The crystal structure of free human hypoxanthineguanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-U-D-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP. Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg2+.PPi complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9 angstrom resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasina gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T gondii). (c) 2005 Elsevier Ltd. All rights reserved.

Language and interdisciplinarity

Since language is multifaceted and heterogeneous, interdisciplinarity is natural to linguistic studies. In this article, after demonstrating that, I present two basic ways of doing science. One is ruled by the principle of exclusion, whereas the other is ruled by the principle of participation. The former leads to specialization, whereas the latter leads to the surpassing of specialization. From that, I discuss the advantages and problems of disciplinarity, and present the reasons why nowadays interdisciplinarity is a positive universal in scientific and pedagogical discourses. Also, based on etymology, I discuss the concepts of interdisciplinarity, multidisciplinarity, pluridisciplinarity and transdisciplinarity. Finally, I examine the bonds between linguistics and other sciences, by drawing a brief history of the relations between linguistics and literature in Brazil.

Solution structure of χ-conopeptide MrIA, a modulator of the human norepinephrine transporter

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.: Schroder, C. L; Kaye, D. M.; Adams, D. I.; Alewood, P. F.; Lewis, R. J. J Biol Client 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hypl2 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency. This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by entailing the Biopolymers editorial office at biopolymers@wiley.com (c) 2005 Wiley Periodicals, Inc.

Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited

Solution structure of the sodium channel antagonist conotoxin GS: A new molecular caliper for probing sodium channel geometry

Background: The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels. Results: The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors. Conclusions: This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding

DsbA is a protein-folding catalyst from the periplasm of Escherichia coli that interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the interaction between DsbA and unfolded substrate is not known. Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 Angstrom, and present a proposal for its interaction with peptide. The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction. A hinge point for domain motion is identified-the typo IV beta-turn Phe 63-Met 64-Gly 65-Gly 66, which connects the two domains. Three unique features on the active site surface of the DsbA molecule-a groove, hydrophobic pocket, and hydrophobic patch-form an extensive uncharged surface surrounding the active-sits disulfide. Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues. Furthermore, the residues immediately surrounding the active-site disulfide are uncharged in all nine DsbA proteins. A model for DsbA-peptide interaction has been derived from the structure of a human thioredoxin:peptide complex. This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin. The active-site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds form a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: The role of side chain rotations and cisoid/transoid interconversions

H-1 NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol(-1). This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is greater than or equal to 37 kJ mol(-1), which is significantly larger than has previously been reported for Csp(2)-Csp(3) bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.

Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes

Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P, and S179C that showed an increase in melting temperature of 20 degrees C. The thermodynamic properties Of a representative subset of nine XylA variants showing a range of thermostabilities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (Delta C(p)) with an increase in the midpoint of the transition temperature (T(m)) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in Delta C(p) on should include effects arising from the prot inlsolvent interface.

Can the durability of one-step self-etch adhesives be improved by double application or by an extra layer of hydrophobic resin?

Objectives: This study evaluated the immediate and 6-month resin-dentin mu-bond strength (mu TBS) of one-step self-etch systems (Adper Prompt L-Pop [AD] 3M ESPE; Xeno III [XE] Dentsply De Trey; iBond [iB] Heraeus Kulzer) under different application modes. Materials and methods: Dentin oclusal surfaces were exposed by grinding with 600-grit SiC paper. The adhesives were applied according to the manufacturer`s directions [MD], or with double application of the adhesive layer [DA] or following the manufacturer`s directions plus a hydrophobic resin layer coating [HL]. After applying the adhesive resins, composite crowns were built up incrementally. After 24-h water storage, the specimens were serially sectioned in ""x"" and ""y"" directions to obtain bonded sticks of about 0.8 mm 2 to be tested immediately [IM] or after 6 months of water storage [6M] at a crosshead speed of 0.5 mm/min. The data from each adhesive was analyzed by a two-way repeated measures ANOVA (mode of application vs. storage time) and Tukey`s test (alpha = 0.05). Results: The adhesives performed differently according to the application mode. The DA and HL either improved the immediate performance of the adhesive or did not differ from the MD. The resin-dentin bond strength values observed after 6 months were higher when a hydrophobic resin coat was used than compared to those values observed under the manufacturer`s directions. Conclusions: The double application of one-step self-etch system can be safety performed however the application of an additional hydrophobic resin layer can improve the immediate resin-dentin bonds and reduce the degradation of resin bonds over time. (c) 2008 Elsevier Ltd. All rights reserved.

Ethanol Wet-bonding Challenges Current Anti-degradation Strategy

The long-term effectiveness of chlorhexidine as a matrix metalloproteinase (MMP) inhibitor may be compromised when water is incompletely removed during dentin bonding. This study challenged this anti-bond degradation strategy by testing the null hypothesis that wet-bonding with water or ethanol has no effect on the effectiveness of chlorhexidine in preventing hybrid layer degradation over an 18-month period. Acid-etched dentin was bonded under pulpal pressure simulation with Scotchbond MP and Single Bond 2, with water wet-bonding or with a hydrophobic adhesive with ethanol wet-bonding, with or without pre-treatment with chlorhexidine diacetate (CHD). Resin-dentin beams were prepared for bond strength and TEM evaluation after 24 hrs and after aging in artificial saliva for 9 and 18 mos. Bonds made to ethanol-saturated dentin did not change over time with preservation of hybrid layer integrity. Bonds made to CHD pre-treated acid-etched dentin with commercial adhesives with water wet-bonding were preserved after 9 mos but not after 18 mos, with severe hybrid layer degradation. The results led to rejection of the null hypothesis and highlight the concept of biomimetic water replacement from the collagen intrafibrillar compartments as the ultimate goal in extending the longevity of resin-dentin bonds.

Mechanical characterization of proanthocyanidindentin matrix interaction

Objectives To characterize the properties of dentin matrix treated with two proanthocyanidin rich cross-linking agents and their effect on dentin bonded interfaces. Methods Sound human molars were cut into 0.5mm thick dentin slabs, demineralized and either treated with one of two cross-linking agents (grape seedGSE and cocoa seedCOE extracts) or left untreated. The modulus of elasticity of demineralized dentin was assessed after 10 or 60min and the swelling ratio after 60min treatment. Bacterial collagenase was also used to assess resistance to enzymatic degradation of samples subjected to ultimate tensile strength. The effect of GSE or COE on the resindentin bond strength was evaluated after 10 or 60min of exposure time. Data were statistically analyzed at a 95% confidence interval. Results Both cross-linkers increased the elastic modulus of demineralized dentin as exposure time increased. Swelling ratio was lower for treated samples when compared to control groups. No statistically significant changes to the UTS indicate that collagenase had no effect on dentin matrix treated with either GSE or COE. Resindentin bonds significantly increased following treatment with GSE regardless of the application time or adhesive system used. Significance Increased mechanical properties and stability of dentin matrix can be achieved by the use of PA-rich collagen cross-linkers most likely due to the formation of a PAcollagen complex. The short term resindentin bonds can be improved after 10min dentin treatment.(C) 2010 Academy of Denta lMaterials. Published by Elsevier Ltd. All rights reserved.

Durability of Enamel Bonding Using One-step Self-etch Systems on Ground and Unground Enamel

Objective: To examine the morphological, early and long-term microtensile bond strengths (mu TBS) of one-step self-etch systems to unground and ground enamel. Materials and Methods: Resin composite (Filtek Z250) buildups were bonded to the buccal and lingual enamel surfaces (unground, bur-cut or SiC-roughened enamel) of third molars after adhesive application using the following adhesives: Clearfil S(3) Bond (CS3); Adper Prompt L-Pop (ADP); iBond (iB) and, as the control, Clearfil SE Bond (CSE). Six tooth halves were assigned for each condition. After storage in water (24 hours/37 degrees C), the bonded specimens were sectioned into beams (0.8 mm(2)) and subjected to pTBS (0.5 mm/min) either immediately (IM) or after six (6M) or 12 months (12M) of water storage. The data were analyzed by three-way repeated measures ANOVA and Tukey`s test (alpha=0.05). Surface conditioning was observed under scanning electron microscopy (SEM). Results: The mu TBS in the Si-C paper and diamond bur groups were similar and higher than the unground group. No significant difference was observed among the different storage periods, except for CS3, which showed an increase in the pTBS after 12M. The etching pattern was more retentive on ground enamel. Conclusions: One-step self-etch adhesives showed higher bond strengths on ground enamel and no reductions in resin-enamel bonds were observed after 12M of water storage.

State of the art etch-and-rinse adhesives

Objectives: The aim of this study was to explore the therapeutic opportunities of each step of 3-step etch-and-rinse adhesives. Methods: Etch-and-rinse adhesive systems are the oldest of the multi-generation evolution of resin bonding systems. In the 3-step version, they involve acid-etching, priming and application of a separate adhesive. Each step can accomplish multiple goals. Acid-etching, using 32-37% phosphoric acid (pH 0.1-0.4) not only simultaneously etches enamel and dentin, but the low pH kills many residual bacteria. Results: Some etchants include anti-microbial compounds such as benzalkonium chloride that also inhibits matrix metalloproteinases (MMPs) in dentin. Primers are usually water and HEMA-rich solutions that ensure complete expansion of the collagen fibril meshwork and wet the collagen with hydrophilic monomers. However, water alone can re-expand dried dentin and can also serve as a vehicle for protease inhibitors or protein cross-linking agents that may increase the durability of resin-dentin bonds. In the future, ethanol or other water-free solvents may serve as dehydrating primers that may also contain antibacterial quaternary ammonium methacrylates to inhibit dentin MMPs and increase the durability of resin-dentin bonds. The complete evaporation of solvents is nearly impossible. Significance: Manufacturers may need to optimize solvent concentrations. Solvent-free adhesives can seal resin-dentin interfaces with hydrophobic resins that may also contain fluoride and antimicrobial compounds. Etch-and-rinse adhesives produce higher resin-dentin bonds that are more durable than most 1 and 2-step adhesives. Incorporation of protease inhibitors in etchants and/or cross-linking agents in primers may increase the durability of resin-dentin bonds. The therapeutic potential of etch-and-rinse adhesives has yet to be fully exploited. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Mechanical stability of adhesives under water storage

Objectives. To evaluate the effects of storage condition (wet or dry) and storage time (24 h and 3 months) on the ultimate tensile strength (UTS) of Single Bond (SB), 3M-ESPE; Opti Bond Solo Plus (OB), Kerr; One Step (OS), Bisco, and Prime & Bond NT (PB), Dentsply adhesive resins. Methods. Hourglass-shaped specimens were obtained from a metallic matrix. Each adhesive was dispensed to fill the molds completely and left undisturbed in a dark chamber for 4 min at 37 degrees C for solvent evaporation. They were individually light-cured for 80 s at 500 mW/cm(2) and randomly divided into three groups: 24 h of water storage; 3 months of water storage; 3 months of dry storage. The specimens were tested in tension at 0.5 mm/min using the microtensile method and data were analyzed by two-way ANOVA and SNK tests for each material. Results. Water storage for 3 months did not cause significant changes in the UTS of any of the adhesives (p-value). Values for water storage ranged from 25.9 MPa for Single Bond at 24 h to 32.7 MPa for Prime & Bond NT after 3 months. Dry storage for 3 months yielded significantly higher UTS for most adhesives, which ranged from approximately 20% for Opti Bond to 160% higher values for Single Bond compared to their 3 months wet storage values. Conclusion. The effects of storage condition and time on the UTS of adhesives were material-dependent. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.