bluuebunny/crossref_metadata_2025 huggingface dataset:
Overactive bladder: Is there a link to the metabolic syndrome in men?
Authors: Kirby Michael G. , Wagg Adrian , Cardozo Linda , Chapple Christopher , Castro‐Diaz David , de Ridder Dirk , Espuna‐Pons Montserrat , Haab Francois , Kelleher Con , Kölbl Heinz , Milsom Ian , Van Kerrebroeck Philip , Vierhout Mark , Salvatore Stefano , Tubaro Andrea
Published: June 2010

It is becoming increasingly clear that a variety of metabolic, cardiovascular, and endocrine factors contribute to male pelvic health. In particular, a growing body of evidence suggests a relationship between lower urinary tract symptoms, benign prostatic hyperplasia, overactive bladder, erectile dysfunction, and the metabolic syndrome. This article explores these relationships, focusing on the role of the autonomic nervous system and hyperinsulinemia, together with their implications for urological practice. Neurourol. Urodynam. 29:1360–1364, 2010. © 2010 Wiley‐Liss, Inc.
Motor deficits and neurofibromatosis type 1 (NF1)‐associated MRI impairments in a mouse model of NF1
Authors: Robinson Ari , Kloog Yoel , Stein Reuven , Assaf Yaniv
Published: June 2010

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized inter alia by cognitive and motor dysfunction and appearance of high‐signal foci on T2‐weighted images in the brain. Nf1+/− mice are useful models for studying aspects of NF1, including cognitive deficits. Here we assessed their motor performance and used quantitative transverse T2 relaxation MRI to identify structural abnormalities in their brains. Nf1+/− mice exhibited both enhanced and reduced T2 signals in distinct brain regions compared to wild‐type mice, and their motor performance was impaired. As in NF1 patients, enhanced T2 signals in Nf1+/− mice were observed in the thalamus and basal ganglia. Reduced T2 signals were seen in motor‐associated regions along the motor pathway, predominantly in the white matter of the cerebral peduncle and the optic tract. Correlation analysis between T2 signals and motor performance suggested that the motor deficits are associated with impairments in the cerebral peduncle and the amygdala. Copyright © 2010 John Wiley & Sons, Ltd.
Fanconi anemia gene mutations are not involved in sporadic Wilms tumor
Authors: Adank Muriel A. , Segers Heidi , van Mil Saskia E. , van Helsdingen Yvette M. , Ameziane Najim , van den Ouweland Ans M.W. , Wagner Anja , Meijers‐Heijboer Hanne , Kool Marcel , de Kraker Jan , Waisfisz Quinten , van den Heuvel‐Eibrink Marry M.
Published: June 2010

Bi‐allelic germline mutations of the Fanconi anemia (FA) genes, PALB2/FANCN and BRCA2/FANCD1, have been reported in a few Wilms tumor (WT) patients with an atypical FA phenotype. Therefore, we screened a random cohort of 47 Dutch WT cases for germline mutations in these two FA‐genes by DNA sequencing and Multiplex Ligation‐dependent Probe Amplification (MLPA). Although several cases appeared to carry missense variants, no bi‐allelic pathogenic mutations were identified, indicating that bi‐allelic mutations in these FA‐genes do not contribute significantly to the occurrence of WT. Pediatr Blood Cancer. 2010;55:742–744. © 2010 Wiley‐Liss, Inc.
Burkitt lymphoma: Residual abdominal tumor volume after induction therapy correlates with outcome
Authors: Hesseling Peter , McCormick Peter , Kouya Francine , Soh Felicite , Katayi Tshimwanga , Noglik Genny , Kidd Martin , Kenyu Ernest , Ngam Ivo , Tata George
Published: June 2010

Staging investigations in the Malawi 2003 BL protocol included abdominal ultrasonography. This sometimes demonstrated tumor that was not palpable. Patients with no palpable tumor following induction with three courses of cyclophosphamide were considered to be in remission, although residual intraabdominal tumor was documented in some by chance. We repeated ultrasonography on day 29 on 22 new patients with non‐palpable abdominal BL following induction. The relapse rate after 1 year correlated with the largest residual tumor volume, and was 17% for tumors 0–35 ml, and 75% for tumors ≥35 ml in volume. Risk patients were thus identified. Pediatr Blood Cancer. 2010;55:761–762. © 2010 Wiley‐Liss, Inc.
Myeloid neoplasm with prominent eosinophilia and PDGFRA rearrangement treated with imatinib mesylate
Authors: Rathe Mathias , Kristensen Thomas Kielsgaard , Møller Michael Boe , Carlsen Niels L.T.
Published: June 2010

The FIP1L1–PDGFRA fusion gene is the most frequent genetic aberration in myeloid neoplasms associated with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1. Affected patients in adult populations are very sensitive to imatinib therapy. Pediatric cases are rare and so far only one case of FIP1L1–PDGFRA positive disease has been reported. We report a 2‐year‐old female with a myeloid neoplasm associated with eosinophilia and rearrangement of PDGFRA. Treatment with imatinib resulted in complete and durable clinical, hematological, and molecular remission within 3 months after starting treatment. Pediatr Blood Cancer. 2010;55:730–732. © 2010 Wiley‐Liss, Inc.
Young age at diagnosis is a risk factor for negative late socio‐economic effects after acute lymphoblastic leukemia in childhood
Authors: Holmqvist Anna Sällfors , Wiebe Thomas , Hjorth Lars , Lindgren Anna , Øra Ingrid , Moëll Christian
Published: June 2010

Background

The increasing number of survivors after childhood cancer requires characterization of the late complications of these diseases and their treatment. We examined a large number of possible socio‐economic late effects following treatment for acute lymphoblastic leukemia (ALL) in order to identify factors leading to a poor outcome.

Procedure

All individuals who had been diagnosed with ALL and who were alive in January 2007 (n = 213; men = 107) were identified from a database of all patients with cancer before the age of 18 in Southern Sweden from 1970 to 1999. For each subject, 50 matched controls were identified from the Swedish Population Register. Information on marital status, children, education, employment, income, and support from the community was obtained from Statistics Sweden.

Results

At the ages of 25 and 30, survivors of ALL had attained a lower level of education than controls. At the age of 30, they were less often employed (70% vs. 82%, P = 0.019), less often married (19% vs. 32%, P = 0.019), and had children to a lesser extent (31% vs. 47%, P = 0.011) than controls. We identified young age at diagnosis as a risk factor for adverse outcome in the majority of the socio‐economic variables studied, apart from the known risk of cranial irradiation treatment. Furthermore, female survivors had a greater risk of achieving a lower level of education than both male survivors and controls.

Conclusions

Young age at diagnosis, as well as treatment with cranial irradiation, is a risk factor for socio‐economic late effects after treatment for ALL in childhood. Pediatr Blood Cancer. 2010;55:698–707. © 2010 Wiley‐Liss, Inc.
Flow/pressure characteristics and modeling of deformation processes of single‐screw extruders
Authors: Borisov A.A. , Berdyshev B.V. , Hosseini H. , Shirkavand‐Hadavand B.
Published: June 2010

One of the most widespread practical methods of polymer processing is the extrusion method that is based on pressing a polymeric melt through channels of the molding tool which have different geometrical cross sections. The principal technical characteristic of extruders, named flow/pressure balance, and also modeling of deformation processes permit to evaluate their practical output in processing various polymers as a function of the hydraulic resistance which depends on the flow of an extruded polymeric material through the channels of the forming tool. The validity of obtained and presented results permits to recommend them reasonably not only for estimation of extruder output but also for making calculations necessary in extruder equipment design: checking calculation of strength and stability of a screw, calculation of driving power of the equipment being designed, strength calculation of a plasticizing cylinder, etc. POLYM. ENG. SCI., 50:2069–2073, 2010. © 2010 Society of Plastics Engineers
Comparison study of graphite nanosheets and carbon black as fillers for high density polyethylene
Authors: Wang Liwei , Hong Jiangbin , Chen Guohua
Published: June 2010

In this work, the properties of high density polyethylene (HDPE)/graphite nanosheets (GN) and HDPE/carbon black (CB) nanocomposites prepared by melt mixing were studied. GN‐filled HDPE nanocomposites exhibited very low percolation threshold (ca. 6 wt%) as compared with that of HDPE/CB (ca. 22 wt%). Moreover, the impact strength and processing properties of HDPE/GN nanocomposites were superior to those of HDPE/CB within a certain content of fillers. The large aspect ratio of GN plays an important role in reducing the percolation threshold of HDPE/GN nanocomposites. Furthermore, the tensile fracture morphology revealed the formation of continuous conducting networks after the content of GN reached the critical percolation threshold. Differential scanning calorimetry (DSC) analysis illustrated a decreasing degree of crystallinity of HDPE/GN nanocomposites, while the crystalline temperature varied slightly. POLYM. ENG. SCI., 2010. © 2010 Society of Plastics Engineers
Viscoelastic flow analysis of surface morphology on injection‐molded polypropylene
Authors: Kobayashi Yutaka , Otsuki Yasuhiko , Kanai Toshitaka
Published: June 2010

The molecular orientation of a frozen layer in an injection‐molded specimen of a polypropylene–rubber blend was investigated. A typical V‐shaped pattern of birefringence was observed from the surface to the core in a crosscut section. From the comparison of the V‐patterns near the gate to the flow end, it was assumed that a frozen layer formed from the surface to a depth of 0.06 mm in a plaque (3 mm thickness) during the injection molding filling process. Numerical viscoelastic analysis of the fountain flow was carried out using an original 2D unsteady flow simulation program and ignored crystallization. A large extensional deformation formed just when the molten polymer contacted the cavity wall and the deformation immediately froze. A layer with a small birefringence between the surface and the shear‐oriented layer was divided into two parts. The depth profile of birefringence was compared to the principal stress difference calculated by numerical analysis. POLYM. ENG. SCI., 2010. © 2010 Society of Plastics Engineers
Orthogonal design study on factors effecting on fibers diameter of melt electrospinning
Authors: Liu Yong , Deng Rongjian , Hao Mingfeng , Yan Hua , Yang Weimin
Published: June 2010

Melt electrospinning is a much more simple and safe method to produce ultra fine fibers than solution electrospinning. The diameters of melt electrospinning fibers are thicker. To find the factors that affect the fibers diameter in melt electrospinning, orthogonal design was used to examine melt temperature, spinning voltage, spinning distance, and melt flow rate (MFR) of polymer. Results showed that MFR at present three levels has the most important impact both on the average diameters and standard deviations of fiber diameters. The scanning electron microscopy pictures show that all the fibers have smooth surface, which means the melt electrospinning fibers have good mechanical properties. POLYM. ENG. SCI., 50:2074–2078, 2010. © 2010 Society of Plastics Engineers
Turbulenz bändigen
Authors: Hof Björn , Schneider Tobias M.
Published: June 2010

Turbulente Strömungen haben einen höheren Reibungswiderstand als laminare. Insbesondere bei hohen Durchflussraten können Turbulenzen zum Beispiel in Ölpipelines oder städtischen Wasserleitungen die Transportkosten auf das 100‐Fache ansteigen lassen. Neue Einsichten in die physikalischen Mechanismen, die turbulente Wirbel erzeugen, haben es unserer Gruppe aus Göttingen und Harvard erstmalig ermöglicht, im Experiment eine turbulente Strömung kontrolliert wieder in den laminaren Zustand zurückzuführen.
Solvatochromism as an efficient tool to study N,N‐dimethylamino‐ and cyano‐substituted π‐conjugated molecules with an intramolecular charge‐transfer absorption
Authors: Bureš Filip , Pytela Oldřich , Kivala Milan , Diederich François
Published: June 2010

A representative data set has been gained by the measurement of the electronic absorption spectra of 12 systematically selected push–pull systems with an intramolecular charge‐transfer (CT) absorption and the general structure D–π–A (D = donor, A = acceptor) featuring electron‐withdrawing CN groups, electron‐donating N(CH3)2 groups, and various π‐conjugated backbones in 32 solvents with different polarities. The longest‐wavelength absorption maxima λmax and the corresponding wavenumbers $\tilde {v}_{{\rm max}} $ were evaluated from the UV/Vis spectra measured in 32 well‐selected solvents. The D–π–A push–pull systems were further characterized by quantum‐chemical quantities and simple structural parameters. Structure–solvatochromism relationships were evaluated by multidimensional statistic methods. Whereas solvent polarizability and solvent cavity size proved to be the most important factors affecting the position of λmax, the solvent polarity was less important. The most important characteristics of organic CT compounds are the energy of the LUMO, the permanent dipole moment, the COSMO (COnductor‐like Screening MOdel) area, the COSMO volume, the number, and ratio of N,N‐dimethylamino and cyano groups, and eventually the number of triple bonds (π‐linkers). A relation between the first‐order polarizability α, the longest‐wavelength absorption maxima λmax, and the structural features has also been found. The higher‐order polarizabilities β and γ are not related to the observed solvatochromism. Copyright © 2010 John Wiley & Sons, Ltd.
Solifluction and mudflow on a limestone periglacial slope in the Swiss Alps: 14 years of monitoring
Authors: Matsuoka Norikazu
Published: June 2010

The dynamics of stone‐banked lobes were investigated by monitoring soil movements and environmental parameters over 14 years on a limestone slope subject to deep seasonal frost. The monitored parameters included downslope soil movement, frost heave, soil temperature, moisture and snow depth. Despite being composed of the same soil, lobes on the upper slope (∼20°) display pressure ridges on the tread and terminate in a high riser (∼1 m), whereas lobes on the lower slope (∼10°) have a flat tread and a low riser (∼0.5 m). The long‐term monitoring demonstrates that gelifluction, resulting from seasonal frost heave of approximately 5 cm and thaw settlement, occurs annually in the lower lobes at a mean surface rate of 4 cm a−1 and with a movement base at approximately 40‐cm depth. The rate of gelifluction shows some interannual fluctuation depending primarily on the annual frost heave amount, but overall regularity of the rate and depth of movement regardless of seasonal frost depth results in lobes having similar morphologies. On the upper, steeper lobes located just below late‐lying snow patches, rapid, shallow mudflows (∼20 cm thick) are often superimposed on gelifluction. The rate of surface movement varies spatially, in places exceeding 2 m per event. The prolonged water supply and low consistency limits of the limestone soil favour mudflows on seasonal thawing. The transported sediments pile up near the lobe front, resulting in a high riser. Such modification of solifluction lobes by mudflows can occur in other slopes with similar gradients, soils, frost and snow conditions. Copyright © 2010 John Wiley & Sons, Ltd.
Domain motion and interdomain hot spots in a multidomain enzyme
Authors: Chuang Gwo‐Yu , Mehra‐Chaudhary Ritcha , Ngan Chi‐Ho , Zerbe Brandon S. , Kozakov Dima , Vajda Sandor , Beamer Lesa J.
Published: June 2010

The aim of this article is to analyze conformational changes by comparing 10 different structures of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase (PMM/PGM), a four‐domain enzyme in which both substrate binding and catalysis require substantial movement of the C‐terminal domain. We focus on changes in interdomain and active site crevices using a method called computational solvent mapping rather than superimposing the structures. The method places molecular probes (i.e., small organic molecules containing various functional groups) around the protein to find hot spots. One of the most important hot spots is in the active site, consistent with the ability of the enzyme to bind both glucose and mannose phosphosugar substrates. The protein has eight additional hot spots at domain‐domain interfaces and hinge regions. The locations and nature of six of these hot spots vary between the open, half‐open, and closed conformers of the enzyme, in good agreement with the ligand‐induced conformational changes. In the closed structures the number of probe clusters at the hinge region significantly depends on the position of the phosphorylated oxygen in the substrate (e.g., glucose 1‐phosphate versus glucose 6‐phosphate), but the protein remains almost unchanged in terms of the overall RMSD, indicating that computational solvent mapping is a more sensitive approach to detect changes in binding sites and interdomain crevices. Focusing on multidomain proteins we show that the subresolution conformational differences revealed by the mapping are in fact significant, and present a general statistical method of analysis to determine the significance of rigid body domain movements in X‐ray structures.
Rearranging the domains of pepsinogen
Authors: Lin Xinli , Koelsch Gerald , Loy Jeffrey A. , Tang Jordan
Published: June 2010

Most eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation. In pepsinogen, the pro peptide precedes the N‐terminal lobe (designated pep) and the C‐terminal lobe (designated sin). Based on the three‐dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro‐pep‐sin (native pepsinogen) to sin‐pro‐pep. The domain‐rearranged zymogen also contains a 10‐residue linker designed to connect sin and pro domains. Recombinant sin‐pro‐pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified. Upon acidification, sin‐pro‐pep autoactivates to a two‐chain enzyme. However, the emergence of activity is much slower than the conversion of the single‐chain zymogen to a two‐chain intermediate. In the activation of native pepsinogen and sin‐pro‐pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin‐pro‐pep requires an additional cleavage at a third site between residues IP and 2P. In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity. In the activation of sin‐pro‐pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step. The reason for these differences is the result of the presence of activation intermediates bearing pro peptide 1P‐16P, which is still covalently attached to the sin domain after the first and second cleavages. This peptide is known to have affinity to the enzyme moiety. Its presence apparently prevents the full expression of proteolytic activity, which catalyzes the cleavage of sites 2 and 3. A mechanism of intramolecular cleavage of site 1 is proposed that involves the local conformational change only near site 1 in the N‐terminal region of the pro peptide.
Heterogeneity of the covalent structure of the blue copper protein umecyanin from horseradish roots
Authors: Driessche Gonzalez Van , Beeumen Jozef Van , Dennison Christopher , Geoffrey Sykes A.
Published: June 2010

The covalent structure of umecyanin has been determined by a combination of classical Edman degradation sequence analysis and plasma desorption, laser desorption, and electrospray ionization mass spectrometry. The preparation appeared to contain two isoforms having either a valine (75%) or an isoleucine (25%) residue at position 48. The polypeptide chain of 115 amino acids is strongly heterogeneous at its C‐terminal end as a result of proteolytic cleavages at several places within the last 10 residues. The major fraction of the umecyanin preparation is only 106 residues long. The C‐terminal tail 107–115 contains mainly alanine and glycine residues and a single hydroxyproline residue. In the native protein there is a disulfide bridge between Cys 91 and Cys 57, but in the apoprotein there is a disulfide shift that involves Cys 91 and one of the four copper binding residues (Cys 85). The three other ligand binding residues are His 44, His 90, and Gin 95. This tetrad of amino acids is the same as occurs in other type 1 copper proteins from plants such as cucumber peeling cupredoxin and lacquer tree stellacyanin. The umecyanin isoforms are glycoproteins with a glycan core having the same carbohydrate composition as that of horseradish peroxidase, a fact that is convincingly supported thanks to the high accuracy of the electrospray mass spectrometric technique. We suggest that the glycan may play a role in the association of the protein to the cellular membrane, but the precise functional role of umecyanin remains to be determined. We also discuss the evolutionary position of umecyanin in relation to the type 1 copper proteins in general.
Octameric enolase from the hyperthermophilic bacterium Thermotoga maritima: Purification, characterization, and image processing
Authors: Schurig Hartmut , Rutkat Kerstin , Jaenicke Rainer , Rachel Reinhard
Published: June 2010

Enolase (2‐phospho‐D‐glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N‐terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel‐permeation chromatography, the enzyme is a 345‐kDa homooctamer with a subunit molecular mass of 48 ± 5 kDa. Electron microscopy and image processing yield ring‐shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 °C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol−1 and 43 kJ mol−1 at temperatures below and above 45 °C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2‐phospho‐D‐glycerate and Mg2+ at 75 °C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 °C in the absence and in the presence of Mg2+.
Weakening of the interface between adjacent catalytic chains promotes domain closure in escherichia coli aspartate transcarbamoylase
Authors: Baker Darren P. , Keiser Richard T. , Kantrowitz Evan R. , Fetler Luc , Vachette Patrice
Published: June 2010

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp‐100, from one catalytic chain, is involved in stabilizing the C1‐C2 interface by means of its interaction with Arg‐65 from an adjacent catalytic chain. Replacement of Asp‐100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 52:10150–10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1‐C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp‐100 → Ala mutation was introduced into the Glu‐50 → Ala holoenzyme, a mutant in which domain closure is impaired. The Glu‐50/Asp‐100 → Ala enzyme is fourfold more active than the Glu‐50 → Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp‐100 → Ala mutation restores the ability of the Glu‐50 → Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N‐(phosphonacetyl)‐L‐aspartate (PALA). At subsaturating concentrations of aspartate, the Glu‐50/Asp‐100 → Ala enzyme is activated more by ATP than the Glu‐50 → Ala enzyme and is also inhibited more by CTP than either the wild‐type or the Glu‐50 → Ala enzyme. As opposed to the wild‐type enzyme, the Glu‐50/Asp‐100 → Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu‐50/Asp‐100 → Ala enzyme by solution X‐ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild‐type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu‐50/Asp‐100 → Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu‐50 → Ala and wild‐type enzymes. Solution X‐ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu‐50/Asp‐100 → Ala enzyme.
Divergent evolution of a β/α‐barrel subclass: Detection of numerous phosphate‐binding sites by motif search
Authors: Bork Peer , Gellerich Johannes , Groth Holger , Hooft Rob , Martin Falk
Published: June 2010

Study of the most conserved region in many β/α‐barrels, the phosphate‐binding site, revealed a sequence motif in a few β/α‐barrels with known tertiary structure, namely glycolate oxidase (GOX), cytochrome b2 (Cyb2), tryptophan synthase α subunit (TrpA), and the indoleglycerolphosphate synthase (TrpC). Database searches identified this motif in numerous other enzyme families: (1) IMP dehydrogenase (IMPDH) and GMP reductase (GuaC); (2) phosphoribosylformimino‐5‐aminoimidazol carboxamide ribotide isomerase (HisA) and the cyclase‐producing D‐erythro‐imidazole‐glycerolphosphate (HisF) of the histidine biosynthetic pathway; (3) dihydroorotate dehydrogenase (PyrD); (4) glutamate synthase (GltB); (5) ThiE and ThiG involved in the biosynthesis of thiamine as well as related proteins; (6) an uncharacterized open reading frame from Erwinia herbicola; and (7) a glycerol uptake operon antiterminator regulatory protein (GlpP). Secondary structure predictions of the different families mentioned above revealed an alternating order of β‐strands and α‐helices in agreement with a β/α‐barrel‐like topology. The putative phosphate‐binding site is always found near the C‐terminus of the enzymes, which are all at least about 200 amino acids long. This is compatible with its assumed location between strand 7 and helix 8. The identification of a significant motif in functionally diverse enzymes suggests a divergent evolution of at least a considerable fraction of β/α‐barrels. In addition to the known accumulation of β/α‐barrels in the tryptophan biosynthetic pathway, we observe clusters of these enzymes in histidine biosynthesis, purine metabolism, and apparently also in thiamine biosynthesis. The substrates are mostly heterocyclic compounds. Although the marginal sequence similarities do not allow a reconstruction of the barrel spreading, they support the idea of pathway evolution by gene duplication.
Structural basis of substrate specificity in the serine proteases
Authors: Perona John J. , Craik Charles S.
Published: June 2010

Structure‐based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system.
Approaches to labeling and identification of active site residues in glycosidases
Authors: Withers Stephen G. , Aebersold Ruedi
Published: June 2010

Glycosidases play a key role in a number of biological processes and, as such, are of considerable clinical and bio‐technological importance. Knowledge of the identities of catalytically important active site residues is essential for understanding the catalytic mechanism, for enzyme classification, and for targeted bioengineering of glycosidases with altered characteristics. Here we review and discuss traditional strategies and novel approaches based on tandem mass spectrometry for the identification of the key active site residues in glycosidases.
Conservative and nonconservative mutations in proteins: Anomalous mutations in a transport receptor analyzed by free energy and quantum chemical calculations
Authors: Cannon William R. , Briggs James M. , Shen Jian , McCammon J. Andrew , Quiocho Florante A.
Published: June 2010

Experimental studies on a bacterial sulfate receptor have indicated anomalous relative binding affinities for the mutations Ser130 → Cys, Ser130 → Gly, and Ser130 → Ala. The loss of affinity for sulfate in the former mutation was previously attributed to a greater steric effect on the part of the Cys side chain relative to the Ser side chain, whereas the relatively small loss of binding affinity for the latter two mutations was attributed to the loss of a single hydrogen bond. In this report we present quantum chemical and statistical thermodynamic studies of these mutations. Qualitative results from these studies indicate that for the Ser130 → Cys mutation the large decrease in binding affinity is in part caused by steric effects, but also significantly by the differential work required to polarize the Cys thiol group relative to the Ser hydroxyl group. The Gly mutant cobinds a water molecule in the same location as the Ser side chain resulting in a relatively small decrease in binding affinity. Results for the Ala mutant are in disagreement with experimental results but are likely to be limited by insufficient sampling of configuration space due to physical constraints applied during the simulation.
Stability of ribonuclease T2 from Aspergillus oryzae
Authors: Kawata Yasushi , Hamaguchi Kozo
Published: June 2010

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal‐mol−1 at 25 °C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 °C and 119.1 kcal‐mol−1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNase T2 in the native state is predominantly due to the slow rate of unfolding.
Comparative modeling of the three‐dimensional structure of Type II antifreeze protein
Authors: Sönnichsen Frank D. , Sykes Brian D. , Davies Peter L.
Published: June 2010

Type II antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C‐type lectins. The structure of this domain from rat mannose‐binding protein (MBP‐A) has been solved by X‐ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 560:127–134) and provided the coordinates for constructing the three‐dimensional model of the 129‐amino acid Type II AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type II AFPs, pancreatic stone protein, MBP‐A, and as many as 50 carbohydrate‐recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two α‐helices, two β‐sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type II AFP and MBP‐A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 111, and Cys 101 and Cys 117. Based on the predicted features of this model, a site for proteinice interaction is proposed.
Stabilization of a strained protein loop conformation through protein engineering
Authors: Hodel Alec , Fox Robert O. , Kautz Roger A.
Published: June 2010

Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116‐ Pro 117 peptide bond, resulting in two different conformations of the residue 112–117 loop. Thecisform is favored over thetranswith an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, atransconformation is stabilized relative to thecisconformation by the release of steric strain in thetransform. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild‐type protein in its structure and in the dominance of thecisconfiguration. The results of these studies suggested that any nuclease variant with a non‐glycine residue at position 116 should also favor thecisform because of steric requirements of the β‐carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the “β‐carbon” hypothesis by favoring thecisform. Furthermore, the crystal structure of K116E is nearly identical to that of the wild‐type protein. Two additional variants, K116D and K16N, provide exceptions to this simple “β‐carbon” rule in that thetransconformation is stabilized relative to thecisconfiguration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailedtransconformation of the K116D variant appears to be similar to thetransconformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.
Simultaneous modeling of multiple loops in proteins
Authors: Rosenbach Dror , Rosenfeld Rakefet
Published: June 2010

The most reliable methods for predicting protein structure are by way of homologous extension, using structural information from a closely related protein, or by “threading” through a set of predefined protein folds (“inverse folding”). Both sets of methods provide a model for the core of the protein —the structurally conserved secondary structures. Due to the large variability both in sequence and size of the loops that connect these secondary structures, they generally cannot be modeled using these techniques. Loop‐closure algorithms are aimed at predicting loop structures, given their end‐to‐end distance. Various such algorithms have been described, and all have been tested by predicting the structure of a single loop in a known protein. In this paper we propose a method, which is based on the bond‐scaling‐relaxation loop‐closure algorithm, for simultaneously predicting the structures of multiple loops, and demonstrate that, for two spatially close loops, simultaneous closure invariably leads to more accurate predictions than sequential closure. The accuracy of the predictions obtained for pairs of loops in the size range of 5–7 residues each is comparable to that obtained by other methods, when predicting the structures of single loops: the RMS deviations from the native conformations of various test cases modeled are approximately 0.6–1.7 Å for backbone atoms and 1.1–3.3 Å for all‐atoms.
Transmembrane helices predicted at 95% accuracy
Authors: Rost Burkhard , Sander Chris , Casadio Rita , Fariselli Piero
Published: June 2010

We describe a neural network system that predicts the locations of transmembrane helices in integral membrane proteins. By using evolutionary information as input to the network system, the method significantly improved on a previously published neural network prediction method that had been based on single sequence information. The input data were derived from multiple alignments for each position in a window of 13 adjacent residues: amino acid frequency, conservation weights, number of insertions and deletions, and position of the window with respect to the ends of the protein chain. Additional input was the amino acid composition and length of the whole protein. A rigorous cross‐validation test on 69 proteins with experimentally determined locations of transmembrane segments yielded an overall two‐state per‐residue accuracy of 95%. About 94% of all segments were predicted correctly. When applied to known globular proteins as a negative control, the network system incorrectly predicted fewer than 5% of globular proteins as having transmembrane helices. The method was applied to all 269 open reading frames from the complete yeast VIII chromosome. For 59 of these, at least two transmembrane helices were predicted. Thus, the prediction is that about one‐fourth of all proteins from yeast VIII contain one transmembrane helix, and some 20%, more than one.
Principles of protein folding — A perspective from simple exact models
Authors: Dill Ken A. , Bromberg Sarina , Yue Kaizhi , Chan Hue Sun , Ftebig Klaus M. , Yee David P. , Thomas Paul D.
Published: June 2010

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two‐state cooperativity, secondary and tertiary structures, and multistage folding kinetics —fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating “foldable” chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent‐averse.
Proline cis‐trans isomerization in staphylococcal nuclease: Multi‐substate free energy perturbation calculations
Authors: Hodel Alec , Rice Luke M. , Simonson Thomas , Fox Robert O. , Brünger Axel T.
Published: June 2010

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116‐Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (δDLGcis‐trans ≈ 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (ΔGcis‐trans (ΔTScis‐trans ≈ 10 kcal/mol). There is evidence from X‐ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112–117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substates. By partitioning the search space into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X‐ray structures of nuclease variants and yield a model of the unknown minor trans conformation.
NMR studies of novel inhibitors bound to farnesyl‐protein transferase
Authors: Koblan Kenneth S. , Mosser Scott D. , Omer Charles A. , Christopher Culberson J. , Jane Desolms S. , Giuliani Elizabeth A. , Pitzenberger Steven M. , Bogusky Michael J.
Published: June 2010

Farnesyl‐protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl‐terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell‐transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras‐dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase‐bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L‐739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two‐dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active‐site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse‐turn conformations most closely approximating a type III β‐turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.
Carbohydrate binding sites in a pancreatic α‐amylase‐substrate complex, derived from X‐ray structure analysis at 2.1 Å resolution
Authors: Qian Minxie , Haser Richard , Payan FranÇoise
Published: June 2010

The X‐ray structure analysis of a crystal of pig pancreatic α‐amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high‐resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duée E, Haser H, Payan F, 1994, Biochemistry 55:6284–6294). One of the binding sites previously identified in a 5‐Å‐resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R‐factor of 17.5%, with an RMS deviation in bond distances of 0.007 Å. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N‐terminal pyrrolidonecarboxylic acid in PPA.
Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two‐dimensional NMR
Authors: Zhang Yu‐Zhu , Roder Heinrich , Paterson Yvonne
Published: June 2010

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein‐ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO‐D2O mixtures was investigated by NMR methods. As in water, H‐D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid‐ and base‐catalyzed processes and negligible water catalysis. However, the base‐catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is ∼ 100‐fold reduced relative to that in D2O. The solvent‐dependent decrease in base‐catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKw seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent‐quenching method, melittin was exchanged in D2O (pH 3.2, 12 °C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.