bluuebunny/crossref_metadata_2025 huggingface dataset:
Ontogeny and phagocytic function of baboon lung dendritic cells
Authors: Awasthi Shanjana , Wolf Roman , White Gary
Published: February 2009

Dendritic cells (DCs) are the most potent antigen‐presenting cells, but the ontogeny and functions of lung DCs are not known during prenatal period. Here, we isolated lung DC population from fetal (125–175 days of gestation age) and adult baboons. The cells were stained with fluorochrome‐conjugated‐HLA‐DP, DQ, DR, CD1a, CD11c, CD14, CD40, CD80, CD86, CD209, CMKLR1, ILT7‐specific antibodies, and staining was analyzed by flow cytometry. The phagocytic function was investigated by incubating the cells with fluorescent‐labeled Escherichia coli bioparticles and analyzed by flow cytometry and fluorescence microscopy. The fetal baboon lung DCs expressed low levels of HLA‐DP, DQ, DR, CD11c and CD86 as compared to adult baboon lung DCs and showed distinct DC morphology. The fetal lung DCs were also less capable of phagocytosing E. coli as compared to the adult lung DCs (P<0.05). In conclusion, the fetal lung DCs are not only phenotypically immature, but also less efficient in phagocytosing E. coli.
Simulating Knowledge Sharing in Spatial Planning: An Agent-Based Approach
Authors: Ligtenberg Arend , Beulens Adrie , Kettenis Dik , Bregt Arnold K , Wachowicz Monica
Published: February 2009

This paper presents a multiagent system (MAS) that simulates a multiactor interactive spatial-planning process. The MAS extends an existing approach with the principle of sharing knowledge between participating actors while trying to create a shared vision. In the simulation, actors are modelled as agents. They have desires and preferences regarding the future development of their environment. These are used to develop their individual views on what areas are eligible for change. A facilitator agent coordinates the exchange of information by indicating possible solutions and conflicts to the actor agents. The simulation is demonstrated for an allocation problem in a pilot area in the southeast of the Netherlands. Four different scenarios are implemented, which demonstrate the impact of cooperation and hierarchy during an interactive spatial-planning process. Although the model is kept limited in terms of input data, the results show its potential for providing insight into the relations and interaction between actors, rather than predicting the results of an interactive spatial-planning process.
Quality Assurance in Construction by Independent Experts: A Case Study of the Efficiency Performance of State-Owned Enterprises in China
Authors: Yung Ping , Lai Lawrence W C
Published: February 2009

As a contribution to the wider theoretical discussion on quality assurance in team production, this case study describes, using the capital–output ratio as a measure of efficiency performance, the quality control problems of Chinese construction state-owned enterprises and explains, with reference to relevant scholarly opinions, why the outcome of legislative requirements for experts falls short of expectations in terms of neo-institutional economic reasoning. This is so, even though the law apparently addresses the principal-agent problem in teamwork by strengthening monitoring.
Visibility and Dominance Analysis: Assessing a High-Rise Building Project in Trondheim
Authors: Rød Jan Ketil , van der Meer Diana
Published: February 2009

This article presents a GIS-aided visibility and dominance analysis used for a visual-impact assessment of a planned high-rise building located in a central area in Trondheim, Norway. The visibility analysis calculates fields of intervisibility between the high-rise building and locations in the urban landscape. Visual obstacles such as existing buildings and trees are included. The dominance analysis adds to the visibility analysis a measure of how visually dominant the high-rise building would be. The dominance measure is based on the distance to the building and how much of the building would be visible from any observation point. The result is an assessment of visibility and dominance throughout the entire study area as opposed to using photomontages for assessing visibility and dominance from single standpoints.
Self‐ and other‐estimates of multiple abilities in Britain and Turkey: A cross‐cultural comparison of subjective ratings of intelligence
Authors: Furnham Adrian , Arteche Adriane , Chamorro‐Premuzic Tomas , Keser Askin , Swami Viren
Published: February 2009

This study is part of a programmatic research effort into the determinants of self‐assessed abilities. It examined cross‐cultural differences in beliefs about intelligence and self‐ and other‐estimated intelligence in two countries at extreme ends of the European continent. In all, 172 British and 272 Turkish students completed a three‐part questionnaire where they estimated their parents', partners' and own multiple intelligences (Gardner (10) and Sternberg (3)). They also completed a measure of the ‘big five’ personality scales and rated six questions about intelligence. The British sample had more experience with IQ tests than the Turks. The majority of participants in both groups did not believe in sex differences in intelligence but did think there were race differences. They also believed that intelligence was primarily inherited. Participants rated their social and emotional intelligence highly (around one standard deviation above the norm). Results suggested that there were more cultural than sex differences in all the ratings, with various interactions mainly due to the British sample differentiating more between the sexes than the Turks. Males rated their overall, verbal, logical, spatial, creative and practical intelligence higher than females. Turks rated their musical, body–kinesthetic, interpersonal and intrapersonal intelligence as well as existential, naturalistic, emotional, creative, and practical intelligence higher than the British. There was evidence of participants rating their fathers' intelligence on most factors higher than their mothers'. Factor analysis of the ten Gardner intelligences yield two clear factors: cognitive and social intelligence. The first factor was impacted by sex but not culture; it was the other way round for the second factor. Regressions showed that five factors predicted overall estimates: sex (male), age (older), test experience (has done tests), extraversion (strong) and openness (strong). Results are discussed in terms of the growing literature in the field.
Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ
Authors: Hsiao Susan J. , Smith Susan
Published: February 2009

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.
Follicular helper T cells are required for systemic autoimmunity
Authors: Linterman Michelle A. , Rigby Robert J. , Wong Raphael. K. , Yu Di , Brink Robert , Cannons Jennifer L. , Schwartzberg Pamela L. , Cook Matthew C. , Walters Giles D. , Vinuesa Carola G.
Published: February 2009

Production of high-affinity pathogenic autoantibodies appears to be central to the pathogenesis of lupus. Because normal high-affinity antibodies arise from germinal centers (GCs), aberrant selection of GC B cells, caused by either failure of negative selection or enhanced positive selection by follicular helper T (TFH) cells, is a plausible explanation for these autoantibodies. Mice homozygous for the san allele of Roquin, which encodes a RING-type ubiquitin ligase, develop GCs in the absence of foreign antigen, excessive TFH cell numbers, and features of lupus. We postulated a positive selection defect in GCs to account for autoantibodies. We first demonstrate that autoimmunity in Roquinsan/san (sanroque) mice is GC dependent: deletion of one allele of Bcl6 specifically reduces the number of GC cells, ameliorating pathology. We show that Roquinsan acts autonomously to cause accumulation of TFH cells. Introduction of a null allele of the signaling lymphocyte activation molecule family adaptor Sap into the sanroque background resulted in a substantial and selective reduction in sanroque TFH cells, and abrogated formation of GCs, autoantibody formation, and renal pathology. In contrast, adoptive transfer of sanroque TFH cells led to spontaneous GC formation. These findings identify TFH dysfunction within GCs and aberrant positive selection as a pathway to systemic autoimmunity.
Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves
Authors: Choi Jae-Hoon , Do Yoonkyung , Cheong Cheolho , Koh Hyein , Boscardin Silvia B. , Oh Yong-Seok , Bozzacco Leonia , Trumpfheller Christine , Park Chae Gyu , Steinman Ralph M.
Published: February 2009

Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter–enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP+ cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c+ cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8+ TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.
Effect of Methicillin-Resistant Staphylococcus aureus Colonization in the Neonatal Intensive Care Unit on Total Hospital Cost
Authors: Schultz Eric D. , Tanaka David T. , Goldberg Ronald N. , Benjamin Daniel K. , Smith P. Brian
Published: February 2009

The rate of methicillin-resistant Staphylococcus aureus (MRSA) infection is increasing in neonatal intensive care units. We determined the economic impact of isolating and cohorting MRSA-colonized neonates on total hospital cost at a 49-bed, level III-IV neonatal intensive care unit.
First principles perspective on the microscopic model for Cs2CuCl4 and Cs2CuBr4
Authors: K Foyevtsova  , Y Zhang  , H O Jeschke  , R Valentí
Published: February 2009

We investigate the microscopic model for the frustrated layered antiferromagnets Cs2CuCl4 and Cs2CuBr4 by performing ab initio density functional theory (DFT) calculations and with the help of the tight-binding method. The combination of both methods provide the relevant interaction paths in these materials, and we estimate the corresponding exchange constants. We find for Cs2CuCl4 that the calculated ratio of the strongest in-plane exchange constants J′/J between the spin-1/2 Cu ions agrees well with neutron scattering experiments. The microscopic model based on the derived exchange constants is tested by comparing the magnetic susceptibilities obtained from exact diagonalization with experimental data. The electronic structure differences between Cs2CuCl4 and Cs2CuBr4 are also analyzed.
The Workplace and Economic Crisis: Canadian Textile Firms, 1929–1935
Authors: Lewis Robert
Published: February 2009

The devastating conditions of the Great Depression forced manufacturers to rethink their approach to workplace control, economic policy, and production practices. Although we know a great deal about how industries responded to the depression, we know very little about the changes implemented by firms. This is unfortunate as firms in the same industry face quite different problems, possess dissimilar work cultures, construct an array of production formats, and have access to a range of financial resources. Based on a literature that documents the variety of strategies devised by industries and firms, this paper shows how four Canadian textile firms—two cotton and two hosiery and knitting—reacted to the economic crisis of the Great Depression. In the face of a different array of conditions, each firm devised different restructuring strategies. The large cotton corporations responded by combining mechanization, product line change, and a new division of labor. The smaller, more competitive hosiery and knitting firms, on the other hand, imposed either a harsh regime of scientific management or conservative, piecemeal changes. In the midst of restructuring the workplace, manufacturers reasserted their prerogatives of managerial authority, selectively took advantage of the opportunities opened up by economic crisis, and created a new regime of industrial-state regulations.
Fightin' Words: Lexical Feature Selection and Evaluation for Identifying the Content of Political Conflict
Authors: Monroe Burt L. , Colaresi Michael P. , Quinn Kevin M.
Published: February 2009

Entries in the burgeoning “text-as-data” movement are often accompanied by lists or visualizations of how word (or other lexical feature) usage differs across some pair or set of documents. These are intended either to establish some target semantic concept (like the content of partisan frames) to estimate word-specific measures that feed forward into another analysis (like locating parties in ideological space) or both. We discuss a variety of techniques for selecting words that capture partisan, or other, differences in political speech and for evaluating the relative importance of those words. We introduce and emphasize several new approaches based on Bayesian shrinkage and regularization. We illustrate the relative utility of these approaches with analyses of partisan, gender, and distributive speech in the U.S. Senate.
Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N‐terminal domain
Authors: Chiti Fabrizio , Bucciantini Monica , Capanni Cristina , Taddei Niccolò , Dobson Christopher M. , Stefani Massimo
Published: February 2009

The HypF N‐terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3–5 nm and 7–9 nm in width, whereas fibrils of 7–9 nm and 12–20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.
Effects of charged amino‐acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c
Authors: Qian Chengmin , Yao Yong , Ye Keqiong , Wang Jinfeng , Tang Wenxia , Wang Yunhua , Wang Wenhu , Lu Junxia , Xie Yi , Huang Zhongxian
Published: February 2009

The solution structure of oxidized bovine microsomal cytochrome b5 mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045±0.009 nm and 0.088±0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X‐ray and solution structures of wild‐type cytochrome b5. The binding of different surface mutants of cytochrome b5 with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b5 surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c‐cyt b5 complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b5, do occur in the association between cytochrome b5 and cytochrome c.
Motif‐based fold assignment
Authors: Salwiński Łukasz , Eisenberg David
Published: February 2009

Conventional fold recognition techniques rely mainly on the analysis of the entire sequence of a protein. We present an MBA method to improve performance of any conventional sequence‐based fold assignment. The method uses sequence motifs, such as those defined in the Prosite database, and the SwissProt annotation of the fold library. When combined with a simple SDP method, the coverage of MBA is comparable to the results obtained with PSI‐BLAST. However, the set of the MBA predictions is significantly different from that of PSI‐BLAST, leading to a 40% increase of the coverage for the combined MBA/PSI‐BLAST method. The MBA approach can be easily adopted to include the results of sequence‐independent function prediction methods and alternative motif and annotation databases. The method is available through the web server localized at http://www.doe‐mbi.ucla.edu/mba.
Structure and dynamics of translation initiation factor aIF‐1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy
Authors: Li Wei , Hoffman David W.
Published: February 2009

Translation initiation factor 1A (aIF‐1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods. The protein was found to be a member of the OB‐fold family of RNA‐associated proteins, containing a barrel of five beta‐strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF‐1A), as well as the prokaryotic translation initiation factor IF1. External to the β barrel, aIF‐1A contains an α‐helix at its C‐terminal and a flexible loop at its N‐terminal, features that are qualitatively similar to those found in eIF‐1A, but not present in prokaryotic IF1. The structural model of aIF‐1A, when used in combination with primary sequence information for aIF‐1A in divergent species, permitted the most‐conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus. Several of the conserved surface residues appear to be unique to the archaea. Nitrogen‐15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF‐1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible. A model is proposed, suggesting some specific interactions that may occur between aIF‐1A and the small subunit of the archaeal ribosome.
Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by 1H‐NMR chemical shift analysis
Authors: Bergkvist Anders , Ejdebäck Mikael , Ubbink Marcellus , Karlsson B. Göran
Published: February 2009

A combination of site‐directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side‐chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the 1H chemical shifts by use of two‐dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild‐type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild‐type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.
Crystal structure of calcium‐free human sorcin: A member of the penta‐EF‐hand protein family
Authors: Xie Xiaoling , Dwyer Maureen D. , Swenson Lora , Parker Matthew H. , Botfield Martyn C.
Published: February 2009

Sorcin is a 22 kD calcium‐binding protein that is found in a wide variety of cell types, such as heart, muscle, brain and adrenal medulla. It belongs to the penta‐EF‐hand (PEF) protein family, which contains five EF‐hand motifs that associate with membranes in a calcium‐dependent manner. Prototypic members of this family are the calcium‐binding domains of calpain, such as calpain dVI. Full‐length human sorcin has been crystallized in the absence of calcium and the structure determined at 2.2 Å resolution. Apart from an extended N‐terminal portion, the sorcin molecule has a globular shape. The C‐terminal domain is predominantly α‐helical, containing eight α‐helices and connecting loops incorporating five EF hands. Sorcin forms dimers through the association of the unpaired EF5, confirming this as the mode of association in the dimerization of PEF proteins. Comparison with calpain dVI reveals that the general folds of the individual EF‐hand motifs are conserved, especially that of EF1, the novel EF‐hand motif characteristic of the family. Detailed structural comparisons of sorcin with other members of PEF indicate that the EF‐hand pair EF1–EF2 is likely to correspond to the two physiologically relevant calcium‐binding sites and that the calcium‐induced conformational change may be modest and localized within this pair of EF‐hands. Overall, the results derived from the structural observations support the view that, in sorcin, calcium signaling takes place through the first pair of EF‐hands.
Structure and function of the bacterial mechanosensitive channel of large conductance
Authors: Oakley Aaron J. , Martinac Boris , Wilce Matthew C.J. , Martinac Boris
Published: February 2009

Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three‐dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure.
Differences in the roles of conserved glutamic acid residues in the active site of human class 3 and class 2 aldehyde dehydrogenases
Authors: Mann Craig J. , Weiner Henry
Published: February 2009

Although the three‐dimensional structure of the dimeric class 3 rat aldehyde dehydrogenase has recently been published (Liu ZJ et al., 1997, Nature Struct Biol 4:317–326), few mechanistic studies have been conducted on this isoenzyme. We have characterized the enzymatic properties of recombinant class 3 human stomach aldehyde dehydrogenase, which is very similar in amino acid sequence to the class 3 rat aldehyde dehydrogenase. We have determined that the rate‐limiting step for the human class 3 isozyme is hydride transfer rather than deacylation as observed for the human liver class 2 mitochondrial enzyme. No enhancement of NADH fluorescence was observed upon binding to the class 3 enzyme, while fluorescence enhancement of NADH has been previously observed upon binding to the class 2 isoenzyme. It was also observed that binding of the NAD cofactor inhibited the esterase activity of the class 3 enzyme while activating the esterase activity of the class 2 enzyme. Site‐directed mutagenesis of two conserved glutamic acid residues (209 and 333) to glutamine residues indicated that, unlike in the class 2 enzyme, Glu333 served as the general base in the catalytic reaction and E209Q had only marginal effects on enzyme activity, thus confirming the proposed mechanism (Hempel J et al., 1999, Adv Exp Med Biol 436:53‐59). Together, these data suggest that even though the subunit structures and active site residues of the isozymes are similar, the enzymes have very distinct properties besides their oligomeric state (dimer vs. tetramer) and substrate specificity.
Pressure response of protein backbone structure. Pressure‐induced amide 15N chemical shifts in BPTI
Authors: Akasaka Kazuyuki , Li Hua , Yamada Hiroaki , Li Renhao , Thoresen Todd , Woodward Clare K.
Published: February 2009

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N‐labeled basic pancreatic trypsin inhibitor (BPTI) in 90%H2O/10%H2O, pH 4.6, by H‐15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 ± 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0‐1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three‐dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 Å/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site‐specific changes in Φ, ψ angles. For β‐sheet residues, a decrease in ψ angles by 1‐2°/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 ± 0.343 and 0.519 ± 0.261 ppm/2 kbar, respectively) than for β‐sheet (0.295 ± 0.195 ppm/2 kbar), suggesting that the pressure‐induced structural changes (local compressibilities) are larger in helical and loop regions than in β‐sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in β‐sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.
Solution structure of the receptor tyrosine kinase EphB2 SAM domain and identification of two distinct homotypic interaction sites
Authors: Smalla Maika , Schmieder Peter , Kelly Mark , Laak Antonius Ter , Krause Gerd , Ball Linda , Wahl Martin , Bork Peer , Oschkinat Hartmut
Published: February 2009

The sterile alpha motif (SAM) is a protein interaction domain of around 70 amino acids present predominantly in the N‐ and C‐termini of more than 60 diverse proteins that participate in signal transduction and transcriptional repression. SAM domains have been shown to homo‐ and hetero‐oligomerize and to mediate specific protein‐protein interactions. A highly conserved subclass of SAM domains is present at the intracellular C‐terminus of more than 40 Eph receptor tyrosine kinases that are involved in the control of axonal pathfinding upon ephrin‐induced oligomerization and activation in the event of cell‐cell contacts. These SAM domains appear to participate in downstream signaling events via interactions with cytosolic proteins.

We determined the solution structure of the EphB2 receptor SAM domain and studied its association behavior. The structure consists of five helices forming a compact structure without binding pockets or exposed conserved aromatic residues. Concentration‐dependent chemical shift changes of NMR signals reveal two distinct well‐separated areas on the domains' surface sensitive to the formation of homotypic oligomers in solution. These findings are supported by analytical ultracentrifugation studies. The conserved Tyr932, which was reported to be essential for the interaction with SH2 domains after phosphorylation, is buried in the hydrophobic core of the structure.

The weak capability of the isolated EphB2 receptor SAM domain to form oligomers is supposed to be relevant in vivo when the driving force of ligand binding induces receptor oligomerization. A formation of SAM tetramers is thought to provide an appropriate contact area for the binding of a low‐molecular‐weight phosphotyrosine phosphatase and to initiate further downstream responses.
Local water bridges and protein conformational stability
Authors: Petukhov Michael , Cregut David , Soares Cláaaudio M. , Serrano Luis
Published: February 2009

Recent studies have pointed out the important role of local water structures in protein conformational stability. Here, we present an accurate and computationally effective way to estimate the free energy contribution of the simplest water structure motif–the water bridge. Based on the combination of empirical parameters for accessible protein surface area and the explicit consideration of all possible water bridges with the protein, we introduce an improved protein solvation model. We find that accounting for water bridge formation in our model is essential to understand the conformational behavior of polypeptides in water. The model formulation, in fact, does not depend on the polypeptide nature of the solute and is therefore applicable to other flexible biomolecules (i.e., DNAs, RNAs, polysaccharides, etc.).
Pig heart short chain L‐3‐hydroxyacyl‐CoA dehydrogenase revisited: Sequence analysis and crystal structure determination
Authors: Barycki Joseph J. , O'Brien Laurie K. , Birktoft Jens J. , Strauss Arnold W. , Banaszak Leonard J.
Published: February 2009

Short chain L‐3‐hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 Å; Rcryst = 22.4%, Rfree = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.
X‐ray crystal structures of a severely desiccated protein
Authors: Bell Jeffrey A.
Published: February 2009

Unlike most protein crystals, form IX of bovine pancreatic ribonuclease A diffracts well when severely dehydrated. Crystal structures have been solved after 2.5 and 4 days of desiccation with CaSO4, at 1.9 and 2.0 Å resolution, respectively. The two desiccated structures are very similar. An RMS displacement of 1.6 Å is observed for main‐chain atoms in each structure when compared to the hydrated crystal structure with some large rearrangements observed in loop regions. The structural changes are the result of intermolecular contacts formed by strong electrostatic interactions in the absence of a high dielectric medium. The electron density is very diffuse for some surface loops, consistent with a very disordered structure. This disorder is related to the conformational changes. These results help explain conformational changes during the lyophilization of protein and the associated phenomena of denaturation and molecular memory.
Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent
Authors: Kleinschmidt Jörg H. , Wiener Michael C. , Tamm Lukas K.
Published: February 2009

Outer membrane protein A (OmpA) of Escherichia coli is a β‐barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in β‐barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS‐PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.
Barrel structures in proteins: Automatic identification and classification including a sequence analysis of TIM barrels
Authors: Nagano Nozomi , Gail HUTCHINSON E. , Thornton Janet M.
Published: February 2009

Automated methods for identifying and characterizing regular β‐barrels from coordinate data have been developed to analyze and classify various kinds of barrel structures based on geometric parameters such as the barrel strand number (n) and shear number (S). In total, we find 1,316 barrels in the January 1998 release of Protein Data Bank. Of 1,316 barrels, 1,277 barrels had an even shear number, corresponding to 50 nonhomologous families. The (βα)8 triose phosphate isomerase (TIM) barrel (n = 8, S = 8) fold has the largest number of apparently nonhomologous entries, 16, although the trypsin like antiparallel (n = 6, S = 8) barrels (representing only three families) are the most common with 527 barrels. Of all the protein families that exhibit barrel structures, 68% are found to be various kinds of enzymes, the remainder being binding proteins and transport membrane proteins. In addition, the layers of side chains, which form the cores of barrels with S = n and S = 2n, are also analyzed. More sophisticated methods were developed for detecting TIM barrels specifically, including consideration of the amino acid propensities for the side chains that form the layers. We found that the residues on the outside of the eight stranded parallel β‐barrel, buried by the α‐helices, are much more hydrophobic than the residues inside the barrel.
Mutants of ETS domain PU.1 and GGAA/T recognition: Free energies and kinetics
Authors: Pio F. , Assa‐Munt N. , Yguerabide J. , Maki R. A.
Published: February 2009

The ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS‐1 recognize different DNA sequences with a core element centered over 5′‐GGAA‐3′ and 5′‐GGAA/T‐3′, respectively. To understand the molecular basis of this recognition, we carried out site‐directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5′‐GGAA‐3′ and 5′‐GGAT‐3′ containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein‐DNA interaction with the 5′‐GGAA‐3′ containing oligonucleotide caused by a faster release of the protein from the DNA (koff tenfold higher than the wild‐type protein). With the double mutant A231S‐N236Y, we obtained an increase in binding affinity and stability toward both 5′‐GGAA‐3′ and 5′‐GGAT‐3′ containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5′‐GGAA‐3′ and 5′‐GGAT‐3′ oligonucleotides. The free energy of dissociation for the double mutant A231S‐N236Y with 5′‐GGAA‐3′ (ΔΔG((A231S‐N236Y) – (N236Y)) = ‐ 1.2 kcal mol−1) confirm the stabilizing effect of this mutant in the protein‐DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5′‐GGAA‐3′ and 5′‐GGAT‐3′ sequences, while A231S mutation modulates the degree of specificity toward 5′‐GGAA‐3′ and 5′GGAT‐3′ sequences. This study explains why wild‐type PU.1 does not recognize 5′‐GGAT‐3′ sequences and in addition broadens our understanding of 5′‐GGAA/T‐3′ recognition by ETS protein family members.
Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7‐Azido‐4‐methylcoumarin
Authors: Chen Guangping , Battaglia Eric , Senay Claire , Falany Charles N. , Radominska‐Pandya Anna
Published: February 2009

A novel fluorescent photoactive probe 7‐azido‐4‐methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P‐PST‐1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed inEscherichia colias a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki= 0.47 ± 0.05 mM). Moreover, enzyme activity toward 2‐naphthol was inactivated in a timeand concentration‐dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.
Mutagenic analysis of conserved arginine residues in and around the novel sulfate binding pocket of the human Theta class glutathione transferase T2‐2
Authors: Flanagan J. U. , Rossjohn J. , Parker M. W. , Board P. G. , Chelvanayagam G.
Published: February 2009

The human Theta class glutathione transferase GSTT2‐2 has a novel sulfatase activity that is not dependent on the presence of a conserved hydrogen bond donor in the active site. Initial homology modeling and the crystallographic studies have identified three conserved Arg residues that contribute to the formation of (Arg107 and Arg239), and entry to (Arg242), a sulfate binding pocket. These residues have been individually mutated to Ala to investigate their potential role in substrate binding and catalysis. The mutation of Arg107 had a significant detrimental effect on the sulfatase reaction, while the Arg242 mutation caused only a small reduction in sulfatase activity. Surprisingly, the Arg239 had an increased activity resulting from a reduction in stability. Thus, Arg239 appears to play a role in maintaining the architecture of the active site. Electrostatic calculations performed on the wild‐type and mutant forms of the enzyme are in good agreement with the experimental results. These findings, along with docking studies, suggest that prior to conjugation, the location of 1‐menaphthyl sulfate, a model substrate for the sulfatase reaction, is approximately midway between the position ultimately occupied by the naphthalene ring of 1‐menaphthylglutathione and the free sulfate. It is further proposed that the Arg residues in and around the sulfate binding pocket have a role in electrostatic substrate recognition.
Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor
Authors: Filippis Vincenzo De , Russo Ilaria , Vindigni Alessandro , Cera Enrico Di , Salmaso Stefano , Fontana Angelo
Published: February 2009

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2‐1.0 pM) and selectivity. Hirudin is composed of a compact N‐terminal region (residues 1‐47, cross‐linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C‐terminal tail (residues 48‐64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N‐terminal fragments have been prepared as potential anti‐coagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30‐400 nM). The aim of the present study is to obtain a derivative of the N‐terminal domain 1‐47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1‐47 of hirudin HM2 in which Val1 has been replaced by tert‐butylglycine, Ser2 by Arg, and Tyr3 by β‐naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6–Cys14, Cys16–Cys28, Cys22–Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507‐13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670‐ and 6,818‐fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full‐length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 μM).
Molecular dynamics investigation of the effect of an antiviral compound on human rhinovirus
Authors: Phelps Donald K. , Post Carol Beth
Published: February 2009

The factors that influence the enhanced stability observed experimentally of human rhinovirus 14 (HRV14) upon binding a hydrophobic antiviral drug have been investigated by molecular dynamics. Simulations centered about the HRV14 drug‐binding pocket allow the reliable assessment of differences in capsid protein motions of HRV14 and drug‐bound HRV14. We propose that the experimentally observed stabilization of the ligated virus arises from higher entropy, rather than enthalpy. Time‐averaged interaction energies between the viral protein and molecules occupying the pocket are less favorable in the presence of the drug, consistent with the proposal that the observed stability arises from entropie effects. Interaction energies characterizing subunit‐subunit contacts within one viral protomer are found to be substantially stronger than those between two protomers. Such distinction in subunit interaction would have clear implications on assembly and disassembly. Drug binding is found to affect large‐scale, collective properties, while leaving local atomic properties unperturbed. Specifically, the simulations reveal a weakening of long‐range correlations in atomic motions upon drug binding. On the other hand, neither the fast time scale RMS fluctuations of individual atomic positions nor the fluctuation build‐up curves from the capsid β‐sandwich forming the drug‐binding pocket show a consistent distinction between the drug‐bound and drug‐free viral simulations. Collectively, the detailed description available from the simulations provides an understanding of the experimental observations on the drug‐induced changes in thermal stability and protease sensitivity reported for picornaviruses. The predicted significance of binding entropy can be explored experimentally and should be considered in the design of new antiviral compounds.