bluuebunny/crossref_metadata_2025 huggingface dataset:
“Wir wollen ein Zeichen setzen”
Authors: Remenyi Christian
Published: July 2010

Mit Helmut Schwarz, Professor für organische Chemie an der TU Berlin, steht seit Anfang des Jahres wieder ein Chemiker als Präsident an der Spitze der Alexander‐von‐Humboldt‐Stiftung. Die Nachrichten aus der Chemie sprachen mit ihm über die Humboldt‐Professur und welche Herausforderungen die Stiftung in den nächsten Jahren meistern muss.
Biomimetische Solarzellen: Von der Photosynthese lernen
Authors: Wörmke Stephan , Mackowski Sebastian , Scheer Hugo , Bräuchle Christoph
Published: July 2010

Stephan Wörmke, Sebastian Mackowski, Hugo Scheer, Christoph Bräuchle

Wesentlicher Grund für den evolutionären Erfolg der Photosynthese ist die funktionale Trennung der Primärreaktionen in Lichtsammelkomplexe und Reaktionszentren. Mit Silbernanopartikeln gekoppelte Komplexe steigern die Effizienz der Lichtsammlung um mehr als eine Größenordnung.
Flüssig vorhergesagt
Authors: Hellweg Arnim , Klamt Andreas
Published: July 2010

Arnim Hellweg, Andreas Klamt

Das Kontinuumsolvensmodell behandelt Lösungsmittel nicht als aus einzelnen Molekülen bestehend, sondern als gleichmäßiges Medium. Ein verbessertes Modell berücksichtigt zudem gerichtete Wechselwirkungen wie Wasserstoffbrückenbindungen. Damit sind physiko‐chemische Eigenschaften von Flüssigkeiten ohne lange Rechenzeit vorherzusagen.
Patentes Kapital
Authors: Pauls Nina
Published: July 2010

Nina Pauls

Alle neun Minuten erhält das Deutsche Patent‐ und Markenamt eine Patentanmeldung. Die Rechte schützen nicht nur vor Plagiaten, sondern können auch in Fonds, geleast oder in Auktionen Gewinne erwirtschaften.
Studierende in der GDCh und im VAA – Ein Zwischenbericht
Authors: Bengs Holger , Siekerkötter Reiner
Published: July 2010

Für einen Jahresbeitrag von nur 30,− Euro können Chemie‐Studierende der GDCh beitreten; die Mitgliedschaft im VAA erhalten sie kostenlos dazu.
PROPELLER for motion‐robust imaging of in vivo mouse abdomen at 9.4 T
Authors: Teh Irvin , Golay Xavier , Larkman David J.
Published: July 2010

In vivo high‐field MRI in the abdomen of small animals is technically challenging because of the small voxel sizes, short T2 and physiological motion. In standard Cartesian sampling, respiratory and gastrointestinal motion can lead to ghosting artefacts. Although respiratory triggering and navigator echoes can either avoid or compensate for motion, they can lead to variable TRs, require invasive intubation and ventilation, or extend TEs. A self‐navigated fast spin echo (FSE)‐based periodically rotated overlapping parallel lines with enhanced reconstruction (PROPELLER) acquisition was implemented at 9.4 T to enable high‐resolution in vivo MRI of mouse abdomen without the use of additional navigators or triggering. T2‐weighted FSE‐PROPELLER data were compared with single‐shot FSE and multi‐shot FSE data with and without triggering. Single‐shot methods, although rapid and robust to motion, demonstrated strong blurring. Multi‐shot FSE data showed better resolution, but suffered from marked blurring in the phase‐encoding direction and motion in between shots, leading to ghosting artefacts. When respiratory triggering was used, motion artefacts were largely avoided. However, TRs and acquisition times were lengthened by up to approximately 20%. The PROPELLER data showed a 25% and 61% improvement in signal‐to‐noise ratio and contrast‐to‐noise ratio, respectively, compared with multi‐shot FSE data, together with a 35% reduction in artefact power. A qualitative comparison between acquisition methods using diffusion‐weighted imaging was performed. The results were similar, with the exception that respiratory triggering was unable to exclude major motion artefacts as a result of the sensitisation to motion by the diffusion gradients. The PROPELLER data were of consistently higher quality. Considerations specific to the use of PROPELLER at high field are discussed, including the selection of practical blade widths and the effects on contrast, resolution and artefacts. Copyright © 2010 John Wiley & Sons, Ltd.
What Motivates Lay Third Parties to Take Sides in a Conflict? Examining the Relationships between the Big Five Personality Traits and Side–Taking Motives
Authors: Yang Huadong , Li Chaoping , Wang Qing , Hendriks A. A. Jolijn
Published: July 2010

Taking sides is one of the reactions available to third parties in handling a dispute. From the perspective of individual differences, this study was aimed at identifying lay third parties’ motives for side taking and exploring their relations with the Big Five personality traits. We tested our assumptions using three samples: A Dutch student sample ( n = 111), a Dutch employee sample ( n = 101) and a Chinese student sample ( n = 124). The findings revealed four types of side–taking motives: Moral, relational, reward–approaching and sanction–avoiding motives. The results also showed that individuals’ personality traits were relevant to the four types of side–taking motives: Agreeableness was associated with the relational motive for side taking, and intellectual autonomy was associated with the reward–approaching motive, the sanction–avoiding motive and the relational motive for side taking. Implications and explanations of these findings are discussed. Copyright © 2010 John Wiley & Sons, Ltd.
Thermotropic Liquid Crystalline Polyesters with Rigid or Flexible Spacer Groups
Authors: Jin J.‐I. , Antoun S. , Ober C. , Lenz R. W.
Published: July 2010

Several different series of rigid and flexible polyesters with main chain liquid crystalline units were prepared and their properties were examined in relation to their structures. The first group of polymers were rigid aromatic copolyesters with mesogenic groups based on either chloro or methyl hydroquinone terephthalate units combined with varying amounts of different types of bisphenol terephthalate units The bisphenol comonomers used contained the structure:
magnified image
in which X was none, C(CH3)2, CH2, O, S, and So2. It was observed that the bisphenols with the bulkier X group were more efficient in destroying thermotropic liquid crystallinity of the resulting copolymers. The second group of polymers studied were flexible polyesters consisting of various types of mesogenic units which were connected together by different lengths of polymethylene flexible spacers. The liquid crystalline behaviours of these polymers, particularly their transition temperatures, were correlated with their structures. A brief review of previous studies on the synthesis of thermotropic liquid crystalline polyesters is included.
Thermotropic polyesters with non‐linear links
Authors: Griffin Brian P. , Cox Michael K.
Published: July 2010

The use of different linking units to promote liquid crystal behaviour in rigid rod like polyarylates is reviewed. By systematically varying the proportion of non linear, meta, ortho, or bent bisphenol units in one particular series based on chloro‐ 1,4 phenylene: 4,4′ oxydibenzoate/terephthalate, complete series of compositions, extending from liquid crystal through to isotropic behaviour, have been prepared, characterised and compared. Using this data, parallels between thermotropic and lyotropic behaviour are illustrated and used to postulate physical models for the structure of liquid crystalline melts and solids.
X‐Ray and Infrared Investigation of the Supramolecular Structure of PVC
Authors: Biais RéGine , Geny Chuong , Mordini Claude , Carrega Marc
Published: July 2010

Two types of crystalline morphology seem to be present in commercial PVC powder, both related to syndiotactic sequences in Natta orthorhombic cell: (1) ribbon‐like crystats formed during the second stage of the mass‐process and in suspension or by annealing PVC samples above Tg. (2) high melting fringed‐micelles are the main crystalline feature of emulsion type powder and of low‐conversion mass PVC.

A second less ordered phase, which does not fit the Natta cell is found also in emulsion powder and in highly strained samples (like fibres).

Ribbon‐like crystals and fringed‐micelles influence the i.r. absorption at 604 and 638 cm−1 respectively. 685cm−1 might be related to the low‐order phase, the X‐ray spacing of which is 3.8‐‐4.0 Å along the chain.
Synthesis of Poly(p‐Benzenesulphonamide) Part I. Preparation of Sulphanilic Acid Derivatives for Use as Intermediates
Authors: Contreras Jordi , Jones J. Idris
Published: July 2010

In view of the unique properties of poly (p‐benzamide), particularly as a fibre, the synthesis of the structurally related poly (p‐benzenesulphonamide) has been investigated. This first paper describes the preparation of two groups of possible precursors of the polymer starting with sulphanilic acid. The first group includes N‐sulphinylaniline‐4‐sulphochloride, aniline‐4‐sulphochloride hydrochloride and aniline‐4‐sulphochloride. The key to the synthesis of these intermediates is the conversion of sulphanilic acid to N‐sulphinylaniline‐4‐sulphochloride by reaction with thionyl chloride in the presence of a catalytic amount of dimethyl formamide. For the synthesis of poly (N‐methyl‐benzenesulphonamide), N‐methylsulphanilic acid has been converted into N‐methylaniline‐4‐sulphochloride hydrochloride by treatment with thionyl chloride.

Some N‐acetylated sulphanilic acid derivatives, CH3CONH‐C6H4‐SO2X, where XOH, OEt, OC6H5 and Cl, and N‐acetyl‐1‐naphthylamine‐4‐sulphochloride have also been prepared for evaluation as monomers in the synthesis of polysulphonamides by melt polymerisation.
Magnesium complexes incorporated by sulfonate phenoxide ligands as efficient catalysts for ring‐opening polymerization of ε‐caprolactone and trimethylene carbonate
Authors: Chen Po‐Sheng , Liu Yi‐Chang , Lin Chia‐Her , Ko Bao‐Tsan
Published: July 2010

Two novel sulfonate phenol ligands—3,3′‐di‐tert‐butyl‐2′‐hydroxy‐5,5′,6,6′‐tetramethyl‐biphenyl‐2‐yl 4‐X‐benzenesulfonate (XCF3, LCF3‐H, and XOCH3, LOMe‐H)—were prepared through the sulfonylation of 3,3′‐di‐tert‐butyl‐5,5′,6,6′‐tetramethylbiphenyl‐2,2′‐diol with the corresponding 4‐substituted benzenesulfonyl chloride (1 equiv.) in the presence of excess triethylamine. Magnesium (Mg) complexes supported by sulfonate phenoxide ligands were synthesized and characterized structurally. The reaction of MgnBu2 with L‐H (2 equiv.) produces the four‐coordinated monomeric complexes (LCF3)2Mg (1) and (LOMe)2Mg (2). Complexes 1 and 2 are efficient catalysts for the ring‐opening polymerization of ε‐caprolactone (ε‐CL) and trimethylene carbonate (TMC) in the presence of 9‐anthracenemethanol; complex 1 catalyzes the polymerization of ε‐CL and TMC in a controlled manner, yielding polymers with the expected molecular weights and narrow polydispersity indices (PDIs). In ε‐CL polymerization, the activity of complex 1 is greater than that of complex 2, likely because of the greater Lewis acidity of Mg2+ metal caused by the electron‐withdrawing substitute trifluoromethyl (CF3) at the 4‐position of the benzenesulfonate group. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3564–3572, 2010
Steric effects and competitive intra‐ and intermolecular host‐guest complexation between beta‐cyclodextrin and adamantyl substituted poly(acrylate)s in water: A 1H NMR, rheological and preparative study
Authors: Guo Xuhong , Wang Jie , Li Li , Pham Duc‐Truc , Clements Philip , Lincoln Stephen F. , May Bruce L. , Chen Qingchuan , Zheng Li , Prud'homme Robert K.
Published: July 2010

A 1H NMR and rheological study of host‐guest complexation interactions between three β‐cyclodextrin and three adamantyl substituted poly(acrylate)s, and also between them and adamantan‐1‐carboxylate and native β‐cyclodextrin, respectively, is reported. A close correllation between molecular level interactions and macroscopic characteristics of polymer networks in aqueous solution exists. It is found that intra‐ and intermolecular host‐guest complexation between the host β‐cyclodextrin and guest adamantyl substituents and the length of the aliphatic tether between them and the poly(acrylate) backbone have important roles. Dominantly, steric effects and competitive intra‐ and intermolecular host‐guest complexation are found to control poly(acrylate) isomeric interstrand linkage in polymer network formation. The preparations of five new 3% randomly substituted poly(acrylate)s are reported. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1818–1825, 2010
Thermodynamic characterization of poly(2,2,3,3,3‐pentafluoropropyl methacrylate)
Authors: Papadopoulou Stella K. , Karapanagiotis Ioannis , Zuburtikudis Ioannis , Panayiotou Costas
Published: July 2010

The thermodynamic characterization of a fluorinated methacrylic homopolymer was conducted by means of inverse gas chromatography (IGC), at infinite dilution. The homopolymer under study, poly(2,2,3,3,3‐pentafluoropropyl methacrylate) (PPFPMA), was synthesized via a free radical polymerization reaction and was characterized by the employment of Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and size exclusion chromatography (SEC) techniques. The specific retention volume of 15 solvents, used as probes, was used for the assessment of the Flory–Huggins interaction parameter, the weight fraction activity coefficient, the molar heat, energy and entropy of sorption, the partial heat of mixing of the probes, as well as the solubility parameter of the polymer. The results demonstrate that PPFPMA is insoluble in most organic solvents even at increased temperatures, with the exception of solvents like 2‐Butanone. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1826–1833, 2010
Meconium exposure dependent cell death and apoptosis in human alveolar epithelial cells
Authors: Jeng Mei‐Jy , Soong Wen‐Jue , Lee Yu‐Sheng , Tsao Pei‐Chen , Yang Chia‐Feng , Chiu Shih‐Yun , Tang Ren‐Bin
Published: July 2010

Alveolar epithelial cells of neonates are directly exposed to aspirated meconium during meconium aspiration syndrome (MAS). This study was designed to investigate the influence of quantity and time of meconium exposure on the cell viability and caspase activity in type II human alveolar epithelial cells. Human alveolar epithelial cells were incubated with human meconium suspension at different concentrations and for different times. Cell viability and DNA fragmentation were investigated together with caspases activity and the amount of Bcl‐2 protein present. We found that cell viability was significantly lower in cells exposed to a higher concentration of meconium. This was also true for cells exposed to meconium for longer. Significantly higher DNA fragmentation, an approximately two‐ to fivefold increase, was observed in cells that had been exposed to higher (5% and 10%) concentration of meconium compared to those treated with lower (0.1% and 1%) concentrations (P < 0.05). The activity of most apoptotic initiators (caspase 2, 8, 9, 10) and effectors (caspase 3, 6) were found to be significantly higher in cells subject to greater meconium exposure compared to cells with no or minor meconium exposure. The level of Bcl‐2 was also found to be significantly decreased in meconium‐exposed cells (P < 0.05). In conclusion, human meconium would seem to induce direct cell death as well as caspase‐dependent apoptosis in alveolar epithelial cells; the amount and period of exposure to meconium are crucial factors in this process. Thus, removing aspirated meconium should alleviate lung cell damage in neonates and improve the outcome with MAS. Pediatr. Pulmonol. 2010; 45:816–823. © 2010 Wiley‐Liss, Inc.
Solution structure of the cysteine‐rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily
Authors: He Fahu , Dang Weirong , Saito Kohei , Watanabe Satoru , Kobayashi Naohiro , Güntert Peter , Kigawa Takanori , Tanaka Akiko , Muto Yutaka , Yokoyama Shigeyuki
Published: July 2010

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF‐like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor‐ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand‐binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine‐rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28‐Ala70) by heteronuclear NMR, with a 13C‐/15N‐labeled sample. The tertiary structure of the CRD comprises a β‐sheet with two strands, followed by a 310 helix and a C‐terminal α‐helix, and is stabilized by three disulfide bonds connecting Cys36‐Cys49, Cys52‐Cys67, and Cys55‐Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1‐C2 module type), but not to the CRD of B‐cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1‐D2 module type). This is the first structural report about the A1‐C2 type CRD that could bind to the known target.
Involvement of a carboxylated lysine in UV damage endonuclease
Authors: Meulenbroek Elisabeth M. , Paspaleva Keti , Thomassen Ellen A. J. , Abrahams Jan Pieter , Goosen Nora , Pannu Navraj S.
Published: July 2010

UV damage endonuclease is a DNA repair enzyme that can both recognize damage such as UV lesions and introduce a nick directly 5′ to them. Recently, the crystal structure of the enzyme from Thermus thermophilus was solved. In the electron density map of this structure, unexplained density near the active site was observed at the tip of Lys229. Based on this finding, it was proposed that Lys229 is post‐translationally modified. In this article, we give evidence that this modification is a carboxyl group. By combining activity assays and X‐ray crystallography on several point mutants, we show that the carboxyl group assists in metal binding required for catalysis by donating negative charge to the metal‐coordinating residue His231. Moreover, functional and structural analysis of the K229R mutant reveals that if His231 shifts away, an increased activity results on both damaged and undamaged DNA. Taken together, the results show that T. thermophilus ultraviolet damage endonuclease is carboxylated and the modified lysine is required for proper catalysis and preventing increased incision of undamaged DNA.
Carboxylic acid‐modified polyethylene: A novel support for the covalent immobilization of polypeptides for C‐terminal sequencing
Authors: Shenoy Narmada R. , Bailey Jerome M. , Shively John E.
Published: July 2010

We have developed a method for the covalent immobilization of peptides, for the purpose of C‐terminal sequencing, to a novel solid support, carboxylic acid‐modified polyethylene (PE‐COOH) film. The peptides are attached by coupling the N‐terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3‐dicyclohexylcarbodiimide (DCC), 1,1′‐carbonyldiimidazole (CDI), 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol‐1‐yl‐oxy‐tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3‐diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly through the α‐amino group for the peptide, SIGSLAK, which after attachment to the PE‐COOH support permitted the C‐terminal lysine residue to be sequenced in good yield, indicating that the ‐amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C‐terminal sequencing including (1) stability to base and the high temperatures (65 °C) employed for C‐terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 × 5‐mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N‐terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517–529). Automated C‐terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68–80).
Primary structure of a protein isolated from reef shark (Carcharhinus springeri) cartilage that is similar to the mammalian C‐type lectin homolog, tetranectin
Authors: Neame Peter J. , Young Carmen N. , Treep James T.
Published: July 2010

During the course of characterization of low molecular weight proteins in cartilage, we have isolated a protein from reef shark (Carcharhinus springeri) cartilage that bears a striking resemblance to the tetranectin monomer originally described by Clemmensen et al. (1986, Eur. J. Biochem. 156, 327–333). The protein was isolated by extraction of neural arch cartilage with 4 M guanidine hydrochloride, dialysis of the extract to bring the guanidine to 0.4 M (reassociating proteoglycan aggregates), followed by cesium chloride density gradient removal of the proteoglycans. The amino acid sequence had 166 amino acids and a calculated molecular weight of 18,430. The shark protein was 45% identical to human tetranectin, indicating that it was in the family of mammalian C‐type lectins and that it was likely to be a shark analog of human tetranectin. The function of tetranectin is unknown; it was originally isolated by virtue of its affinity for the kringle‐4 domain of plasminogen. Sequence comparison of human tetranectin and the shark‐derived protein gives clues to potentially important regions of the molecule.
Environment‐specific amino acid substitution tables: Tertiary templates and prediction of protein folds
Authors: Overington John , Donnelly Dan , Johnson Mark S. , Šali Andrej , Blundell Tom L.
Published: July 2010

The local environment of an amino acid in a folded protein determines the acceptability of mutations at that position. In order to characterize and quantify these structural constraints, we have made a comparative analysis of families of homologous proteins. Residues in each structure are classified according to amino acid type, secondary structure, accessibility of the side chain, and existence of hydrogen bonds from the side chain. Analysis of the pattern of observed substitutions as a function of local environment shows that there are distinct patterns, especially for buried polar residues. The substitution data tables are available on diskette with Protein Science. Given the fold of a protein, one is able to predict sequences compatible with the fold (profiles or templates) and potentially to discriminate between a correctly folded and misfolded protein. Conversely, analysis of residue variation across a family of aligned sequences in terms of substitution profiles can allow prediction of secondary structure or tertiary environment.
pH dependence of the reverse reaction catalyzed by phosphofructokinase I from Escherichia coli: Implications for the role of Asp 127
Authors: Auzat Isabelle , Garel Jean‐Renaud
Published: July 2010

The kinetics of the reverse reaction catalyzed by Escherichia coli phosphofructokinase, i.e., the synthesis of ATP and fructose‐6‐phosphate from ADP and fructose‐1,6‐bisphosphate, have been studied at different pH values, from pH 6 to pH 9.2. Hyperbolic saturations of the enzyme are observed for both substrates. The affinity for fructose‐1,6‐bisphosphate decreases with pH following the ionization of a group with a pK of 6.6, whereas the catalytic rate constant and perhaps the affinity for ADP are controlled by the ionization of a group with a pK of 6. Several arguments show that the pK of 6.6 is probably that of the carboxyl group of Asp 127, whereas the pK of 6 is tentatively attributed to the carboxyl group of Asp 103.

The pK of 6.6 is assigned to the carboxyl group of Asp 127 in the free enzyme, and a simple model suggests that the same group would have an abnormally high pK, above 9.6, in the complex between phosphofructokinase and fructose‐1,6‐bisphosphate. It is proposed that the large pK shift of more than 3 pH units upon binding of fructose‐1,6‐bisphosphate is due to an electrostatic repulsion that could exist between the 1‐phosphate group and the carboxyl group of Asp 127, which are close to each other in the crystal structure of phosphofructokinase (Shirakihara, Y.&Evans, P.R., 1988, J. Mol. Biol. 204, 973–994). The same interpretation would also explain the much higher affinity of the enzyme for fructose‐1,6‐bisphosphate when Asp 127 is protonated. Because of its pK above 9.6, Asp 127 would be protonated in the complex between phosphofructokinase and fructose‐1,6‐bisphosphate, and could act as a proton donor, in agreement with its catalytic role as a proton acceptor in the forward reaction (Hellinga, H.W.&Evans, P.R., 1987, Nature 327, 437–439).
Substrate polarization by residues in Δ5‐3‐ketosteroid isomerase probed by site‐directed mutagenesis and UV resonance Raman spectroscopy
Authors: Austin Janina C. , Spiro Thomas G. , Kuliopulos Athan , Mildvan Albert S.
Published: July 2010

Δ5‐3‐Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of Δ5‐3‐ketosteroids to Δ4‐3‐ketosteroids by the stereospecific transfer of the steroid 4β‐proton to the 6β‐position, using Tyr‐14 as a general acid and Asp‐38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr‐14 as the only tyrosine residue (referred to as the Y140 mutant), and the Y14F mutant, which has 50,000‐fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19‐nortestosterone or 4‐fluoro‐19‐nortestosterone to the Y140 mutant does not result in the formation of deprotonated Tyr‐14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y140 enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19‐nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm−1, respectively, on binding to Y140 KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp‐38 side chain to the vinyl group and the directional hydrogen bond between Tyr‐14 and the 3‐carbonyl oxygen of the steroid enone. The 230‐nm‐excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr‐14 oxygen from another protein residue.

UVRR spectra of the Y140 enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in α‐helical structure. The δ‐helix content was estimated to be in the range of 0–25% (i.e., 10 ± 15%).
Yeast ferrochelatase: Expression in a baculovirus system and purification of the expression protein
Authors: Eldridge Marlies G. , Dailey Harry A.
Published: July 2010

The terminal step of the heme biosynthetic pathway is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). In eukaryotes this enzyme is bound to the inner mitochondrial membrane with its active site facing the matrix side of the membrane. Previously this laboratory has characterized this enzyme via kinetic and protein chemical modification techniques, and with the recent cloning of the enzyme from yeast, mouse, and human sources it now becomes possible to approach structure‐function questions by using site‐directed mutagenesis. Of primary significance to this is the development of an efficient expression vector. This is of particular significance for ferrochelatase, as it is a low‐abundance protein whose DNA coding sequence has a very low codon bias. In the current work we describe the production of yeast ferrochelatase in a baculovirus system. This system is shown to be an excellent one in which to produce large quantities of active ferrochelatase. The expressed enzyme is membrane associated and is not released into the growth medium either during or after virus development and cell lysis. The expressed protein can be purified in a procedure that requires only 1 day and makes use of a Pharmacia Hi Trap blue affinity column. The measured Km's for the substrates mesoporphyrin and iron are the same as those reported previously for the yeast enzyme. To our knowledge this is the first example of a mitochondrial membrane protein that has been expressed in a baculovirus system.
Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins
Authors: Viitanen Paul V. , Gatenby Anthony A. , Lorimer George H.
Published: July 2010

In vitro experiments employing the soluble proteins from Escherichia coli reveal that about half of them, in their unfolded or partially folded states, but not in their native states, can form stable binary complexes with chaperonin 60 (groEL). These complexes can be isolated by gel filtration chromatography and are efficiently discharged upon the addition of Mg · ATP. Binary complex formation is substantially reduced if chaperonin 60 is presaturated with Rubisco‐I, the folding intermediate of Rubisco, but not with native Rubisco. Binary complex formation is also reduced if the transient species that interact with chaperonin 60 are permitted to progress to more stable states. This implies that the structural elements or motifs that are recognized by chaperonin 60 and that are responsible for binary complex formation are only present or accessible in the unfolded states of proteins or in certain intermediates along their respective folding pathways. Given the high‐affinity binding that we have observed in the present study and the normal cellular abundance of chaperonin 60, we suspect that the folding of most proteins in E. coli does not occur in free solution spontaneously, but instead takes place while they are associated with molecular chaperones.
Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi, and eubacteria
Authors: Reizer Jonathan , Reizer Aiala , Saier Milton H. , Finley Kim , Kakuda Donald , Macleod Carol L.
Published: July 2010

We demonstrate that three integral membrane receptors of mammals — the ecotropic retroviral leukemia receptor (ERR), the human retroviral receptor (HRR), and the T‐cell early activator (Tea) — are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC), which catalyze solute uniport, solute:cation symport, or solute:solute antiport in yeast, fungi, and eubacteria. Interestingly, the ERR membrane protein was recently shown to function as a cation:amino acid cotransporter. A binary sequence similarity matrix and an evolutionary tree of the 14 members of this family, illustrating their sequence similarities and divergences, were constructed. Other proteins, including the developmentally controlled GerAII spore germination protein of Bacillus subtilis and the acetylcholine receptor of Drosophila melanogaster gave sequence comparison scores of a sufficiently large magnitude to suggest (but not to establish) a common evolutionary origin with members of the APC family.

We report an extended and corrected Tea cDNA sequence and show that the mammalian Tea and ERR encoding genes are differentially expressed in tissues and cell lines. Furthermore, the two mammalian cDNA sequences hybridize with other vertebrate and yeast genomic DNAs under stringent conditions. These observations support the notion that cell surface receptor proteins in mammals are transport proteins that share a common origin with transport proteins of single‐celled organisms. Thus, permeases of essential metabolites may function pathologically as viral receptors.
An automated method for modeling proteins on known templates using distance geometry
Authors: Srinivasan Subhashini , March Carl J. , Sudarsanam Sucha
Published: July 2010

We present an automated method incorporated into a software package, FOLDER, to fold a protein sequence on a given three‐dimensional (3D) template. Starting with the sequence alignment of a family of homologous proteins, tertiary structures are modeled using the known 3D structure of one member of the family as a template. Homologous interatomic distances from the template are used as constraints. For nonhomologous regions in the model protein, the lower and the upper bounds for the interatomic distances are imposed by steric constraints and the globular dimensions of the template, respectively. Distance geometry is used to embed an ensemble of structures consistent with these distance bounds. Structures are selected from this ensemble based on minimal distance error criteria, after a penalty function optimization step. These structures are then refined using energy optimization methods.

The method is tested by simulating the α‐chain of horse hemoglobin using the α‐chain of human hemoglobin as the template and by comparing the generated models with the crystal structure of the α‐chain of horse hemoglobin. We also test the packing efficiency of this method by reconstructing the atomic positions of the interior side chains beyond Cβ atoms of a protein domain from a known 3D structure. In both test cases, models retain the template constraints and any additionally imposed constraints while the packing of the interior residues is optimized with no short contacts or bond deformations. To demonstrate the use of this method in simulating structures of proteins with nonhomologous disulfides, we construct a model of murine interleukin (IL)‐4 using the NMR structure of human IL‐4 as the template. The resulting geometry of the nonhomologous disulfide in the model structure for murine IL‐4 is consistent with standard disulfide geometry.
Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3‐hydroxynorcamphor product
Authors: Paulsen Mark D. , Ornstein Rick L. , Filipovic Djordje , Sligar Stephen G.
Published: July 2010

Cytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1‐methyl‐norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1‐methyl‐norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%, but for both norcamphor and 1‐methyl‐norcamphor, the efficiency is dramatically lowered to 12% and 50%, respectively. Based on an examination of the active site of P450cam, it appeared that mutating position 185 might dramatically alter the product specificity and coupling of hydroxylation of norcamphor by P450cam. Analysis of molecular dynamics trajectories of norcamphor bound to the T185F mutant of cytochrome P450cam predicted that hydroxylation at the 3 position should be abolished and that the coupling should be dramatically increased. This mutant was constructed and the product profile and coupling experimentally determined. The coupling was doubled, and hydroxylation at the 3 position was essentially abolished. Both of these results are in agreement with the prediction.
Orientational constraints as three‐dimensional structural constraints from chemical shift anisotropy: The polypeptide backbone of gramicidin A in a lipid bilayer
Authors: Mai W. , Hu W. , Wang C. , Cross T.A.
Published: July 2010

Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high‐resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these constraints have been used to evaluate models for the channel conformation of gramicidin A. Of the three models used, the one experimentally derived model of gramicidin in sodium dodecyl sulfate micelles fits the data least well.
G proteins: Critical control points for transmembrane signals
Authors: Neer Eva J.
Published: July 2010

Heterotrimeric GTP‐binding proteins (G proteins) that are made up of α and βγy subunits couple many kinds of cell‐surface receptors to intracellular effector enzymes or ion channels. Every cell contains several types of receptors, G proteins, and effectors. The specificity with which G protein subunits interact with receptors and effectors defines the range of responses a cell is able to make to an external signal. Thus, the G proteins act as a critical control point that determines whether a signal spreads through several pathways or is focused to a single pathway. In this review, I will summarize some features of the structure and function of mammalian G protein subunits, discuss the role of both a and by subunits in regulation of effectors, the role of the βγ subunit in macromolecular assembly, and the mechanisms that might make some responses extremely specific and others rather diffuse.
A two‐domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase
Authors: Chen Shiuan , Deng Paulis S.K. , Bailey Jerome M. , Swiderek Kristine M.
Published: July 2010

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT‐diaphorase) is a FAD‐containing reductase that catalyzes a unique 2‐electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl‐terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino‐terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2‐domain structure, i.e., an aminoterminal compact domain and a carboxyl‐terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino‐terminal and carboxyl‐terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, andStaphylococcus aureusprotease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.
Kinetics and thermodynamics of thermal denaturation in acyl carrier protein
Authors: Horvath L.A. , Sturtevant J.M. , Prestegard J.H.
Published: July 2010

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable‐temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.
On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)
Authors: Balme Alexis , Lederer Florence
Published: July 2010

The family of FMN‐dependent, α‐hydroxy acid‐oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate α‐proton (so‐called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins & flavoproteins, Berlin: Walter de Gruyter & Co., pp 513‐526; Urban P, Lederer F, 1985, J Biol Chem 260:11115‐11122). This suggested the occurrence of a pKa increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and O4, a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild‐type enzyme, whereas it is about 3‐fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pKa drop of about half a pH unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pKa increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.
Improving the sensitivity of the sequence profile method
Authors: Lüthy Roland , Xenarios Ioannis , Bucher Philipp
Published: July 2010

The sequence profile method (Gribskov M, McLachlan AD, Eisenberg D, 1987, Proc Natl Acad Sci USA 84:4355‐4358) is a powerful tool to detect distant relationships between amino acid sequences. A profile is a table of posi‐tion‐specific scores and gap penalties, providing a generalized description of a protein motif, which can be used for sequence alignments and database searches instead of an individual sequence. A sequence profile is derived from a multiple sequence alignment. We have found 2 ways to improve the sensitivity of sequence profiles: (1) Sequence weights: Usage of individual weights for each sequence avoids bias toward closely related sequences. These weights are automatically assigned based on the distance of the sequences using a published procedure (Sib‐bald PR, Argos P, 1990, J Mol Biol 216:813‐818). (2) Amino acid substitution table: In addition to the alignment, the construction of a profile also needs an amino acid substitution table. We have found that in some cases a new table, the BLOSUM45 table (Henikoff S, Henikoff JG, 1992, Proc Natl Acud Sci USA 89:10915‐10919), is more sensitive than the original Dayhoff table or the modified Dayhoff table used in the current implementation. Profiles derived by the improved method are more sensitive and selective in a number of cases where previous methods have failed to completely separate true members from false positives.
Identification of succinimide sites in proteins by N‐terminal sequence analysis after alkaline hydroxylamine cleavage
Authors: Kwong May Y. , Harris Reed J.
Published: July 2010

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 °C will specifically cleave on the C‐terminal side of succinimides without cleavage at Asn‐Gly bonds; yields are typically ∼ 50%. N‐terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site.
Structure and dynamics of the active site gorge of acetylcholinesterase: Synergistic use of molecular dynamics simulation and X‐ray crystallography
Authors: Axelsen Paul H. , Harel Michal , Silman Israel , Sussman Joel L.
Published: July 2010

The active site of acetylcholinesterase (AChE) fromTorpedo californicais located 20 Å from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis‐quaternary decamethonium (DECA) ion, acquired during enzyme purification, resides in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.
Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor
Authors: Neumann Ulf , Hofsteenge Jan
Published: July 2010

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisincatalyzed semisynthesis starting from synthetic RNase 1‐20 peptides and S‐protein (RNase 21‐124). The lysyl residue at position 1 was replaced by alanine, whereas Lys‐7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl‐3′,5′‐adenosine and yeast RNA. When Lys‐7 was replaced by S‐methyl‐cysteine or S‐carboxamido‐methyl‐cysteine, binding of recombinant ribonuclease inhibitor (RI) from porcine liver was strongly affected. In contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A‐RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys‐1, Cys‐7‐methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol−1. Modifications that introduced a positive charge in position 7 (S‐aminoethyl‐ or S‐ethylpyridyl‐cysteine) led to much smaller losses. The replacement of Lys‐1 resulted in a 4‐kJ mol−1 loss in binding energy. S‐protein bound to R1 with K1 = 63.4 pM, 800‐fold weaker than RNase A. This corresponded to a 16‐kJ mol−1 difference in binding energy. The results show that the N‐terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys‐7 and to a smaller extent of Lys‐1 provide most of the binding energy.