Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.     

Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance

Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. In this work, we characterized the organic solvent tolerance of Escherichia coli cells that overexpress prefoldin and group II chaperonin from a hyperthermophilic archeaum, Pyrococcus horikoshii OT3. The colony-forming efficiency of E. coli cells overexpressing prefoldin increased by 1,000-fold and decreased the accumulation of intracellular organic solvent. The effect was impaired by deletions of the region responsible for the chaperone function of prefoldin. Therefore, we concluded that prefoldin endows E. coli cells by preventing accumulation of intracellular organic solvent through its molecular chaperone activity.     

Atomistic mechanisms of huntingtin N‐terminal fragment insertion on a phospholipid bilayer revealed by molecular dynamics simulations

The huntingtin protein is characterized by a segment of consecutive glutamines (Q_N) that is responsible for its fibrillation. As with other amyloid proteins, misfolding of huntingtin is related to Huntington's disease through pathways that can involve interactions with phospholipid membranes. Experimental results suggest that the N‐terminal 17‐amino‐acid sequence (htt^NT) positioned just before the Q_N region is important for the binding of huntingtin to membranes. Through all‐atom explicit solvent molecular dynamics simulations, we unveil the structure and dynamics of the htt^NTQ_N fragment on a phospholipid membrane at the atomic level. We observe that the insertion dynamics of this peptide can be described by four main steps—approach, reorganization, anchoring, and insertion—that are very diverse at the atomic level. On the membrane, the htt^NT peptide forms a stable α‐helix essentially parallel to the membrane with its nonpolar side‐chains—mainly Leu‐4, Leu‐7, Phe‐11 and Leu‐14—positioned in the hydrophobic core of the membrane. Salt‐bridges involving Glu‐5, Glu‐12, Lys‐6, and Lys‐15, as well as hydrogen bonds involving Thr‐3 and Ser‐13 with the phospholipids also stabilize the structure and orientation of the htt^NT peptide. These observations do not significantly change upon adding the Q_N region whose role is rather to provide, through its hydrogen bonds with the phospholipids' head group, a stable scaffold facilitating the partitioning of the htt^NT region in the membrane. Moreover, by staying accessible to the solvent, the amyloidogenic Q_N region could also play a key role for the oligomerization of htt^NTQ_N on phospholipid membranes. Proteins 2014; 82:1409–1427. © 2014 Wiley Periodicals, Inc.     

Molecular dynamics study of the metallochaperone Hah1 in its apo and Cu(I)-loaded states: role of the conserved residue M10

Molecular dynamics simulations were performed on both apo and copper forms of the human copper chaperone, Hah1. Wild-type Hah1 and a methionine (M10) to serine mutant were investigated. We have evidenced the central role of residue M10 in stabilizing the hydrophobic core of Hah1 as well as the internal structure of the metal-binding site. When copper(I) is bound, the mobility of Hah1 is reduced whereas mutation of M10 implies a drastic increase of the mobility of apoHah1, stressing the importance of this highly conserved hydrophobic residue for copper sequestration by the apoprotein.     

Carbazole and arylamine functionalized iridium complexes for efficient electro-phosphorescent light-emitting diodes

We report the synthesis and characterization of four cyclometalated iridium complexes based on carbazole and arylamine modified 2-phenylpyridine. The carbazole and arylamine groups are linked to 2-phenyl pyridine backbone to enhance the energy harvesting and transfer from host to guest materials. The electrochemical and photophysical properties of the complexes are studied and electroluminescent devices are fabricated. The results show that the complexes with ligands containing carbazole moieties have desirable phosphorescent properties. The device with complex 3 doped PVK (poly (vinylcarbazole)) as emission layer achieves maximum luminous efficiency of 6.56 cd A⁻¹ and maximum brightness of 14448 cd m⁻².     

The Effects of Organic Solvent on the Ribosyl Transfer Reaction by Thermostable Purine Nucleoside Phosphorylase and Pyrimidine Nucleoside Phosphorylase from Bacillus stearothermophilus JTS 859

The effects of organic solvents on the reaction rate and equilibrium of the ribosyl transfer reaction catalyzed by thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859 were examined at 60°C. The reaction rate in the presence of 10% acetone was 1.6 times higher than that of the control. Acetone was the best organic solvent among those tested for accelerating the reaction rate without denaturing the enzymes. On the other hand, the reaction rate in the presence of 5% ethyl acetate was 1.5 times higher than that of the control. However the enzymes were denatured completely after 1 h incubation. Consequently, the acceleration was not attributed to the stabilization of the enzymes. The equilibrium constants of the reaction were not influenced by the presence of acetone, methyl or ethyl alcohols.     

Direct measurements of carbon and carbonate export from a coral reef ecosystem (Moorea Island , French Polynesia)

The export of carbon and carbonate from coral reefs was investigated through a multidisciplinary investigation of the hydrological, geochemical, sedimentological and biological features of Tiahura reef on the northwestern coast of Moorea Island (French Polynesia). The hydrology of the fore-reef is characterised by prevailing longshore western currents and a strong thermocline. As revealed by turbidity structures (benthic and intermediate nepheloid layers) and by the amount of particles collected by near-bottom sediment traps, horizontal and downslope advections of particles dominate over offshore vertical transport. The exported material is rich in carbonate (ca. 80%) and poor in organic matter (ca. 4%). Sedimentation rates at 430 m depth, i.e. definitive export, reached 209.6 mg m^-2 d^-1 (dry weight). Estimates of carbon and carbonates export for Tiahura reef also reported here represent respectively 47% and 21% of the organic and inorganic carbon produced within the reef.     

Comparison of the capabilities of accelerated solvent extraction and sonication as extraction techniques for the quantification of kavalactones in Piper methysticum (Kava) roots by high performance liquid chromatography with ultra violet detection

A conventional extraction technique of sonication has been compared, in terms of extraction efficiency, extraction time and amount of solvent, with the more novel technique of accelerated solvent extraction for the extraction of kavain from the powdered roots of Piper methysticum (Kava) with acetone. The extracts were analysed using high-performance liquid chromatography with ultra violet detection. The effects of varying solvent volume and extraction time upon the quantity of kavain extracted with sonication, and the effects of varying temperature upon the kavain extraction efficiency by ASE, were investigated. ASE was found to be more efficient with respect to time and solvent volume required; however, a good agreement was found between the kavain concentration obtained using both extraction techniques.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.