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This study uses differential scanning calorimetry, X-ray crystallography and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5 °C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25 and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22 °C. Crystal structures of the wild type and a thermally destabilized (∆Tm -19.7 °C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the wild type were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins. This article is protected by copyright. All rights reserved.

Carbon sequestration and utilization are currently gaining attention as they help to reduce the emission of greenhouse gases in the atmosphere. This study explores the possibility of using carbon dioxide as a feedstock for the production of ammonium carbamate, a precursor molecule for urea production. Waste cooking oil was used as the indispensable nonaqueous medium for the formation of pure ammonium carbamate. This makes the process completely ecofriendly and cost‐effective. A continuous process of ammonium carbamate production at 20°C results in 88.86% consumption of carbon dioxide. The highest yield of ammonium carbamate was achieved at an NH3/CO2 ratio of 2.5. A three‐stage setup was also found to increase the yield of ammonium carbamate. The reusability of oil after each cycle was checked and it was found that the formation of the product is comparatively less compared to the previous cycle. Lower yield was observed when the same experiment was carried out with fresh oil (sunflower oil). The exit gas from the process was found to be rich in ammonia that can be further used for the production of struvite, a phosphate fertilizer. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd.

Microtubule‐associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF‐tau). AT8 is a PHF‐tau‐specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30‐fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF‐tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202–209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR‐L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody. Proteins 2016; 84:427–434. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Journal of applied polymer science e49630 (2020). doi:10.1002/app.49630, Published by Wiley, New York, NY [u.a.]

The crystal structure of DARPin 44C12V5 that neutralizes IL‐4 signaling has been determined alone and bound to human IL‐4. A significant conformational change occurs in the IL‐4 upon DARPin binding. The DARPin binds to the face of IL‐4 formed by the A and C α‐helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL‐4 include a reorientation of the C‐helix Trp91 side chain and repositioning of CD‐loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL‐4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical “hotspot.” The structures also reveal how the DARPin neutralizes IL‐4 signaling. Comparing the IL‐4:DARPin complex structure with the structures of IL‐4 bound to its receptors (Hage et al., Cell 1999; 97, 271‐281; La Porte et al., Cell 2008, 132, 259‐272), it is found that the DARPin binds to the same IL‐4 face that interacts with the junction of the D1 and D2 domains of the IL‐4Rα receptors. Signaling is blocked since IL‐4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL‐13α1 or the γ c receptor. Proteins 2015; 83:1191–1197. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

To assess the state of the art in antibody 3D modeling, 11 unpublished high-resolution x-ray Fab crystal structures from diverse species and covering a wide range of antigen-binding site conformations were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. The participants included: Accerlys Inc, Chemical Computer Group (CCG), Schrodinger, Jeff Gray's lab at John Hopkins University, Macromoltek, Astellas Pharma/Osaka University and Prediction of ImmunoGlobulin Structure (PIGS). The sequences of benchmark structures were submitted to the modelers and PIGS, and a set of models were generated for each structure. We provide here an overview of the organization, participants and main results of this second antibody modeling assessment (AMA-II). Also, we compare the results with the first antibody assessment published in this journal (Almagro et al., 2011;79:3050).

To assess the state-of-the-art in antibody structure modeling, a blinded study was conducted. Eleven unpublished Fab crystal structures were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. In the first round, each participant submitted three non-ranked complete Fv models for each target. In the second round, CDR-H3 modeling was performed in the context of the correct environment provided by the crystal structures with CDR-H3 removed. In this report we describe the reference structures and present our assessment of the models. Some of the essential sources of errors in the predictions were traced to the selection of the structure template, both in terms of the CDR canonical structures and VL/VH packing. On top of this, the errors present in the Protein Data Bank structures were sometimes propagated in the current models, which emphasized the need for the curated structural database devoid of errors. Modeling non-canonical structures, including CDR-H3, remains the biggest challenge for antibody structure prediction.

Three Fab structures used as targets in the Antibody Modeling Assessment presented a challenge for modeling CDR-L3 due to deviations from the most typical canonical structure. In all three antibodies CDR-L3 has eight residues, which is one residue shorter than usual, and has a conformation that is rarely observed in crystal structures. We analyzed the sequence and structural determinants of this conformation and found that the “short” CDR-L3 is remarkably rigid and retains the conformation in the interactions with antigens and neighboring CDRs. Proteins 2014; 82:1679–1683. © 2014 Wiley Periodicals, Inc.

The crystal structure of an N-terminal β-strand-swapped consensus-derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N-terminal β-strand swapping, leading to the formation of a stable compact dimer.

The crystal structures of six different fibronectin Type III consensus-derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C-terminal β-strand resulting in the formation of Tencon dimers in which the target-binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β-strand-swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non-glycine residue at position 77 and insertions of

A blinded study to assess the state of the art in three-dimensional structure modeling of the variable region (Fv) of antibodies was conducted. Nine unpublished high-resolution x-ray Fab crystal structures covering a wide range of antigen-binding site conformations were used as benchmark to compare Fv models generated by four structure prediction methodologies. The methodologies included two homology modeling strategies independently developed by CCG (Chemical Computer Group) and Accerlys Inc, and two fully automated antibody modeling servers: PIGS (Prediction of ImmunoGlobulin Structure), based on the canonical structure model, and Rosetta Antibody Modeling, based on homology modeling and Rosetta structure prediction methodology. The benchmark structure sequences were submitted to Accelrys and CCG and a set of models for each of the nine antibody structures were generated. PIGS and Rosetta models were obtained using the default parameters of the servers. In most cases, we found good agreement between the models and x-ray structures. The average rmsd (root mean square deviation) values calculated over the backbone atoms between the models and structures were fairly consistent, around 1.2 A. Average rmsd values of the framework and hypervariable loops with canonical structures (L1, L2, L3, H1, and H2) were close to 1.0 A. H3 prediction yielded rmsd values around 3.0 A for most of the models. Quality assessment of the models and the relative strengths and weaknesses of the methods are discussed. We hope this initiative will serve as a model of scientific partnership and look forward to future antibody modeling assessments.

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β-amyloid (Aβ) plaques. Aggregation of the Aβ42 peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti-Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 A resolution. The structure was determined in two crystal forms, P21 and C2. Although the Fab was crystallized in the presence of Aβ16, no peptide was observed in the crystals. The antigen-binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly-His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen-binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his-tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B-cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.

Background and purpose: P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport. Experimental approach: Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine. Key results: In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells. Conclusions and implications: Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport.

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which hasmore » a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length ECD. Quite unexpectedly, ND1 forms a dimer mediated through the exchange of its last {beta}-strand (strand G). {beta}-strand swapping, which is a subset of 3D domain swapping, has been found to mediate cell-cell adhesion by cadherins. 3D domain swapping has been proposed to be a mechanism of protein oligomerization, aggregation, evolution of oligomeric proteins from single domains and amyloidogenesis. In domain swapped proteins, the same structural elements are involved in the final 3D structure, and so there is little overall energetic difference between the monomer and the swapped oligomers. However, there is often a high energy barrier for the conversion as it often goes through an unfolded state. It is also possible that strand-swapping occurs during folding of nascent polypeptide chains. Frequently, the exchange hinges contain proline-rich motifs which are often in high strain conformations. Domain swapping appears to be a strategy to resolve such local structural strain. The exchange hinge of ND1 contains a Pro-Glu-Pro tripeptide motif. Both of the proline residues adopt extended trans conformations, when compared with cis in the full-length ECD structure. Proline cis-trans isomerization may be the driving force for this exchange. Strand-exchanged dimerization may be a mechanism for the oligomerization of EMMPRIN ECD and its cis-dependent homophilic interactions in cell-cell adhesion.« less

Purpose: Frequent epileptic seizures or prolongedseizureactivity (status epilepticus, SE) isknowntoincrease the brain expression of drug efflux trans-porter genes and proteins, such as P-glycoprotein(Pgp) and members of the multidrug resistanceprotein (MRP) family, which might reduce brainlevels of antiepileptic drugs and, therefore, beinvolved in drug resistance. However, the timecourse of alterations in Pgp or MRPsafterseizuresorSEisonlyincompletelyknown.Methods: This prompted us to study the timecourse of alterations in the expression of differentefflux transporter genes (Mdr1a, Mdr1b, MRP1,MRP2, MRP5) at various times after a pilocarpine-induced SE in limbic brain regions, usingquantitative real-time polymerase chain reaction(RT-PCR)(qPCR).Results: Unexpectedly, between 6 and 24 h afteronset of SE, genes encoding Pgp (Mdr1a, Mdr1b),Mrp1, and Mrp5 were downregulated in hippo-campus, amygdala, or piriform cortex. This initialdecrease in expression was followed by normali-zation and then increased expression, whichbecame maximal 2 days after SE. One explana-tion for the initial decrease in transporter expres-sion could be SE-induced acute inflammatoryprocesses, because proinflammatory cytokinesare known to suppress the expression of Pgp andother efflux transporters. To directly address thispossibility, we quantified the hippocampal mRNAexpression of interleukin-1b, interleukin-6, andtumor necrosis factor-a, showing a markedSE-induced increase in these cytokines, whichparalleled the decreased expression of effluxtransporters.Discussion: Taken together, these findings indi-cate that alterations in expression of drug effluxtransporters after prolonged seizure activity aremorecomplexthanpreviouslythought.KEY WORDS: P-glycoprotein, Multidrug resis-tant proteins, Cytokines, Cyclooxygenase-2,Inflammation,Seizures.Active drug efflux transporters of theATP-binding cas-sette (ABC) family, such as P-glycoprotein (Pgp; MDR1;ABCB1) or members of the multidrug resistance protein(MRP; ABCC) family, play important roles in regulatingdrug concentrations in various cells and tissues, includingthe brain (Schinkel & Jonker, 2003). In the brain, suchtransporters are expressed at the blood–brain barrier(BBB) and by parenchymal cells such as astrocytes,affecting the brain disposition and hence clinical efficacyof a variety of drugs (Begley, 2004). In antiepileptic drug(AED) resistant epilepsy, particularly temporal lobe epi-lepsy, an overexpression of Pgp and several MRPs hasbeen determined in epileptogenic brain tissue resectedduring epilepsy surgery (Kwan & Brodie, 2005; Lcscher& Potschka, 2005a, 2005b; Tate & Sisodiya, 2007). Thislocalized overexpression of efflux transporters has beenproposed to reduce the penetration of AEDs into thebrain and thereby contribute to AED resistance, but this

Glucosamine 6-phosphate synthase converts fructose-6P into glucosamine-6P or glucose-6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40-kDa C-terminal domain, which has already been characterized crystallographically. Now the three-dimensional structures of the complexes with the reaction product glucose-6P and with the transition state analog 2-amino-2-deoxyglucitol-6P have been determined. Glucose-6P binds in a cyclic form whereas 2-amino-2-deoxyglucitol-6P is in an extended conformation. The information on ligand-protein interactions observed in the crystal structures together with the isotope exchange and site-directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine-6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the epsilon-amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate.

A series of relatively short (GCC)n triplet repeats (n = 3–30) located within regulatory regions of many mammalian genes may be considered as putative cis-acting transcriptional elements (GCC-elements). Fragile X-mental retardation syndrome is caused by an expansion of (GCC)n triplet repeats within the 5′-untranslated region of the human fragile X-mental retardation 1 (FMR1) gene. The present study aimed to characterize a novel human (GCC)n-binding protein and investigate its possible role in the regulation of the FMR1 gene. A novel human (GCC)n-binding protein, p56, was isolated and identified as a Kruppel-like transcription factor, ZF5, by MALDI-TOF analysis. The capacity of ZF5 to specifically interact with (GCC)n triplet repeats was confirmed by the electrophoretic mobility shift assay with purified recombinant ZF5 protein. In cotransfection experiments, ZF5 overexpression repressed activity of the GCC-element containing mouse ribosomal protein L32 gene promoter. Moreover, RNA interference assay results showed that endogenous ZF5 acts as a repressor of the human FMR1 gene. Thus, these data identify a new class of ZF5 targets, a subset of genes containing GCC-elements in their regulatory regions, and raise the question of whether transcription factor ZF5 is implicated in the pathogenesis of fragile X syndrome.

Composite films based on the polyvinyltrimethylsilane (PVTMS) with polyaniline (PANI) coating were obtained by borderline polymerization of aniline. The obtained coating was shown to differ from the polymer forming in the reaction mixture bulk both in chemical structure and morphology. Ratio and concentration of the reagents, mixing rate, reaction time, and condition of support surface were among the investigated factors affecting the growth and quality of PANI coating. The gas separation characteristics of composite membranes, as affected by the process conditions and the type of PANI, were investigated. It is shown that the proposed method provides a means for obtaining composite membranes that combine high selectivity, especially in O2/N2, He/CH4, and CO2/CH4 separation, with permeability higher than that of known composite materials. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1379–1384, 2003

A hypothetical protein encoded by the gene YjeE of Haemophilus influenzae was selected as part of a structural genomics project for X-ray analysis to assist with the functional assignment. The protein is considered essential to bacteria because the gene is present in virtually all bacterial genomes but not in those of archaea or eukaryotes. The amino acid sequence shows no homology to other proteins except for the presence of the Walker A motif G-X-X-X-X-G-K-T that indicates the possibility of a nucleotide-binding protein. The YjeE protein was cloned, expressed, and the crystal structure determined by the MAD method at 1.7-A resolution. The protein has a nucleotide-binding fold with a four-stranded parallel β-sheet flanked by antiparallel β-strands on each side. The topology of the β-sheet is unique among P-loop proteins and has features of different families of enzymes. Crystallization of YjeE in the presence of ATP and Mg2+ resulted in the structure with ADP bound in the P-loop. The ATPase activity of YjeE was confirmed by kinetic measurements. The distribution of conserved residues suggests that the protein may work as a “molecular switch” triggered by ATP hydrolysis. The phylogenetic pattern of YjeE suggests its involvement in cell wall biosynthesis. Proteins 2002;48:220–226. © 2002 Wiley-Liss, Inc.

A reaction of 4-(2-mercaptophenyl)-1,2,3-thiadiazoles with an oxidant in the presence of 1.1 equiv of base afforded good yields of benzo[4,5]thieno[3,2-d][1,2,3]thiadiazoles via the intramolecular oxidative nucleophilic substitution of a hydrogen (ONHS) pathway. The reaction of 4-(2-mercaptophenyl)-1,2,3-thiadiazoles in the presence of ≥2 equiv of base gave 2-mercaptobenzo[b]thiophenes via an anionic ring-opening/ring-closure pathway.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

A new method for the synthesis of benzo[b]furan-2-thiols is developed using the base-mediated decomposition of title o-hydroxyaryl-thiadiazoles, which is accompanied by liberation of nitrogen, and subsequent recyclization.

New polycondensation network polymers containing cobalticinium fragments have been synthesized using two different methods: (i) polycondensation of di- and monofunctional monomers [1,1′-diacetylcobalticinium hexafluorophosphate (1a) or a mixture of 1a and 1-acetyl-1′- ethylcobalticinium hexafluorophosphate (1b)] with acetophenone, in the melt at 140–200°C in the presence of triethyl orthoformate (TEOF) and p-toluenesulfonic acid (p-TSA); (ii) introduction of cobalticinium fragments in the polymer chain through the interaction of acetyl derivatives of cobalticinium with acetyl-terminated oligophenylenes at 200°C in the presence of p-TSA. The resulting polymers reveal the properties of strongly, middle and weakly basic anionites and retain ion-exchange properties after heating at 125°C.

The three-dimensional structure of apo-neocarzinostatin, an antitumour antibiotic protein isolated from Streptomyces carzinostaticus, has been determined by X-ray diffraction at 0.15-nm resolution and refined to R = 17.2%. The crystal structure of neocarzinostatin is similar to that of the related proteins actinoxanthin and macromomycin. It is also in good agreement with the solution structure determined by NMR spectroscopy. The protein molecule consists of a seven-stranded antiparallel beta-sandwich and a smaller lobe formed by two beta-ribbons. A deep cleft between the two lobes is a putative chromophore binding site. Side chains of Trp39, Leu45, Phe52, Phe78 and the disulphide Cys37-Cys47 aligning the binding cleft in neocarzinostatin suggest the importance of hydrophobic interactions in stabilizing the chromophore molecule. Comparison of the atomic models of neocarzinostatin, actinoxanthin and macromomycin reveals functional residues which might determine specificity towards different chromophores.

As the cornerstone of multiple practical applications, silicon single crystal surfaces have attracted the interest of scientific and engineering communities for several decades. The most recent advances employ the surfaces precovered with a specific functionality to extend into the realm of organic and metal–organic films with well-defined interfaces, to protect the surfaces from oxidation and other contaminations, and to build the components of present and future molecular electronics and sensing devices. This critical review will focus on the reactivity of the selectively terminated Si(100) and Si(111) surfaces. The hydrogen and halogen-terminated surfaces are the most widely used and most heavily reviewed previously, thus only a brief summary will be given here with the emphasis of the most recent thermal approaches to functionalization of hydrogen-terminated silicon. The silicon surfaces precovered with NHx functionality are emerging as a very likely candidate both for the production of sharp interfaces and for coadsorption, co-assembly, and potential molecular templating of patterns on single crystalline surfaces. A brief overview of recent advances in achieving control over the hydroxyl-termination of silicon will be given. Some future directions for further development of chemistry, reactivity, and assembly on these surfaces, as well as potential applications, are highlighted in the last section (152 references).

Synthesis and X-ray structural investigation of 1,3,5-tris[4-(2-propynyloxy)phenyl]benzene (1) were carried out. Compound1 is a trifunctional monomer for the synthesis of cross-linked polymers and a model of a section of polymeric network of this kind. In a crystal, the molecule of1 is located on the threefold symmetry axis and has a propeller-like conformation (the dihedral angle between the central and peripheral benzene rings is ∼35°). An essential feature of the crystal structure of1 is the proximity of the ethynyl groups of the neighboring molecules, which may be favorable to cyclotrimerization in the crystal. The spatial prerequisite to this process is the simultaneous rotation of the neighboring molecules1 about their symmetry axes by ∼10°. The displacements of the atoms in the reacting groups should amount to ∼1.6–1.8 A.

Derivatives of benzo[b]furan-2-thiols exhibit a biological action [1] and are employed in the organic synthesis [1–7]. Now only unsubstituted benzo[b]-furan2-thiol and several 2-sulfanylbenzo[b]furan-3-aldehydes are known. The benzo[b]furan-2-thiol proper is obtained by the metallation of benzo[b]furan with butyllithiun followed by treating with sulfur and isolating the benzo[b]furan-2-thiol by acidification [1, 4]. 2-Sulfanylbenzo[b]furan-3-aldehydes were prepared from 2-halobenzo[b]furan-3-aldehydes and sodium hydrosulfite [6, 7]. We developed a new method of preparation of both unsubstituted and previously unknown substituted in the aromatic ring benzo[b]furan-2-thiol from easily available 4-(2-hydroxyaryl)-1,2,3-thiadiazoles. The initial 4-(2hydroxyaryl)-1,2,3-thiadiazoles we prepared from 2-hydroxyacetophenones through the corresponding ethoxycarbonylhydrazones with subsequent treatment with thionyl chloride [8]. 4-(2-Hydroxyaryl)-1,2,3-thiadiazoles Ia–Ic under the action of a base, potassium carbonate, in anhydrous DMF decomposed with the nitrogen elimination and the forma

Covering: up to June 2001This review summarizes the state of knowledge on D-glucosamine-6P synthesis catalyzed by glucosamine-6P synthase. The mechanisms of L-glutamine hydrolysis, ammonia transfer and fructose-6P conversion into D-glucosamine-6P are analyzed with the E. coli enzyme in light of recent X-ray structures. With 92 references this paper covers the literature up to June 2001 and emphasizes the potential implication of the mammalian glucosamine-6P synthase in type 2 diabetes.

The crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the three-dimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed beta sheet. The active site is located at the C-edge of the central (parallel) part of the beta sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.