Chemistry and Chemical Engineering

An NMR-based approachm arries the two traditional screening technologies (phenotypic and target-based screening) to find compounds inhibiting as pecific enzymatic reaction in bacterial cells.B uilding on ap revious study in which it was demonstrated that hydrolytic decomposition of meropenem in living Escherichia coli cells carrying New Delhi metallo-b-lactamase subclass 1(NDM-1) can be monitored in real time by NMR spectroscopy, we designed acell-based NMR screening platform. As trong NDM-1 inhibitor was identified with cellular IC 50 of 0.51 mm,w hich is over 300-fold more potent than captopril, ak nownN DM-1 inhibitor.T his new screening approach has great potential to be applied to targets in other cell types,such as mammalian cells,and to targets that are only stable or functionally competent in the cellular environment.

The effects of temperature, polymer composition, and concentration on the micellization and gelation properties of poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) (PEO−PPO−PEO) block copolymers in aqueous solutions were investigated by 1 H NMR ...

The effects of temperature, polymer composition, and concentration on the micellization and gelation properties of poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide) (PEO−PPO−PEO) block copolymers in aqueous solutions were investigated by 1 H NMR ...

Chemical shift perturbation mapping of backbone amides is one of the most widely employed techniques in biomolecular NMR, providing residue-by-residue information on interaction interfaces, ligand binding and chemical modification sites, even for samples where poor solubility, short lifetime, or large size precludes more sophisticated experimental approaches. Significant changes can also occur in the amide one-bond 15 N-1 H scalar coupling constants for glutamine binding protein (GlnBP) due to ligand binding. Like chemical shift perturbations, large changes (> 1 Hz) are seen near the site of glutamine binding, though perturbations also occur distant to the site. The coupling constant perturbations correlate with significant structural changes, especially changes in backbone hydrogen bonding. Thus, amide scalar coupling perturbation can serve as an adjunct to chemical shift perturbation, providing additional information on both short-range and longer-range, allosteric structural changes. Chemical shift perturbation mapping of backbone amides is one of the most widely employed techniques in biomolecular NMR 1,2. It provides residue-by-residue information on interaction interfaces, ligand binding and chemical modification sites. It is applicable even for samples where poor solubility, short lifetime, or large size precludes more sophisticated experimental approaches. Here, we show that significant changes can also occur in the backbone amide 15 N-1 H scalar coupling constants for glutamine binding protein (GlnBP) due to ligand binding. Like chemical shift perturbations, large changes (> 1 Hz) are seen near the site of glutamine binding, though perturbations also occur distant to the site. The coupling constant perturbations correlate with significant structural changes, especially changes in backbone hydrogen bonding 3-6. More importantly some of the changes in the Jcouplings do not correspond to changes in the chemical shifts. Thus, amide scalar coupling perturbation can serve as an adjunct to chemical shift perturbation, providing additional information on both short-range and longer-range, allosteric structural changes. Nuclear spin-spin scalar coupling between nuclei is relayed via their interactions with the electrons of the system 7. The interaction is dominated by the four Ramsey terms, the diamagnetic and paramagnetic spin-orbit, spin-dipole, and Fermi contact mechanisms 8. The first two, diamagnetic and paramagnetic spin-orbit mechanisms are related to the diamagnetic and paramagnetic shielding terms that lead to chemical shift perturbation, thus the perturbation in coupling values arising from these terms should resemble the pattern seen for chemical shift perturbation. The second two terms have no shielding counterparts, and for one-bond scalar coupling, the Fermi contact term dominates. The largest contribution

Dissipative particle dynamics (DPD) was used to simulate the formation and stabilization of gold nanoparticles in poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer micelles. Primary gold clusters that were experimentally observed in the early stage of gold nanoparticle formation were modeled as gold bead in DPD simulation. It showed that gold beads were wrapped by the block copolymer and aggregated into spherical particles inside the micelles and forming stable Pluronic-gold colloids with two-layer structures. Increasing Pluronic concentration, molecular weight, and PPO block length led to the formation of more uniform and more stable gold nanoparticles. Density profiles of water beads suggested that the micelles, especially the hydrophobicity of the micellar cores, played an important role in stabilizing gold nanoparticles. Dynamic process indicated that the formation of gold nanoparticles was controlled by the competition between aggregation of primary gold clusters and the stabilization by micelles of block copolymers.. The DPD simulation results of gold-copolymer-water system agree well with previous experiments, while more structure information on microscopic level could be provided.

The human protein Bax sits at a critical regulatory junction of apoptosis, or programmed cell death. Bax exists in equilibrium between cytosolic and mitochondria-associated forms that shifts toward the latter when Bax is activated by proapoptotic proteins. Activated Bax changes conformation, inserts into the mitochondrial outer membrane (MOM), oligomerizes, and induces MOM permeabilization, causing the release of cytochrome c , which effectively commits the cell to die. Because apoptosis is also a basic defense mechanism against invading pathogens, many viruses have developed counteractive measures. Such is the case of human cytomegalovirus, the replication of which hinges on vMIA (viral mitochondria-localized inhibitor of apoptosis), a virus-encoded protein with a unique, albeit poorly understood antiapoptotic activity by which it binds and recruits Bax to mitochondria. Here we show, via the structure determination of the complex between Bax and a peptide comprising vMIA’s Bax-bind...

Measurement of residual dipolar couplings (RDCs) between pairs of nuclei has become a routine method for macromolecular structure determinations by solution NMR spectroscopy. 1 Compared to the short-range restraints derived from inter-proton distances, torsion angles, and J couplings the RDCs can provide unique long-range orientation restraints of inter-nuclear vectors to the molecular alignment frame 2 , and thus are very useful in improving the accuracy and quality of protein NMR structure. This type of information is essential when the molecule under study is multidomain. 3 RDCs can also provide important internal dynamic information of biomolecules. 4 To measure the RDCs, a weak alignment of molecule in a magnetic field is required. As such, various technologies for creating the necessary partial alignment in solution have been developed, including direct magnetic alignment, 5 alignment in different liquid crystals, 6 or by dissolving the protein in a highly hydrated, anisotropically compressed polyacrylamide gel. 7 Recently, new detergent-compatible liquid crystals based on DNA nanotubes 8 and nucleic acid G-tetrad structures 9 have been successfully exploited.

Wu (2010). The Fas-FADD death domain complex structure reveals the basis of DISC assembly and disease mutations''. Nature Struct. Mol. Biol. in press. [3] Su-Chang Lin, Yu-Chih Lo and Hao Wu (2010). Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signaling. Nature, 465: 885-90.

The micellar behavior of a polyethylene oxide block-polypropylene oxide block-polyethylene oxide copolymer Pluronic L64 in aqueous urea solutions (urea concentrations 0, 1, 2, 3, 4, and 6 M) by physical methods, viz. cloud point and viscosity, spectral techniques (Fourier transform infrared, fluorescence, and nuclear magnetic resonance), differential scanning calorimetry, and dynamic light scattering is reported. The presence of urea causes an increase in the clouding temperature, critical micellization temperature, and the structural transition (spherical micelles to ellipsoid/rod) temperature; different methods showed good agreement. Urea induces demicellization of L64, enhancing accumulation of water molecules surrounding micelles. L64 micelles show growth at high temperatures, but this micellar transition is suppressed by urea, as proven by calorimetric and viscometric methods. The results are discussed in terms of the molecular mechanism underlying the effects of urea.

Symmetrical poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEO-PPO-PEO, triblock copolymers with 80% polyethylene oxide (PEO, the hydrophilic end blocks) and polypropylene oxide (PPO, the hydrophobic middle block) usually remain as molecularly dissolved at ambient temperature even at fairly high-concentrations (2 wt.% or more). However, the micellization is induced at lower concentration/temperature in the presence of salts. The results on salt induced micellization from four such hydrophilic copolymers Pluronic Ò F38, F68, F88 and F108 obtained from several independent techniques are described. FTIR and fluorescence results provide essentially identical critical micelle temperatures (CMTs) showing marked decrease with increase in PPO molecular weight and in the presence of salt. These copolymers were weakly surface active and did not show a clear break point in surface tension concentration plot typical of surfactants. While addition of salt decreases the cloud point, no significant micelle growth was observed even at temperature close to cloud point (CP). Marked increased in solubilization of an oil dye was observed in presence of KCl. Different methods showed good agreement in temperature/salt-induced micellization of these hydrophilic copolymers.

The aqueous solution behavior of an ethylene oxide-propylene oxide triblock copolymer Pluronic ® P123 [(EO) 20 (PO) 70 (EO) 20 ] was investigated in the presence of various n-alkanols (C 1-C 6) by cloud-point, viscosity, dynamic light scattering (DLS) and spectroscopic (FTIR, NMR) measurements. For lower alkanols (methanol, ethanol and 1-propanol), the cloud-points (CPs) increased with increase in alkanol concentration. The reverse eVect was found for higher alkanols (C 4-C 6) where both the CPs and critical micelle temperatures (CMTs) decreased with increase in concentration. This behavior is explained in terms of a cooperative association of higher alkanols and block copolymers by replacing water molecules in the PPO core and inducing micellar growth in aqueous P123 solution. Lower alkanols are likely to be good solvents for both PEO and PPO blocks and the eVect on PPO blocks predominates indicating an increase in CP and CMT with increase in alkanol concentration.

Here, we have reported a new approach for utilizing oleic acid-Pluronic L-64 block copolymer coated iron oxide nanoparticles as supports for enzyme immobilization. Iron oxide nanoparticles were prepared by a coprecipitation method and were coated with oleic acid and Pluronic to achieve higher stability and dispersibility. The surface morphology and size of the particle, as determined by transmission electron microscopy (TEM), was (10 nm. X-ray diffraction (XRD) patterns were taken over a range from 10°to 90°2 θ, using Cu KR radiation. Saturation magnetization values, measured at 300 K, varied from 34.6 emu/g to 60.8 emu/g. The possible interaction behavior of oleic acid and Pluronic was observed by Fourier transform infrared (FTIR) analysis and nuclear magnetic resonance (NMR) studies. Further potential of this material as a support for lipase immobilization and enzymatic hydrolysis at the oil/water interface was also investigated. The features of the surface-coated magnetic particles enable the adsorption of lipase from Candida cylindraces via strong hydrophobic interactions, which enhances the stability of the adsorbed enzyme molecules. The stability of the catalyst and, hence, its industrial applicability was tested by performing subsequent reaction cycles for the hydrolysis of olive oil. The activity of the immobilized lipase, pretreated with its substrate, was 510 U/g-matrix and was observed to be maintained at levels as high as 90% of its original activity for up to at least seven reuses.

In this study, temperature-responsive magnetite/polymer nanoparticles were developed from iron oxide nanoparticles and poly(ethyleneimine)-modified poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymer. The particles were characterized by TEM, XRD, DLS, VSM, FTIR, and TGA. A typical product has an ∼20 nm magnetite core and an ∼40 nm hydrodynamic diameter with a narrow size distribution and is superparamagnetic with large saturation magnetization (51.34 emu/g) at room temperature. The most attractive feature of the nanoparticles is their temperature-responsive volume-transition property. DLS results indicated that their average hydrodynamic diameter underwent a sharp decrease from 45 to 25 nm while evaluating the temperature from 20 to 35°C. The temperature-dependent evolution of the CO stretching band in the FTIR spectra of the aqueous nanoparticles solution revealed that thermo-induced self-assembly of the immobilized block copolymers occurred on the magnetite solid surfaces, which is accompanied by a conformational change from a fully extended state to a highly coiled state of the copolymer. Consequently, the copolymer shell could act as a temperature-controlled "gate" for the transit of guest substance. The uptake and release of both hydrophobic and hydrophilic model drugs were well controlled by switching the transient opening and closing of the polymer shell at different temperatures. A sustained release of about 3 days was achieved in simulated human body conditions. In primary mouse experiments, drugentrapped magnetic nanoparticles showed good biocompatibility and effective therapy for spinal cord damage. Such intelligent magnetic nanoparticles are attractive candidates for widespread biomedical applications, particularly in controlled drug-targeting delivery.

By using a combination of 1 H NMR spectroscopy, two-dimensional heteronuclear single-quantum coherenceresolved 1 H{ 13 C} and homonuclear rotating-frame Overhauser enhancement NMR correlation experiments with diffusion ordered spectroscopy (DOSY), the location and distribution of a hydrophobic drug, paeonol, have been established with respect to the methyl groups of the poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer. The interaction between them is adjustable according to the different temperature-dependent hydrophilicities or hydrophobicities of the triblock copolymer components. On the other hand, such interactions influence the self-assembly properties of the block copolymer amphiphiles in solution. The amount of anhydrous methyl groups of PPO segments shows an increase with increasing paeonol concentration. It was also demonstrated that the shell-crosslinking of the Pluronic polymer has an effect in increasing the amount of anhydrous methyl groups and thus increasing the hydrophobicity of Pluronic micelles. This might be the deeper reason underlying the increase in drug-loading capacity and prolongation in release time of Pluronic micelles for drug delivery after the shell-crosslinking. Changes in self-diffusion coefficients of paeonol with varying copolymer concentrations and types were also determined by the diffusion-based NMR DOSY technique, and values of K a , ∆G, and n were calculated.