Waals and hydrogen bonding forces as the major forces responsible for interactions between compounds and Lysozymes. The binding site for 2′-MeO-BDE-3 and 5-MeO-BDE-47 in lysozyme was determined to be at the active pocket, where these ligands bound strongly with lysozyme tryptophan (TRP) residues TRP62, TRP63 and GLU35. In the 3-MeO-BDE-100−lysozyme system, F-shaped and T-shaped interactions between ligand and TRP62 (TRP108) were present, which suggested the stability of the combination. Fluorescence lifetime measurements suggested that be interactions between MeO-PBDEs and lysozyme could be present, and the microenvironment around the TRP residues was slightly altered during the binding process. Circular dichroism (CD) spectroscopy and UV–vis absorption spectra revealed interactions between lysozyme and MeO-PBDEs, thus inducing changes in the lysozyme microenvironment and an increase in its content of α-helix structures.

Formation of Al + (C 6 H 6 ) 13 : The Origin of Magic Number in Metal–Benzene Clusters Determined by the Nature of the Core

The identification of highly abundant, “magic” species in the mass spectra of clusters have proven to be valuable in nanoscience, leading to the discovery of new stable species such as fullerenes a...

Hydantoin and Its Derivatives Reduce the Viscosity of Concentrated Antibody Formulations by Inhibiting Associations via Hydrophobic Amino Acid Residues

Therapeutic antibodies for subcutaneous (SC) injection must be formulated at high concentrations because of the large therapeutic dose and volume restriction. However, concentrated antibody solutions have undesirably high viscosity, which hampers SC injection. In this study, we demonstrated that hydantoin and its derivatives suppressed the viscosity of concentrated antibody and bovine serum albumin solutions. Hydantoin derivatives, in particular 1-methylhydantoin, appeared more effective. Both hydantoin and 1-methylhydantoin suppressed the viscosity of proteins more effectively when combined with a physiological concentration of NaCl. Moreover, hydantoin rings exhibited thermodynamically favorable interactions with hydrophobic amino acids, as demonstrated using solubility measurements. Molecular dynamics simulations indicated planar stacking interaction or T-shaped interaction between the hydantoin ring structure and the aromatic rings of tryptophan. Thus, the effects of hydantoin compounds in the presence of NaCl on the high viscosity of concentrated protein solutions result from the combined effects between hydantoin and NaCl in suppressing multiple interactions (electrostatic, hydrophobic, π–π, and cation−π interactions) between protein molecules. The obtained data here should be useful for developing therapeutic antibody formulations.

Eckol as a Potential Therapeutic against Neurodegenerative Diseases Targeting Dopamine D₃/D₄ Receptors.

The G protein-coupled receptor (GPCR) family of proteins comprises signaling proteins that mediate cellular responses to various hormones and neurotransmitters, and serves as a prime target for drug discovery. Towards our goal of discovering secondary metabolites from natural sources that can function as neuronal drugs, we evaluated the modulatory effect of eckol on various GPCRs via cell-based functional assays. In addition, we conducted in silico predictions to obtain molecular insights into the functional effects of eckol. Functional assays revealed that eckol had a concentration-dependent agonist effect on dopamine D3 and D4 receptors. The half maximal effective concentration (EC50) of eckol for the dopamine D3 and D4 receptors was 48.62 ± 3.21 and 42.55 ± 2.54 µM, respectively, while the EC50 values of dopamine as a reference agonist for these two receptors were 2.9 and 3.3 nM, respectively. In silico studies revealed that a low binding energy in addition to hydrophilic, hydrophobic, π–alkyl, and π–π T-shaped interactions are potential mechanisms by which eckol binds to the dopamine receptors to exert its agonist effects. Molecular dynamics (MD) simulation revealed that Phe346 of the dopamine receptors is important for binding of eckol, similar to eticlopride and dopamine. Our results collectively suggest that eckol is a potential D3/D4 agonist for the management of neurodegenerative diseases, such as Parkinson’s disease.

Adsorption and structure of benzene, toluene, and p-xylene in carbon slit pores: A Monte Carlo simulation study

The adsorption of benzene (B), toluene (T), and p-xylene (X) at ambient temperature in carbon slit pores was studied using the grand canonical Monte Carlo simulation. We focused on how the structure of the adsorbed B, T, and X molecules is influenced by pore size, in particular the effects of the methyl groups in the cases of toluene and p-xylene. The packing density of the compounds in the pores was found to follow the order B < T < X at low loadings but X < T < B at high loadings. Furthermore, various molecular orientations were observed in the different pore sizes, including parallel, perpendicular, and parallel combined with perpendicular, and the nearest neighboring molecules assumed to undergo an edge-wise or T-shaped interaction. For parallel molecules, two adjacent molecules were aligned in an edge-wise interaction, which is the most favorable for solid–fluid interactions. However, for perpendicular molecules, two adjacent molecules were arranged in a T-shape, which is more favorable for fluid-fluid interactions. For the perpendicular toluene and p-xylene molecules, the angle between the methyl group and the pore walls was found to be 30°. In the smallest pore studied, the benzene molecules were found to adopt a 2D hexagonally packed solid-like state, whereas the toluene and p-xylene molecules were in a liquid-like state. Consequently, the microscopic structures of B, T, X in various sized pores were investigated. The molecular models showed that the fluid-fluid interactions were significant for B, T, X adsorption, except for the cases of toluene and p-xylene in pores that can accommodate more than one molecular layer, where the solid–fluid interactions play a more dominant role because of the enhanced solid–fluid interactions of the toluene and p-xylene molecules oriented in parallel to each other. In addition, we have compared the results of our simulation with experimental data from previous research. The simulated isotherm agrees reasonably well with the experimentally obtained isotherm, and the simulated nearest-neighbor distance between the adsorbed molecules is almost the same as the experimental value.

Warfarin and vitamin K epoxide reductase: a molecular accounting for observed inhibition

AbstractVitamin K epoxide reductase (VKOR), an endoplasmic reticulum membrane protein, is the key enzyme for vitamin K–dependent carboxylation, a posttranslational modification that is essential for the biological functions of coagulation factors. VKOR is the target of the most widely prescribed oral anticoagulant, warfarin. However, the topological structure of VKOR and the mechanism of warfarin's inhibition of VKOR remain elusive. Additionally, it is not clear why warfarin-resistant VKOR mutations identified in patients significantly decrease warfarin's binding affinity, but have only a minor effect on vitamin K binding. Here, we used immunofluorescence confocal imaging of VKOR in live mammalian cells and PEGylation of VKOR's endogenous cytoplasmic-accessible cysteines in intact microsomes to probe the membrane topology of human VKOR. Our results show that the disputed loop sequence between the first and second transmembrane (TM) domain of VKOR is located in the cytoplasm, supporting a 3-TM topological structure of human VKOR. Using molecular dynamics (MD) simulations, a T-shaped stacking interaction between warfarin and tyrosine residue 139, within the proposed TY139A warfarin-binding motif, was observed. Furthermore, a reversible dynamic warfarin-binding pocket opening and conformational changes were observed when warfarin binds to VKOR. Several residues (Y25, A26, and Y139) were found essential for warfarin binding to VKOR by MD simulations, and these were confirmed by the functional study of VKOR and its mutants in their native milieu using a cell-based assay. Our findings provide new insights into the dynamics of the binding of warfarin to VKOR, as well as into warfarin's mechanism of anticoagulation.