bluuebunny/crossref_metadata_2025_split huggingface dataset:
Allergic bronchopulmonary aspergillosis in a 2‐year‐old asthmatic boy with immune dysregulation, polyendocrinopathy, enteropathy, X‐linked
Authors: Ohshima Mihoko , Futamura Masaki , Kamachi Yoshiro , Ito Komei , Sakamoto Tatsuo 
Published: February 2009

AbstractImmune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) is caused by impaired function of CD4+CD25+ regulatory T cells that play an important role in controlling exaggerated Th2 responses. The pathogenesis of allergic bronchopulmonary aspergillosis (ABPA) appears to be closely associated with the Th2 response. We report the first case of ABPA in a 2‐year‐old asthmatic boy with IPEX, this being an unusually young age for the development of ABPA. Pediatr Pulmonol. 2009; 44:297–299. © 2009 Wiley‐Liss, Inc.
Lower airway obstruction is associated with increased morbidity in children with sickle cell disease
Authors: Boyd Jessica H. , DeBaun Michael R. , Morgan Wayne J. , Mao Jingnan , Strunk Robert C. 
Published: February 2009

AbstractRationaleThe association between pulmonary function and morbidity in children with sickle cell disease (SCD) has not been previously evaluated. Our objective was to study the relationship between abnormalities in pulmonary function and morbidity as represented by the rate of hospitalizations for pain or acute chest syndrome (ACS) in children with SCD.MethodsResults of pulmonary function tests obtained for clinical indications in children ages 6–18 years were classified as lower airway obstruction (forced expiratory volume in 1 sec/forced volume capacity <95% confidence interval adjusted for age, gender, race, and height), restriction (total lung capacity <80% predicted adjusted for gender, age, race, and height), and normal lung function. Incidence rates of pain or ACS were compared between children with lower airway obstruction or restriction and children with normal lung function.ResultsA total of 102 children, mean age at evaluation 12.0 years with follow‐up of 3.8 years, were included. Children with lower airway obstruction had twice the rate of morbidity compared to children with normal lung function (2.5 vs. 1.2 hospitalizations for pain or ACS per patient‐year, P = 0.003) (Risk ratio: 2.0; 95% CI: 1.3–3.3). Children with restriction did not have different rates of future morbidity compared to children with normal lung function (1.4 vs. 1.2 hospitalizations for pain or ACS per patient‐year, P = 0.68) (Rate ratio: 1.1; 95% CI: 0.6–2.1).ConclusionsWe conclude that children with SCD who have lower airway obstruction should have increased surveillance for future morbidity. Pediatr Pulmonol. 2009; 44:290–296. © 2009 Wiley‐Liss, Inc.
Thiol‐disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum)
Authors: Weiner Lev , Shin Irina , Shimon Linda J. W. , Miron Talia , Wilchek Meir , Mirelman David , Frolow Felix , Rabinkov Aharon 
Published: February 2009

AbstractAlliinase, an enzyme found in garlic, catalyzes the synthesis of the well‐known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold‐type I family of pyridoxal‐5′‐phosphate‐dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a SS bridge located near the C‐terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate‐cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored SH reagent N‐(4‐dimethylamino‐3,5‐dinitrophenyl) maleimide yielded chromophore‐bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free SH groups. This allowed the oriented conjugation of alliinase, via the SH groups, with low‐ or high‐molecular‐weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
Structural modification of acyl carrier protein by butyryl group
Authors: Wu Bai‐Nan , Zhang Yong‐Mei , Rock Charles O. , Zheng Jie J. 
Published: February 2009

AbstractFatty acid synthesis in bacteria is catalyzed by a set of individual enzymes known as the type II fatty acid synthase. Acyl carrier protein (ACP) shuttles the acyl intermediates between individual pathway enzymes. In this study, we determined the solution structures of three different forms of ACP, apo‐ACP, ACP, and butyryl‐ACP under identical experimental conditions. The structural studies revealed that attachment of butyryl acyl intermediate to ACP alters the conformation of ACP. This finding supports the more general notion that the attachment of different acyl intermediates alters the ACP structure to facilitate their recognition and turnover by the appropriate target enzymes.
High‐resolution NMR characterization of a spider‐silk mimetic composed of 15 tandem repeats and a CRGD motif
Authors: McLachlan Glendon D. , Slocik Joseph , Mantz Robert , Kaplan David , Cahill Sean , Girvin Mark , Greenbaum Steve 
Published: February 2009

AbstractMultidimensional solution NMR spectroscopic techniques have been used to obtain atomic level information about a recombinant spider silk construct in hexafluoro‐isopropanol (HFIP). The synthetic 49 kDa silk‐like protein mimics authentic silk from Nephila clavipes, with the inclusion of an extracellular matrix recognition motif. 2D 1H‐15N HSQC NMR spectroscopy reveals 33 cross peaks, which were assigned to amino acid residues in the semicrystalline repeat units. Signals from the amorphous segments in the primary sequence were weak and broad, suggesting that this region is highly dynamic and undergoing conformational exchange. An analysis of the deviations of the 13Cα, 13Cβ, and 13CO chemical shifts relative to the expected random coil values reveals two highly α‐helical regions from amino acid 12–19 and 26–32, which comprise the polyalanine track and a GGLGSQ sequence. This finding is further supported by ϕ‐value analysis and sequential and medium‐range NOE interactions. Pulsed field gradient NMR measurements indicate that the topology of the silk mimetic in HFIP is nonglobular. Moreover, the 3D 15N‐NOESY HSQC spectrum exhibits few long‐range NOEs. Similar spectral features have been observed for repeat modules in other polypeptides and are characteristic of an elongated conformation. The results provide a residue‐specific description of a silk sequence in nonaqueous solution and may be insightful for understanding the fold and topology of highly concentrated, stable silk before spinning. Additionally, the insights obtained may find application in future design and large‐scale production and storage of synthetic silks in organic solvents.
Insights from multiple structures of the shell proteins from the β‐carboxysome
Authors: Tanaka Shiho , Sawaya Michael R. , Phillips Martin , Yeates Todd O. 
Published: February 2009

AbstractCarboxysomes are primitive bacterial organelles that function as a part of a carbon concentrating mechanism (CCM) under conditions where inorganic carbon is limiting. The carboxysome enhances the efficiency of cellular carbon fixation by encapsulating together carbonic anhydrase and the CO2‐fixing enzyme ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO). The carboxysome has a roughly icosahedral shape with an outer shell between 800 and 1500 Å in diameter, which is constructed from a few thousand small protein subunits. In the cyanobacterium Synechocystis sp. PCC 6803, the previous structure determination of two homologous shell protein subunits, CcmK2 and CcmK4, elucidated how the outer shell is formed by the tight packing of CcmK hexamers into a molecular layer. Here we describe the crystal structure of the hexameric shell protein CcmK1, along with structures of mutants of both CcmK1 and CcmK2 lacking their sometimes flexible C‐terminal tails. Variations in the way hexamers pack into layers are noted, while sulfate ions bound in pores through the layer provide further support for the hypothesis that the pores serve for transport of substrates and products into and out of the carboxysome. One of the new structures provides a high‐resolution (1.3 Å) framework for subsequent computational studies of molecular transport through the pores. Crystal and solution studies of the C‐terminal deletion mutants demonstrate the tendency of the terminal segments to participate in proteinprotein interactions, thereby providing a clue as to which side of the molecular layer of hexameric shell proteins is likely to face toward the carboxysome interior.
Crystal structures of the X‐domains of a Group‐1 and a Group‐3 coronavirus reveal that ADP‐ribose‐binding may not be a conserved property
Authors: Piotrowski Yvonne , Hansen Guido , Boomaars‐van der Zanden A. Linda , Snijder Eric J. , Gorbalenya Alexander E. , Hilgenfeld Rolf 
Published: February 2009

AbstractThe polyproteins of coronaviruses are cleaved by viral proteases into at least 15 nonstructural proteins (Nsps). Consisting of five domains, Nsp3 is the largest of these (180–210 kDa). Among these domains, the so‐called X‐domain is believed to act as ADP‐ribose‐1″‐phosphate phosphatase or to bind poly(ADP‐ribose). However, here we show that the X‐domain of Infectious Bronchitis Virus (strain Beaudette), a Group‐3 coronavirus, fails to bind ADP‐ribose. This is explained on the basis of the crystal structure of the protein, determined at two different pH values. For comparison, we also describe the crystal structure of the homologous X‐domain from Human Coronavirus 229E, a Group‐1 coronavirus, which does bind ADP‐ribose.
Single molecule effects of osteogenesis imperfecta mutations in tropocollagen protein domains
Authors: Gautieri Alfonso , Vesentini Simone , Redaelli Alberto , Buehler Markus J. 
Published: February 2009

AbstractOsteogenesis imperfecta (OI) is a genetic disease characterized by fragile bones, skeletal deformities and, in severe cases, prenatal death that affects more than 1 in 10,000 individuals. Here we show by full atomistic simulation in explicit solvent that OI mutations have a significant influence on the mechanical properties of single tropocollagen molecules, and that the severity of different forms of OI is directly correlated with the reduction of the mechanical stiffness of individual tropocollagen molecules. The reduction of molecular stiffness provides insight into the molecular‐scale mechanisms of the disease. The analysis of the molecular mechanisms reveals that physical parameters of side‐chain volume and hydropathy index of the mutated residue control the loss of mechanical stiffness of individual tropocollagen molecules. We propose a model that enables us to predict the loss of stiffness based on these physical characteristics of mutations. This finding provides an atomistic‐level mechanistic understanding of the role of OI mutations in defining the properties of the basic protein constituents, which could eventually lead to new strategies for diagnosis and treatment the disease. The focus on material properties and their role in genetic diseases is an important, yet so far only little explored, aspect in studying the mechanisms that lead to pathological conditions. The consideration of how material properties change in diseases could lead to a new paradigm that may expand beyond the focus on biochemical readings alone and include a characterization of material properties in diagnosis and treatment, an effort referred to as materiomics.
Using affinity chromatography to engineer and characterize pH‐dependent protein switches
Authors: Sagermann Martin , Chapleau Richard R. , DeLorimier Elaine , Lei Margarida 
Published: February 2009

AbstractConformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione‐S‐transferase to convert this protein into a pH‐dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH‐dependent GSH‐binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector‐specific allostery into a protein structure.
Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR
Authors: Rajapaksha Maheshinie , Eichler Jack F. , Hajduch Jan , Anderson David E. , Kirk Kenneth L. , Bann James G. 
Published: February 2009

AbstractThe binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional 13C‐edited 1H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.
The oligomerization of OxyR in Escherichia coli
Authors: Knapp Gwendowlyn S. , Tsai Jerry W. , Hu James C. 
Published: February 2009

AbstractWe examine the contribution of residues at the dimer interface of the transcriptional regulator OxyR to oligomerization. Residues in contact across the dimer interface of OxyR were identified using the program Quaternary Contacts (QContacts). Site‐directed mutagenesis was performed on the non‐alanine or glycine residues identified in the resultant contact profile and the oligomerization ability of the mutant proteins was tested using the λcI repressor system to identify residues that are hot spots in OxyR. We compared the properties of these hot spots to those described in the literature from other systems. The hot spots identified in this study are not especially conserved amongst a set of OxyR orthologs.
Characterization of the structure and properties of the His 62 → Ala and Arg 38 → Ala mutants of yeast phosphoglycerate kinase: An investigation of the catalytic and activatory sites by site‐directed mutagenesis and NMR
Authors: Sherman Mark A. , Mas Maria T. , Fairbrother Wayne J. 
Published: February 2009

AbstractThe role of two “basic patch” residues, Arg‐38 and His‐62, in the catalytic function and anion‐dependent activation of yeast 3‐phosphoglycerate kinase (PGK) was investigated by site‐directed mutagenesis. Steady‐state kinetics and NMR experiments were conducted to characterize the functional properties and structural integrity of the R38A and H62A mutants. The results of these studies, in combination with earlier mutagenesis experiments, suggest that Arg‐38 is the only catalytically essential residue among the conserved histidines and arginines of the basic patch. It appears that, similar to the remaining basic patch residues, His‐62 is important for anion‐dependent activation but not for enzyme activity. Cumulative evidence from this study and from previous mutagenesis experiments suggests that the basic patch region is in effect an extended anion binding site that encompasses both the catalytic and the general anion‐binding site. It is proposed that substitution of any one of the basic patch residues results in an increased localization of the catalytic site. Substrate and product may still bind to this site, but a simultaneous binding of activatory anions, required for activation, has been impaired. NMR experiments suggest that the conformational changes observed upon binding of 3‐PG to wild‐type PGK are necessary for anion‐and substrate‐dependent activation.
Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability
Authors: Zhang X.‐J. , Baase W. A. , Matthews B. W. 
Published: February 2009

AbstractIn a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within α‐helix 126–134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild‐type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the α‐helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions.All of the variants could be crystallized isomorphously with wild‐type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent‐exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (≤0.4 Å) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133 → Ala made α‐helix 126–134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta‐alanine variant a distinct change occurred in a crystal‐packing contact, and the “hinge‐bending angle” between the amino‐ and carboxy‐terminal domains changed by 3.6°. This tends to confirm that such hinge‐bending in T4 lysozyme is a low‐energy conformational change.
A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains
Authors: Pochapsky Thomas C. , Gopen Quinton 
Published: February 2009

AbstractA liquid chromatographic stationary phase was prepared by covalently binding to the surface of microparticulate silica gel functionality (benzylsilane), which mimics the side chain of the amino acid phenylalanine. The chromatographic retentions of the N‐acetyl C‐(N′‐methyl) amides of various hydrophobic and amphiphilic amino acids on this stationary phase were measured using an aqueous mobile phase. A retention order of Gly < Ala < Cys < Val < Met < Pro < Ile < Leu < Tyr < Phe < Trp is seen at room temperature. Chromatographic retentions were used to derive free energies of adsorption of the amino acid derivatives on the chromatographic support relative to that of the glycine derivative. The temperature dependencies of the retention of aromatic and aliphatic amino acid derivatives differ in curvature, indicating a qualitative difference in the absorption mechanism. An adsorption model for retention is proposed, and arguments are made as to the suitability of an adsorption model for describing the contacts between amino acid side chains during the initial steps of protein folding.
Reversible dissociation and unfolding of the dimeric protein thymidylate synthase
Authors: Perry Kathy M. , Pookanjanatavip Manee , Zhao Jia , Santi Daniel V. , Stroud Robert M. 
Published: February 2009

AbstractConditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding–refolding conditions, demonstrating a monomer to dimer association step.
Characteristics of murine protoporphyrinogen oxidase
Authors: Proulx Karen L. , Dailey Harry A. 
Published: February 2009

AbstractProtoporphyrinogen oxidase (EC 1.3.3.4) (PPO) is the penultimate enzyme of the heme biosynthetic pathway. Mouse PPO has been purified in low yield and kinetically characterized by this laboratory previously. A new more rapid purification procedure is described herein, and with this protein we detect a noncovalently bound flavin moiety. This flavin is present at approximately stoichiometric amounts in the purified enzyme and has been identified by its fluorescence spectrum and high performance liquid chromatography as flavin mononucleotide (FMN). Fluorescence quenching studies on the flavin yielded a Stern–Volmer quenching constant of 12.08 M−1 for iodide and 1.1 M−1 for acrylamide. Quenching of enzyme tryptophan fluorescence resulted in quenching constants of 6 M−1 and 10 M−1 for iodide and acrylamide, respectively. Plasma scans performed on purified enzyme preparations did not reveal the presence of stoichiometric amounts of protein‐bound metal ions, and we were unable to detect any protein‐associated pyrroloquinoline quinone (PQQ). Data from circular dichroism studies predict a secondary structure of the native protein consisting of 30.5% alpha helix, 40.5% beta sheet, 13.7% turn, and 15.3% random coil. Denaturation of PPO with urea resulted in a biphasic curve when ellipticity is plotted against urea concentration, typical of amphipathic proteins.