bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling of transpiration reduction in van Genuchten–Mualem type soils
Authors: de Jong van Lier Quirijn , Dourado Neto D. , Metselaar Klaas
Published: February 2009

We derive an analytic expression for the matric flux potential (M) for van Genuchten–Mualem (VGM) type soils which can also be written in terms of a converging infinite series. Considering the first four terms of this series, the accuracy of the approximation was verified by comparing it to values of M estimated by numerical finite difference integration. Using values of the parameters for three soils from different texture classes, the proposed four‐term approximation showed an almost perfect match with the numerical solution, except for effective saturations higher than 0.9. Including more terms reduced the discrepancy but also increased the complexity of the equation. The four‐term equation can be used for most applications. Cases with special interest in nearly saturated soils should include more terms from the infinite series. A transpiration reduction function for use with the VGM equations is derived by combining the derived expression for M with a root water extraction model. The shape of the resulting reduction function and its dependency on the derivative of the soil hydraulic diffusivity D with respect to the soil water content θ is discussed. Positive and negative values of dD/dθ yield concave and convex or S‐shaped reduction functions, respectively. On the basis of three data sets, the hydraulic properties of virtually all soils yield concave reduction curves. Such curves based solely on soil hydraulic properties do not account for the complex interactions between shoot growth, root growth, and water availability.
Electronic Waste Management
Authors: Goosey Martin , Stevens Gary , Herman Henryk , Baird Patrick , Holmes Ian , Kell Darren , Kellner Rod , Butler Scott , Shayler Mark , Holloway Leigh , Matharu Avtar , McIntyre Kirsty , Dempsey Mark
Published: February 2009

Electronic waste includes such items as TVs, computers, LCD and plasma displays, and mobile phones, as well as a wide range of household, medical and industrial equipment which are simply discarded as new technologies become available. Huge and growing quantities of waste are discarded every year and this waste contains toxic and carcinogenic compounds which can pose a risk to the environment. However, if handled correctly, electronic waste presents a valuable source of secondary raw materials. This book brings together a group of leading experts in the management of electrical and electronic waste to provide an up-to-date review of the scale of the waste problem, the impact of recent legislation such as the Waste Electrical and Electronic Equipment Directive (WEEE) and the "restriction of the use of certain hazardous substances in electrical and electronic equipment" directive (RoHS), and of current and future methods for treatment, recycling and disposal of this waste. The book discusses these latest directives, examines current worldwide legislation and considers the opportunities and threats posed by this form of waste. While the emphasis is on European practice, comparisons with other countries such as the USA, Japan and China are made. The book deals with the full range of waste management issues, including recycling and recovery of materials, design considerations for waste minimisation, and contains a wide variety of illustrative case studies eg: LCD displays. With detailed and comprehensive coverage of the subject matter it also contains an extensive bibliography with each chapter. Key chapters cover areas such as: -electronic waste -materials -EU directives -landfill and incineration -recycling and recovery -'cradle to grave' design considerations -engineering thermoplastics It is essential reading for all involved with electrical and electronic waste management through its comprehensive review of recent EU legislation and the subsequent impact on manufacturers and users of electronic equipment.
Improving the Synthetic Data Generation Process in Spatial Microsimulation Models
Authors: Smith Dianna M , Clarke Graham P , Harland Kirk
Published: February 2009

Simulation models are increasingly used in applied research to create synthetic micro-populations and predict possible individual-level outcomes of policy intervention. Previous research highlights the relevance of simulation techniques in estimating the potential outcomes of changes in areas such as taxation and child benefit policy, crime, education, or health inequalities. To date, however, there is very little published research on the creation, calibration, and testing of such micro-populations and models, and little on the issue of how well synthetic data can fit locally as opposed to globally in such models. This paper discusses the process of improving the process of synthetic micropopulation generation with the aim of improving and extending existing spatial microsimulation models. Experiments using different variable configurations to constrain the models are undertaken with the emphasis on producing a suite of models to match the different sociodemographic conditions found within a typical city. The results show that creating processes to generate area-specific synthetic populations, which reflect the diverse populations within the study area, provides more accurate population estimates for future policy work than the traditional global model configurations.
Applying an exemplar model to the serial reaction-time task: Anticipating from experience
Authors: Jamieson Randall K. , Mewhort D. J. K.
Published: February 2009

We present a serial reaction time (SRT) task in which participants identified the location of a target by pressing a key mapped to the location. The location of successive targets was determined by the rules of a grammar, and we varied the redundancy of the grammar. Increasing both practice and the redundancy of the grammar reduced response time, but the participants were unable to describe the grammar. Such results are usually discussed as examples of implicit learning. Instead, we treat performance in terms of retrieval from a multitrace memory. In our account, after each trial, participants store a trace comprising the current stimulus, the response associated with it, and the context provided by the immediately preceding response. When a target is presented, it is used as a prompt to retrieve the response mapped to it. As participants practise the task, the redundancy of the series helps point to the correct response and, thereby, speeds retrieval of the response. The model captured performance in the experiment and in classic SRT studies from the literature. Its success shows that the SRT task can be understood in terms of retrieval from memory without implying implicit learning.
Dbf2–Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis
Authors: Mohl Dane A. , Huddleston Michael J. , Collingwood Therese S. , Annan Roland S. , Deshaies Raymond J.
Published: February 2009

Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2–Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.
Characterization of a PLLA-Collagen I Blend Nanofiber Scaffold with Respect to Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells
Authors: Schofer Markus D. , Boudriot Ulrich , Leifeld Irini , Sütterlin Romina I. , Rudisile Markus , Wendorff Joachim H. , Greiner Andreas , Paletta Jürgen R. J. , Fuchs-Winkelmann Susanne
Published: February 2009

The aim of this study was to enhance synthetic poly(L-lactic acid) (PLLA) nanofibers by blending with collagen I (COLI) in order to improve their ability to promote growth and osteogenic differentiation of stem cellsin vitro. Fiber matrices composed of PLLA and COLI in different ratios were characterized with respect to their morphology, as well as their ability to promote growth of human mesenchymal stem cells (hMSC) over a period of 22 days. Furthermore, the course of differentiation was analyzed by gene expression of alkaline phosphatase (ALP), osteocalcin (OC), and COLI. The PLLA-COLI blend nanofibers presented themselves with a relatively smooth surface. They were more hydrophilic as compared to PLLA nanofibers alone and formed a gel-like structure with a stable nanofiber backbone when incubated in aqueous solutions. We examined nanofibers composed of different PLLA and COLI ratios. A composition of 4:1 ratio of PLLA:COLI showed the best results. When hMSC were cultured on the PLLA-COLI nanofiber blend, growth as well as osteoblast differentiation (determined as gene expression of ALP, OC, and COLI) was enhanced when compared to PLLA nanofibers alone. Therefore, the blending of PLLA with COLI might be a suitable tool to enhance PLLA nanofibers with respect to bone tissue engineering.
Metabolic Syndrome May Not be a Good Predictor of Coronary Artery Disease in the Iranian Population: Population-Specific Definitions are Required
Authors: Ebrahimi Mahmoud , Kazemi-Bajestani Seyyed Mohammad Reza , Ghayour-Mobarhan Majid , Moohebati Mohsen , Paydar Roghaye , Azimi-Nezhad Mohsen , Esmaily Habib Ollah , Ferns Gordon A. A.
Published: February 2009

Metabolic syndrome (MS) is associated with an increased risk of coronary artery disease (CAD) in Western populations. We have investigated the relationship between the presence of MS and other conventional risk factors, and angiographically defined CAD, in a Middle Eastern population. Patients (n = 431) attending a hospital cardiology clinic for angiography were assessed. Each patient subsequently underwent routine angiography. Anthropometric and biochemical data were used to establish whether patients had MS, using either IDF or NCEP-ATP III criteria. The relationship between the presence of MS, or other individual coronary risk factors, and angiographically defined CAD was assessed by logistic regression analysis. A further reference group of individuals without overt CAD (n = 1276) was used as an additional comparator group. Of the 431 patients, 327 (75.9%) were found to have angiographically defined CAD. There was no significant relationship between MS, using either the IDF or NCEP-ATP III definitions, and CAD in this population. Of the parameters assessed, age, total cholesterol, and low serum HDL cholesterol were the strongest independent predictors of angiographically defined CAD (p< 0.01,p< 0.01,p< 0.05, respectively). It appears that within an Iranian population, the presence of MS defined by either the NCEP-ATP III or IDF criteria fails to identify individuals with established, angiographically defined CAD. However, a low serum HDL cholesterol, a component of MS, was an important independent predictor of CAD in this population. It is possible that the criteria for defining MS as a risk predictor of CAD in Middle Eastern populations may need to be revised.
Association between HIV status and Positive Prostate Biopsy in a Study of U.S. Veterans
Authors: Hsiao Wayland , Anastasia Katrina , Hall John , Goodman Michael , Rimland David , Ritenour Chad W. M. , Issa Muta M.
Published: February 2009

HIV infection is associated with increased incidence of malignancies, such as lymphomas and testicular cancers. We reviewed the relationship between HIV infection and prostate cancer in a contemporary series of prostate biopsy patients. The study is a retrospective analysis of consecutive prostate biopsies performed at a VA Medical Center. The indications for performing a prostate biopsy included an abnormal digital rectal examination and/or an elevated PSA. Patients were categorized according to their HIV status, biopsy results, and various demographic and clinical characteristics. Univariate and multivariate analyses compared distributions of HIV status, and various clinical and demographic characteristics. The adjusted measures of association between HIV status and positive biopsy were expressed as odds ratios (ORs) and corresponding 95% confidence intervals (CI). The likelihood of positive biopsy was significantly higher among 18 HIV-positive patients compared to patients with negative HIV tests (adjusted OR = 3.9; 95% CI: 1.3–11.5). In analyses restricted to prostate cancer patients, HIV-positive patients were not different from the remaining group with respect to their prostate cancer stage, PSA level, PSA velocity, PSA density, or Gleason grade. There is an association between HIV infection and prostate biopsy positive for carcinoma in a population referred for urologic workup. Further confirmation of this association by prospective studies may impact the current screening practices in HIV patients.
Thermal Analysis of Whole Bacterial Cells Exposed to Potassium Permanganate Using Differential Scanning Calorimetry: a Biphasic Dose-Dependent Response to Stress
Authors: Abuladze Marina K. , Sokhadze Victor M. , Namchevadze Emma N. , Kiziria E. , Tabatadze Leila V. , Lejava Lia V. , Gogichaishvili Sh. , Bakradze Nugzar B.
Published: February 2009

Differential scanning calorimetry (DSC) was applied to estimate the impact of the toxic oxidant potassium permanganate (PM) on the intracellular structural and functional alterations at whole cell level using soil bacteriaArthrobacter oxydansas a model culture. Differential scanning calorimetry (DSC) was applied in order to estimate the impact of the toxic oxidant potassium permanganate (PM) on the intracellular structural and functional alterations at the whole cell level using the soil bacteria Arthrobacter oxydans as a model culture. We compared the total melting heat and the temperature of DNA-protein complex (DNP) melting at the PM application prior to the calorimetry measurement and after 24-h exposure at the concentration range 0.02–1.4 mM. The initial oxidative effect caused changes in the pattern of the whole cell melting spectra (mainly at the temperature range 56–78°C), the decrease of Tmax°C DNP melting, and did not influence significantly the total heat of bacterial melting at different concentrations of PM. The prolonged effect of permanganate up to 24 h was characterized by a biphasic dose-dependent response to stress estimated by the DSC technique and the colony-forming assay. The low doses of PM (0.02 and 0.2 mM) stimulated cell proliferation, and increased the total whole cell melting heat and the temperature of DNP melting. The toxic effect of PM up to 0.04 mMreduced cell viability, changed the character of multipeaked thermograms, and lowered the total melting heat and the temperature of DNP melting in a concentration-dependent manner. This study presents the DSC method for evaluating and monitoring the effects of exposure to potential human and environmental toxicants.
Substitution rates in α‐helical transmembrane proteins
Authors: Stevens Timothy J. , Arkin Isaiah T.
Published: February 2009

It has been shown previously that some membrane proteins have a conserved core of amino acid residues. This idea not only serves to orient helices during model building exercises but may also provide insight into the structural role of residues mediating helix–helix interactions. Using experimentally determined high‐resolution structures of α‐helical transmembrane proteins we show that, of the residues within the hydrophobic transmembrane spans, the residues at lipid and subunit interfaces are more evolutionarily variable than those within the lipid‐inaccessible core of a polypeptide's transmembrane domain. This supports the idea that helix–helix interactions within the same polypeptide chain and those at the interface between different polypeptide chains may arise in distinct ways. To show this, we use a new method to estimate the substitution rate of an amino acid residue given an alignment and phylogenetic tree of closely related proteins. This method gives better sensitivity in the otherwise‐conserved transmembrane domains than a conventional similarity analysis and is relatively insensitive to the sequences used.
Structure determination of human and murine β‐defensins reveals structural conservation in the absence of significant sequence similarity
Authors: Bauer Finn , Schweimer Kristian , Klüver Enno , Conejo‐Garcia Jose‐Ramon , Forssmann Wolf‐Georg , Rösch Paul , Adermann Knut , Sticht Heinrich
Published: February 2009

Defensins are cationic and cysteine‐rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian β‐defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human β‐defensins, hBD‐1 and hBD‐2, as well as those of two novel murine defensins, termed mBD‐7 and mBD‐8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent‐exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that β‐defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the β‐defensin‐specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because β‐defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.
Structure and function of the N‐linked glycans of HBP/CAP37/azurocidin: Crystal structure determination and biological characterization of nonglycosylated HBP
Authors: Iversen L. F. , Kastrup J. S. , Bjørn S. E. , Wiberg F. C. , Larsen I. K. , Flodgaard H. J. , Rasmussen P. B.
Published: February 2009

The three N‐glycosylation sites of human heparin binding protein (HBP) have been mutated to produce a nonglycosylated HBP (ng‐HBP) mutant. ng‐HBP has been crystallized and tested for biological activity. Complete X‐ray data have been collected to 2.1 Å resolution, and the structure has been fully refined to an R‐factor of 18.4% (Rfree 27.7%). The ng‐HBP structure reveals that neither the secondary nor tertiary structure have changed due to the removal of the glycosylation, as compared to the previously determined glycosylated HBP structure. Although the primary events in N‐linked glycosylation occurs concomitant with polypeptide synthesis and therefore possesses the ability to influence early events in protein folding, we see no evidence of glycosylation influencing the structure of the protein. The root‐mean‐square deviation between the superimposed structures was 0.24 Å (on Cα atoms), and only minor local structural differences are observed. Also, the overall stability of the protein seems to be unaffected by glycosylation, as judged by the B‐factors derived from the two X‐ray structures. The flexibility of a glycan site may be determined by the local polypeptide sequence and structure rather than the glycan itself. The biological in vitro activity assay data show that ng‐HBP, contrary to glycosylated HBP, mediates only a very limited stimulation of the lipopolysaccharide induced cytokine release from human monocytes. In animal models of fecal peritonitis, glycosylated HBP treatment rescues mice from and an otherwise lethal injury. It appears that ng‐HBP have significant effect on survival, and it can be concluded that ng‐HBP can stimulate the host defence machinery albeit to a lesser extent than glycosylated HBP.
N5‐(L‐1‐carboxyethyl)‐L‐ornithine synthase: Physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues
Authors: Sackett Dan L. , Ruvinov Sergei B. , Thompson John
Published: February 2009

N5‐(L‐1‐carboxyethyl)‐L‐ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH‐dependent enzyme is a homotetramer (Mr ≡ 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far‐ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the α,β family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N‐acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of ∼8.5, 11 have pKa ∼10.75, and the remaining six titrate with pKa ∼11.3. The two residues with pKa ∼8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr‐Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the βαβ fold of the nucleotide‐binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single‐site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.
A molecular dynamics study of the 41‐56 β‐hairpin from B1 domain of protein G
Authors: Roccatano Danilo , Amadei Andrea , Nola Alfredo Di , Berendsen Herman J. C.
Published: February 2009

The structural and dynamical behavior of the 41‐56 β‐hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300 K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin‐like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.
Cooperative effects of potassium, magnesium, and magnesium‐ADP on the release of Escherichia coli dihydrofolate reductase from the chaperonin GroEL
Authors: Clark A. Clay , Karon Brad S. , Frieden Carl
Published: February 2009

Previous investigation has shown that at 22 °C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512‐525). In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL. The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four‐ to fivefold. In the absence of potassium, magnesium, and ADP, ∼80‐90% of GroEL resides in the form with the faster rate of release. Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium‐induced transition is observed only in the presence of potassium. MgADP at low concentrations (0‐50 μM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent. Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL. In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild‐type GroEL at 22 °C reaches a limiting value of 0.014 s−1. For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s−1, suggesting that inter‐ring negative cooperativity exists for MgADP‐induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein.
Protein histidine phosphorylation: Increased stability of thiophosphohistidine
Authors: Lasker Michael , Bui Cuong D. , Besant Paul G. , Sugawara Kiyoshi , Thai Philip , Medzihradszky Gergely , Turck Christoph W.
Published: February 2009

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine‐containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes.
Identification of ligand effector binding sites in transmembrane regions of the human G protein‐coupled C3a receptor
Authors: Sun Jianzhong , Ember Julia A. , Chao Ta‐Hsiang , Hugli Tony E. , Fukuoka Yoshihiro , Ye Richard D.
Published: February 2009

The human C3a anaphylatoxin receptor (C3aR) is a G protein‐coupled receptor (GPCR) composed of seven transmem‐brane α‐helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site‐directed mutagenesis, and mutant receptors were stably expressed in the RBL‐2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a‐induced receptor activation.
Arginine 197 of the cholecystokinin‐A receptor binding site interacts with the sulfate of the peptide agonist cholecystokinin
Authors: Gigouj Véronique , Escrieut Chantal , Silvente‐Poirot Sandrine , Bouisson Michèle , Vaysse Nicole , Fourmy Daniel , Maigret Bernard , Fehrentz Jean‐Alain , Martinez Jean , Moroder Luis , Gully Danielle
Published: February 2009

The knowledge of the binding sites of G protein‐coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin‐A receptor (CCK‐AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three‐dimensional model of the [CCK‐AR‐CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site‐directed mutagenesis. Wild‐type and mutated CCK‐AR were transiently expressed in COS‐7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I‐BH‐[Thr, Nle]‐CCK‐9; however, it bound the nonpeptide antagonist [3H]‐SR27,897 as the wild‐type receptor. The mutant was ≅1,470‐ and 3,200‐fold less potent than the wild‐type CCK‐AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500‐fold lower potency and 800‐fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild‐type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK‐AR, strongly support an interaction between Arg 197 of the CCK‐AR binding site and the sulfate of CCK. In addition, the mutated CCK‐AR resembled the low affinity state of the wild‐type CCK‐AR, suggesting that Arg 197‐sulfate interaction regulates conformational changes of the CCK‐AR that are required for its physiological activation.
Mapping of ATP binding regions in poly (A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases
Authors: Jenö Paul , Keller Walter , Martin Georges
Published: February 2009

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross‐linking of recombinant bovine and yeast poly(A) polymerases to 8‐azido‐ATP. Mn2+ strongly stimulated this reaction due to a 50‐fold lower Ki for 8‐azido‐ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross‐linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8‐azido‐ATP crosslinking were mapped in different poly(A) polymerases by CNBr‐cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross‐link in Schizosaccharomyces pombe poly (A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167‐177). Database searches with sequences surrounding the cross‐link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide‐binding fold among these families of enzymes. Mutations in the region of the “helical turn motif” (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide‐binding helix of bovine poly (A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.
The novel fluorescent CDP‐analogue (Pβ)MABA‐CDP is a specific probe for the NMP binding site of UMP/CMP kinase
Authors: Rudolph Markus G. , Veit THOMAS J.H. , Reinstein Jochen
Published: February 2009

Direct thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N‐methylanthraniloyl‐ (mant) group to the β‐phosphate of CDP via a butyl linker, a CDP analogue [(Pβ)MABA‐CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments.
Spectral contribution of the individual tryptophan of αB‐crystallin: A study by site‐directed mutagenesis
Authors: Liang Jack J‐N. , Sun Tian‐Xiao , Akhtar Nila J.
Published: February 2009

There are two tryptophan residues in the lens αB‐crystallin, Trp9 and Trp60. We prepared two Trp → Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near‐ultraviolet circular dichroism (UV CD) differently. The major difference in the near‐UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near‐UV CD shows an increased intensity in the region of 270–280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a β‐strand.
Synthesis, folding, and structure of the β‐turn mimic modified B1 domain of streptococcal protein G
Authors: Odaert Benoit , Jean Fabienne , Melnyk Oleg , Tartar Andre , Lippens Guy , Boutillon Christophe , Buisine Eric
Published: February 2009

The mechanism of β‐sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long‐range interactions. Here, we present the incorporation of a well‐studied β‐turn mimic, designated as the dibenzofuran‐based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N‐terminal β‐hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147–150). The DBF‐B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native‐like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the β‐turn mimic from acting as a strong β‐sheet nucleator, which reinforces the idea that the native β‐hairpin formation is not driven by the β‐turn formation, but by tertiary interactions.
Mass spectrometric characterization of oat phytochrome A: Isoforms and posttranslational modifications
Authors: Lapko Veniamin N. , Jiang Xiang‐Yu , Smith David L. , Song Pill‐Soon
Published: February 2009

At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N‐terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass‐spectrometry (ESIMS). The total tryptic digest of iodoacetamide‐modified phyA was fractionated by gel filtration chromatography followed by reversed‐phase high‐performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision‐induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1, 128 amino acid residues) was determined in the present study. Mass‐spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light‐induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595–10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark‐grown and red‐light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light‐treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A‐mediated light‐signal transduction.
Auracyanin a from the thermophilic green gliding photosynthetic bacterium chloroflexus aurantiacus represents an unusual class of small blue copper proteins
Authors: Driessche Gonzalez Van , Hu Wei , Werken Gerrit Van De , Selvaraj Fabiyola , Mcmanus James D. , Blankenship Robert E. , Van Beeumen Jozef J.
Published: February 2009

The amino acid sequence of the small copper protein auracyanin A isolated from the thermophilic photosynthetic green bacterium Chloroflexus aurantiacus has been determined to be a polypeptide of 139 residues. His58, Cys123, His128, and Met132 are spaced in a way to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction also indicates that auracyanin has a general β‐barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes. The overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5%. We suggest that auracyanin A, together with the B forms, is the first example of a new class of small copper proteins that may be descendants of an ancestral sequence to both the azurin proteins occurring in prokaryotic nonphotosynthetic bacteria and the plastocyanin proteins occurring in both prokaryotic cyanobacteria and eukaryotic algae and plants. The N‐terminal sequence region 1–18 of auracyanin is remarkably rich in glycine and hydroxy amino acids, and required mass spectrometric analysis to be determined. The nature of the blocking group X is not yet known, although its mass has been determined to be 220 Da. The auracyanins are the first small blue copper proteins found and studied in anoxygenic photosynthetic bacteria and are likely to mediate electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center.
Evidence for a copper‐coordinated histidine–tyrosine cross‐link in the active site of cytochrome oxidase
Authors: Buse Gerhard , Soulimane Tewfik , Dewor Manfred , Meyer Helmut E. , Blüggel Martin
Published: February 2009

Following hints from X‐ray data (Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547–10553; Yoshikawa S et al., 1998, Science 280:1723–1729), chemical evidence is presented from four distantly related cytochrome‐c oxidases for the existence of a copperB‐coordinated His240–Tyr244) cross‐link at the O2‐activating Heme Fea3–CuB center in the catalytic subunit I of the enzyme. The early evolutionary invention of this unusual structure may have prevented demaging OH‐radical release at e–‐transfer to dioxygen and thus have enabled O2 respiration.
Study of the stability and unfolding mechanism of BBA1 by molecular dynamics simulations at different temperatures
Authors: Wang Lu , Duan Yong , Shortle Rebecca , Imperiali Barbara , Kollman Peter A.
Published: February 2009

BBA1 is a designed protein that has only 23 residues. It is the smallest protein without disulfide bridges that has a well‐defined tertiary structure in solution. We have performed unfolding molecular dynamics simulations on BBA1 and some of its mutants at 300, 330, 360, and 400 K to study their kinetic stability as well as the unfolding mechanism of BBA1. It was shown that the unfolding simulations can provide insights into the forces that stabilize the protein. Packing, hydrophobic interactions, and a salt bridge between Asp12 and Lys16 were found to be important to the protein' stability. The unfolding of BBA1 goes through two major steps: (1) disruption of the hydrophobic core and (2) unfolding of the helix. The β‐hairpin remains stable in the unfolding because of the high stability of the type II' turn connecting the two β‐strands.
A novel recombinant single‐chain hepatitis C virus ns3‐ns4a protein with improved helicase activity
Authors: Howe ANITA Y.M. , Chase Robert , Taremi S. Shane , Risano Christine , Beyer Brian , Malcolm Bruce , Lau JOHNSON Y.N.
Published: February 2009

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single‐chain form of the NS3/NS4A complex (His‐NS4A21–32‐GSGS‐NS33–631) was constructed in which the NS4A core peptide is fused to the N‐terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21–32) fused to the full‐length NS3 (a.a. 3–631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels inEscherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single‐chain recombinant NS3‐NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single‐chain recombinant NS3‐NS4A with that of the full‐length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.
Packed protein bilayers in the 0.90 å resolution structure of a designed alpha helical bundle
Authors: Privé Gilbert G. , Anderson Daniel H. , Wesson Laura , Cascio Duilio , Eisenberg David
Published: February 2009

A 12‐residue peptide designed to form an α‐helix and self‐associate into an antiparallel 4‐α‐helical bundle yields a 0.9 Å crystal structure revealing unanticipated features. The structure was determined by direct phasing with the “Shake‐and‐Bake” program, and contains four crystallographically distinct 12‐mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal α‐helices hydrogen bonded head‐to‐tail into columns, which in turn pack side‐by‐side into sheets spanning the width of the crystal. Within each sheet, the α‐helices run antiparallel and are closely spaced (9–10 Å center‐to‐center). The sheets are more loosely packed against each other (13–14 Å between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four‐α‐helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7–11° rather than 18°).
Analysis of the extent of unfolding of denatured insulin‐like growth factor
Authors: Chang Jui‐Yoa , WALTER MÄRKI  , Lai Por‐Hsiung
Published: February 2009

Insulin‐like growth factor (IGF‐1) contains three disulfide bonds. In the presence of denaturant and thiol catalyst, IGF‐1 shuffles its native disulfide bonds and denatures to form a mixture of scrambled isomers. The composition of scrambled IGF varies under different denaturing conditions. Among the 14 possible scrambled IGF isomers, the yield of the beads‐form isomer is shown to be directly proportional to the strength of the denaturing condition. This paper demonstrates a new approach to quantify the extent of unfolding of the denatured protein.
Folding of barstar C40A/C82A/P27A and catalysis of the peptidyl‐prolylcis/transisomerization by human cytosolic cyclophilin (Cyp18)
Authors: Golbik Ralph , Fischer Gunter , Fersht Alan R.
Published: February 2009

Refolding of b*C40A/C82A/P27A is comprised of several kinetically detectable folding phases. The slowest phase in refolding originates fromtrans → cisisomerization of the Tyr47–Pro48 peptide bond being incisconformation in the native state. This refolding phase can be accelerated by the peptidyl‐prolylcis/transisomerase human cytosolic cyclophilin (Cyp18) with akcat/KMof 254, 000 M–1s–1. The fast refolding phase is not influenced by the enzyme.
The disulfide‐coupled folding pathway of apamin as derived from diselenide‐quenched analogs and intermediates
Authors: Pegoraro Stefano , Fiori Stella , Cramer Jörg , Rudolph‐Böhner Sabine , Moroder Luis
Published: February 2009

The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide‐coupled folding into the cystine‐stabilized α‐helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three‐dimensional (3D) structures of the selenocysteine‐containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.