Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 ? resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 ? and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.     

An integrated console for capsule-based,automated organic synthesis

The current laboratory practices of organic synthesis are labor intensive, impose safety and environmental hazards, and hamper the implementation of artificial intelligence guided drug discovery. Using a combination of reagent design, hardware engineering, and a simple operating system we provide an instrument capable of executing complex organic reactions with prepacked capsules. The machine conducts coupling reactions and delivers the purified products with minimal user involvement. Two desirable reaction classes – the synthesis of saturated N-heterocycles and reductive amination – were implemented, along with multi-step sequences that provide drug-like organic molecules in a fully automated manner. We envision that this system will serve as a console for developers to provide synthetic methods as integrated, user-friendly packages for conducting organic synthesis in a safe and convenient fashion.

Using a combination of reagent design, hardware engineering, and a simple operating system we provide an instrument capable of executing complex organic reactions using prepacked capsules with minimal user involvement.     

A stereoelectronic effect on turn formation due to proline substitution in elastin-mimetic polypeptides

Stereoelectronic effects have been identified as contributing factors to the conformational stability of collagen-mimetic peptide sequences. To assess the relevance of these factors within other protein structural contexts, three polypeptide sequences were prepared in which the sequences were derived from the canonical repeat unit (Val-Pro-Gly-Val-Gly) of the protein material elastin. These elastin-mimetic polypeptides, elastin-1, elastin-2, and elastin-3, incorporate (2S)-proline, (2S,4S)-4-fluoroproline, and (2S,4R)-4-fluoroproline, respectively, at the second position of the elastin repeat. Calorimetric and spectroscopic investigations of these three polypeptides indicate that the incorporation of the substituted proline residues had a dramatic effect upon the self-assembly of the corresponding elastin peptide. The presence of (2S,4R)-4-fluoroproline in elastin-3 lowered the temperature of the phase transition and increased the type II beta-turn population with respect to the parent polypeptide, while the presence of (2S,4S)-4-fluoroproline in elastin-2 had the opposite effect. These results suggest that stereoelectronic effects could either enhance or hinder the self-assembly of elastin-mimetic polypeptides, depending on the influence of the proline analogue on the energetics of the beta-turn conformation that develops within the pentapeptide structural repeats above the phase transition. Density functional theory (DFT) was employed to model three possible turn types (betaI-, betaII-, and inverse gamma-turns) derived from model peptide segments (MeCO-Xaa-Gly-NHMe) (Xaa = Pro, 4S-F-Pro, or 4R-F-Pro) corresponding to the turn-forming residues of the elastin repeat unit (Val-Pro-Gly-Val-Gly). The results of the these calculations suggested a similar outcome to the experimental data for the elastin-mimetic polypeptides, in that type II beta-turn structures were stabilized for peptide segments containing (2S,4R)-fluoroproline and destabilized for segments containing (2S,4S)-fluoroproline relative to the canonical proline residue.     

Glutathione modulates the level of free radicals produced in UVA-irradiated cells

We have developed an assay to detect reactive oxygen species (ROS) generated by UVA radiation utilising chemical probes which become fluorescent upon oxidation. Using a human bladder carcinoma cell line (MGH-U1) and spontaneously immortalised keratinocytes (HaCaT), we have shown a UVA (narrow band 365+/-5 nm) dose-dependent increase in fluorescence by flow cytometry following loading of the cells with either dihydrorhodamine 123 (DHR) or 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The UVA response of both DHR and DCFH was enhanced by elevation of intracellular levels of the photosensitiser protoporphyrin IX by incubation for 2.5 h with 5-aminolaevulinic acid. Depletion of the antioxidant glutathione (GSH) using the inhibitor D,L-buthionine-sulphoximine (BSO), resulted in an increase in the UVA-induced fluorescence of DCF but not of rhodamine 123. Conversely, raising intracellular GSH levels with N-acetyl cysteine (NAC) had relatively little protective effect in terms of degree of induced fluorescence.     

Ab initio stochastic optimization of conformational and many-body degrees of freedom

Moderate to large size molecules in solution have complex energy surfaces due to intramolecular (conformational) and intermolecular (many-body) interactions. The first principles Monte Carlo (FPMC) method, previously shown to effectively locate minimum-energy structures for systems with only many-body complexity, has been extended to address conformational flexibility by adding three new Monte Carlo move types. The primary advantage of the FPMC method is the ability to efficiently locate minimum energy structures of molecules with conformational flexibility in the presence of explicit solvent molecules using highly accurate quantum chemical calculations. The additions to FPMC were validated by studying conformers of glycerol, glyceraldehyde, and a large humic acid monomer unit. The structure of glyceraldehyde in the presence of one and two water molecules was also explored to demonstrate the power of FPMC to study systems with both conformational and many-body degrees of freedom.     

Chemistry of materials under extreme high pressure-high-temperature conditions

Most of our knowledge of chemistry is derived from experiments carried at the Earth's surface, at pressures near one atmosphere. However, most elements and compounds in the universe exist under conditions of extremely high pressures, often combined with high temperatures, deep within the planets and stars. Under these conditions, new high-density crystal forms occur, species usually known only as molecules become dense covalent or ionic solids, and insulators and semiconductors become metals and even superconductors. Valency states and coordination numbers are changed, and it is expected that chemical bonding and reactivity is modified. Paul McMillan describes how the field of condensed matter chemistry under extreme high pressure conditions now represents a vast new area to be explored.     

A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays

Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here, we introduce a general technique for patterning nanoparticle arrays using two-dimensional crystals of genetically modified hollow protein structures called chaperonins. Constrained chemical synthesis of transition metal nanoparticles is initiated using templates functionalized with polyhistidine sequences. These nanoparticles are ordered into arrays because the template-driven synthesis is constrained by the nanoscale structure of the crystallized protein. We anticipate that this system may be used to pattern different classes of nanoparticles based on the growing library of sequences shown to specifically bind or direct the growth of materials.     

Amorphous and nanocrystalline luminescent Si and Ge obtained via a solid-state chemical metathesis synthesis route

A new solid-state metathesis synthesis route was applied to obtain bulk samples of amorphous or microcrystalline Si and Ge. The method involves reaction of Zintl phases such as NaSi or NaGe, with ammonium or metal (e.g., CuCl, CoBr₂) halides. The driving force for the solid-state reaction is provided by the formation of alkali halides and the transition metals or metal silicides, or gaseous ammonia and hydrogen. The semiconductors were purified by washing to remove other solid products. The amorphous semiconductors were obtained in bulk form from reactions carried out at 200-300 °C. Syntheses at higher temperatures gave rise to microcrystalline semiconductors, or to micro-/nanocrystalline particles contained within the amorphous material. Similar crystalline/amorphous composites were obtained after heat treatment of bulk amorphous materials.     

Evaluation of PTMSP membranes in achieving enhanced ethanol removal from fermentations by pervaporation

The use of membrane processes for the recovery of fermentation products has been gaining increased acceptance in recent years. Pervaporation has been studied in the past as a process for simultaneous fermentation and recovery of volatile products such as ethanol and butanol. However, membrane fouling and low permeate fluxes have imposed limitations on the effectiveness of the process. In this study, we characterize the performance of a substituted polyacetylene membrane, poly[(l-trimethylsilyl)-l-propyne] (PTMSP), in the recovery of ethanol from aqueous mixtures and fermentation broths. Pervaporation using PTMSP membranes shows a distinct advantage over conventional poly(dimethyl siloxane) (PDMS) membranes in ethanol removal. The flux with PTMSP is about threefold higher and the concentration factor is about twofold higher than the corresponding performance achieved with PDMS under similar conditions. The performance of PTMSP with fermentation broths shows a reduction in both flux and concentration factor relative to ethanol-water mixtures. However, the PTMSP membranes indicate initial promise of increased fouling resistance in operation with cell-containing fermentation broths.