bluuebunny/crossref_metadata_2025 huggingface dataset:
A tunable narrow‐linewidth Er‐doped fiber ring laser based on tunable fiber Bragg gratings
Authors: Liu Peng , Yan Feng Ping , Li Jian , Wang Lin , Gong Tao Rong , Tao Pei Lin
Published: February 2009

A tunable narrow‐linewidth Er‐doped fiber ring laser using tunable fiber Bragger gratings (TFBGs) is proposed and demonstrated. The TFBG is fabricated with a strain‐induced uniform FBG, which acts as an excellent reflecting mirror. By putting longitudinal strain to the TFBG, an approximately continuous tunable lasing output is obtained, the wavelength tunable range covers 8.00 nm (from 1543.5643 to 1551.5108 nm), and the tunable resolution is 1.92 nm/mm. A repeated testing result shows that the linewidth of the laser is stable and less than 0.01 nm, and the side‐mode suppression ratio is better than 50 dB. Moreover, an array waveguide grating is utilized for wavelength preselecting, and a 50‐km transmission performance of the proposed laser is tested experimentally under a 10 Gb/s modulation rate. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 1005–1010, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24245
Analysis and design of novel dual‐band branch line coupler with patterned ground plane
Authors: You Changjiang , Zhu Xiaowei
Published: February 2009

This article presents the novel dual‐band planar 90° branch line coupler with patterned ground plane, sometimes referred to as ‘defected ground structure.’ In the new layout, two cross coupling branches are added to the conventional branch line coupler. The additional branches make the design of dual‐band coupler at two arbitrary bands more flexible. Moreover, the used patterned ground plane makes an implementation of high impedance microstrip line easy and increases the electrical length of microstrip line. The basic principles of the novel coupler are analyzed and the design procedure is presented. For method validation, a dual‐band microstrip coupler operating at 2.1 GHz and 3.6 GHz was designed, tested, and analyzed. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 1000–1005, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24246
Analysis of the small slot‐loaded elliptical patch antenna with a band‐notched for UWB applications
Authors: Peng Lin , Ruan Chengli , Yin Xuncai
Published: February 2009

A small slot‐loaded elliptical antenna with a band‐notched for ultra wide‐band (UWB) applications is presented in this article. Printed on a dielectric substrate with relative permittivity of 4.4, thickness of 1 mm, and fed by a 50 Ω microstrip line, the proposed antenna satisfies the UWB bandwidth requirement of 3.1–10.6 GHz. A slot is used to reject the frequency band (5.15–5.825 GHz) limited by IEEE802.11a. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 973–976, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24247
A compact dual‐band modified T‐shaped CPW‐fed monopole antenna
Authors: Sujith R. , Deepu V. , Laila D. , Aanandan C. K. , Vasudevan K. , Mohanan P.
Published: February 2009

A compact, dual band coplanar waveguide fed modified T‐shaped uniplanar antenna is presented. The antenna has resonances at 1.77 and 5.54 GHz with a wide band from 1.47–1.97 GHz and from 5.13–6.48 GHz with an impedance bandwidth of 34% and 26%, respectively. Also the antenna has an average gain of 3 dBi in lower band and 3.5 dBi in higher band with an average efficiency of 90%. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 937–939, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24249
Design techniques applied to partially reflective surface antenna
Authors: Chang The Nan , Chen Chun‐Hua
Published: February 2009

Partially reflective surface antenna (PRSA) contains a highly reflective sheet and a perfect conductor (or ground plane) separated by a half wavelength. Usually, the reflective sheet is constructed by etching uniformly spaced patch elements on a supporting dielectric. In this article, an inhomogeneous spaced partial reflective sheet with an enlarged dielectric rim is investigated. We found that efficiency of the antenna can be optimized with a suitable dielectric size. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 1112–1116, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24251
Subsurface imaging, FDTD‐based simulations and alternative scan/processing approaches
Authors: Karpat E. , Çakır M. , Sevgi L.
Published: February 2009

Nondestructive and noncontacting subsurface imaging is discussed. A finite‐difference time‐domain (FDTD) based virtual tool is developed to create various imaging scenarios and to generate synthetic scattering data. Matlab‐based imaging algorithms are developed to process recorded FDTD data and to reconstruct objects. Different contour synthetic aperture type scanning configurations and reconstruction techniques are applied and their effects on imaging are reviewed. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 1070–1075, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24253
Erratum: Application of IE‐FFT with combined field integral equation to electrically large scattering problems
Authors: An Xiang , Lü Zhi‐Qing
Published: February 2009

Originally published in Microwave Opt Technol Lett 50: 2561–2566, 2008. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 1153, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24259
Design a new structure 2.4 GHz/5.2 GHz dual‐band bandpass filters on the MgTa1.5Nb0.5O6ceramic
Authors: Yang Cheng‐Fu , Yang Hung‐Chi , Cheng Chien‐Min , Chen Kai‐Huang , Chen Ying‐Chung
Published: February 2009

In this article, a λ/2 U‐shaped hairpin resonator was contained in a modified end‐coupled microstrip line. The increase of the sizes of U‐shaped hairpin resonator and modified end‐coupled microstrip line had no influence on the lower operating frequency and would shift the upper operating frequency to lower values. After finding the best designed parameters, a miniature dual‐band microstrip bandpass filter with three transmission zeros generated in the stop‐bands was developed on the MgTa1.5Nb0.5O6microwave dielectric ceramic substrates. The dual‐band filter had the central frequencies of 2.45 and 5.2 GHz and was suitable for the applications in the modern WLAN communication. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 1085–1087, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24264
A printed ultra‐wideband hexagon monopole antenna with WLAN band‐notched designs for wireless communication
Authors: Li Dong , Quan Shaohui , Wang Zhengpeng
Published: February 2009

A planar monopole antenna is designed for ultra‐wideband applications. A novel feeding structure is proposed to improve the impedance bandwidth. Promising performances are achieved, including wide impedance bandwidth, flat gain, and omnidirectional radiation patterns. Two kinds of band‐notched structure extended from the proposed antenna are designed and verified. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 899–902, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24265
Electromagnetic scattering from a dielectric cylinder and the method of lines
Authors: Hooshyar M. A.
Published: February 2009

A modified method of lines is developed for solving electromagnetic scattering problems, when TM plane waves are incident on cylindrical structures with dielectric properties that are function of two spatial directions. Numerical performance of the proposed method are examined and found to be very satisfactory. © 2009 Wiley Periodicals, Inc. Microwave Opt Technol Lett 51: 1046–1049, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.24266
A robust protocol for regional evaluation of methacholine challenge in mouse models of allergic asthma using hyperpolarized 3He MRI
Authors: Thomas Abraham C. , Potts Erin N. , Chen Ben T. , Slipetz Deborah M. , Foster W. Michael , Driehuys Bastiaan
Published: February 2009

Hyperpolarized (HP) 3He magnetic resonance imaging has been recently used to produce high‐resolution images of pulmonary ventilation after methacholine (MCh) challenge in mouse models of allergic inflammation. This capability presents an opportunity to gain new insights about these models and to more sensitively evaluate new drug treatments in the pre‐clinical setting. In the current study, we present our initial experience using two‐dimensional (2D), time‐resolved 3He MRI of MCh challenge‐induced airways hyperreactivity (AHR) to compare ovalbumin‐sensitized and challenged (N = 8) mice to controls (N = 8). Imaging demonstrated that ovalbumin‐sensitized and challenged animals exhibited many large ventilation defects even prior to MCh challenge (four out of eight) compared to no defects in the control animals. Additionally, the ovalbumin‐sensitized and challenged animals experienced a greater number of ventilation defects (4.5 ± 0.4) following MCh infusion than did controls (3.3 ± 0.6). However, due to variability in MCh delivery that was specific to the small animal MRI environment, the difference in mean defect number was not statistically significant. These findings are reviewed in detail and a comprehensive solution to the variability problem is presented that has greatly enhanced the magnitude and reproducibility of the MCh response. This has permitted us to develop a new imaging protocol consisting of a baseline 3D image, a time‐resolved 2D series during MCh challenge, and a post‐MCh 3D image that reveals persistent ventilation defects. Copyright © 2009 John Wiley & Sons, Ltd.
Conducting polyaniline‐titanium dioxide nanocomposites prepared by inverted emulsion polymerization
Authors: Karim Mohammad Rezaul , Lee Hyun Woo , Cheong In Woo , Park Sung Min , Oh Weontae , Yeum Jeong Hyun
Published: February 2009

Conducting polyaniline (PAni)‐titanium dioxide (TiO2) nanocomposites have been synthesized by the inverted emulsion polymerization method. Aqueous mixtures of aniline, a free‐radical oxidant, and/or TiO2 nanoparticles (∼25 nm in diameter; mixture of anatase and rutile) are utilized to synthesize the hybrid nanocomposites. The polymerization is carried out in an organic solvent (chloroform, CHCl3) in the presence of a protonic acid (hydrochloric acid, HCl) as a dopant and an emulsifier (cetyl trimethylammonium bromide). The resultant PAni‐TiO2 nanocomposites are characterized with their structural, morphological, conducting, and optical properties. SEM and TEM images represent the PAni‐TiO2 nanocomposites with the diameter range of 50–200 nm. Electrical conductivities are checked by standard four‐point probes method and found to be 0.38 S/cm for bulk PAni and 0.11 S/cm for PAni‐TiO2 nanocomposites. UV–visible absorption shows two electronic bands at about 320 and 596 nm for bulk PAni and the blue‐shifted bands with the intensity changes due to the formation of PAni‐TiO2 composites. Thermogravimetric analysis reveals that the composites have a higher degradation temperature than the PAni alone. POLYM. COMPOS., 2010. © 2009 Society of Plastics Engineers
Effect of annealing on structural and optical properties of polypyrrole doped with different acids
Authors: Saxena Rashmi , Sharma Kananbala , Saxena N.S. , Sharma T.P.
Published: February 2009

Polypyrrole (pure) and polypyrrole doped with different protonic acids (HCl and H3PO4) have been synthesized using NaOH as reducing agent in aqueous HCl medium and ammonium persulphate (APS) as an oxidant, respectively. These pure and doped samples were named as S1, S2, and S3 for further investigations. These pure and doped samples were annealed at 200oC for 4 hr. The amorphous nature of these annealed and as‐prepared polypyrrole samples was confirmed by XRD. FTIR spectra and SEM images of all the samples were taken at room temperature, which indicate: significant change in annealed samples. To calculate the energy band gap of all the samples, the absorption spectra were recorded by USB‐2000 spectrophotometer at room temperature in the wavelength range 300–800 nm. From the analysis of absorption spectra, optical band gap of as‐prepared samples (S1–S3) was determined to be 2.64, 2.43, and 2.29 eV, respectively and for annealed samples A1–A3 values obtained were 2.30, 2.25, and 2.17 eV. POLYM. COMPOS., 2009. © 2009 Society of Plastics Engineers
Privileged communication in the school psychologist–client relationship
Authors: Jacob Susan , Powers Katie E.
Published: February 2009

In the years since the 1996 Supreme Court Jaffee v. Redmond decision, many states broadened the scope of their laws governing privilege to include non‐doctoral school psychologists. This article addresses the meaning of the term privileged communication and identifies common exceptions to privilege. Findings from a computerized search of the status of privileged communication between school psychologists and their clients in all states are reported. Implications of privilege communication laws for the delivery of school psychological services are discussed. © 2009 Wiley Periodicals, Inc.
Intermittent gaseous bowel distention: Atypical sign of congenital tracheoesophageal fistula
Authors: Gardella Chiara , Tomà Paolo , Sacco Oliviero , Girosi Donata , Panigada Serena , Battistini Elena , Mattioli Girolamo , Jasonni Vincenzo , Rossi Giovanni A.
Published: February 2009

Three girls, 5‐, 9‐, and 15‐year‐old, were evaluated for recurrent airway infections and pneumonia. Chest X‐rays, which included the upper portion of the abdomen, showed marked gaseous bowels distention, while computed tomography scans of the chest demonstrated the presence of tracheoesophageal fistula (TEF), confirmed by fiberoptic bronchoscopy. Abdominal gaseous distension, a known possible clinical manifestation of TEF in the neonatal period generated by airflow through the fistula into the oesophagus, has not been reported as a clue to the diagnosis in older children. When detected in patients with recurrent respiratory infection, should raise the suspicion of unrecognized TEF. Pediatr Pulmonol. 2009; 44:244–248. © 2009 Wiley‐Liss, Inc.
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

Immune 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

Rationale

The 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.

Methods

Results 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.

Results

A 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).

Conclusions

We 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

Alliinase, 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

Fatty 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

Multidimensional 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

Carboxysomes 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

The 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

Osteogenesis 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

Conformational 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

The 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

We 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

The 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

In 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

A 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

Conditions 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

Protoporphyrinogen 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.
An energy‐based approach to packing the 7‐helix bundle of bacteriorhodopsin
Authors: Chou Kuo‐Chen , Carlacci Louis , Maggiora Gerald M. , Parodi L. A. , Schulz M. W.
Published: February 2009

Based on the heavy‐atom coordinates determined by the electron microscopy for the seven main helical regions of bacteriorhodopsin with the all‐trans retinal isomer, energy optimizations were carried out for helix bundles containing the all‐trans retinal and 13‐cis retinal chromophores, respectively. A combination of simulated annealing and energy minimization was utilized during the process of energy optimization. It was found that the 7‐helix bundle containing the all‐trans isomer is about 10 kcal/mol lower in conformational energy than that containing the 13‐cis isomer. An energetic analysis indicates that such a difference in energy is consistent with the observation that absorption of a 570‐nm proton is required for the conversion of a bacteriorhodopsin from its all‐trans to 13‐cis form.

It was also found that the above conversion process is accompanied by a significant conformational perturbation around the chromophore, as reflected by the fact that the β‐ionone ring of retinal moves about 5.6 Å along the direction perpendicular to the membrane plane. This is consistent with the observation by Fodor et al. (Fodor, S.P.A., Ames, J.B., Gebhard, R., van der Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., & Mathies, R.A., 1988, Biochemistry 27, 7097–7101). Furthermore, it is interesting to observe that although the retinal chromophore undergoes a significant change in its spatial position, the orientation of its transition dipole changes only slightly, in accord with experimental observations. In other words, even though orientation of the retinal transition dipole is very restricted, there is sufficient room, and degrees of freedom, for the retinal chromophore to readjust its position considerably. This finding provides new insight into the subtle change of the retinal microenvironment, which may be important for revealing the proton‐pumping mechanism of bacteriorhodopsin.

The importance of electrostatic and nonbonded interactions in stabilizing the 7‐helix bundle structure has also been analyzed. Electrostatic interactions favor an antiparallel arrangement among adjacent helices. Nonbonded interactions, however, drive most of the closely packed helices into an arrangement in which the packing angles lie around – 160°, a value very near the – 154° value computed earlier as the most favorable packing arrangement of two poly(Ala) α‐helices (Chou, K.‐C., Némethy, G., & Scheraga, H.A., 1983, J. Phys. Chem. 87, 2869–2881).

The structural features of the 7‐helix bundle and their relationship to those found in typical 4‐helix bundle proteins are also discussed. The knowledge thus obtained suggests that many 7‐helix membrane proteins might probably be built in a stepwise fashion from 4‐helix bundle groups.
Structural homology between rbs repressor and ribose binding protein implies functional similarity
Authors: Mauzy C.A. , Hermodson M.A.
Published: February 2009

The deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C‐terminal 272 residues to the entire sequence of the periplasmic ribose binding protein. RbsR is also homologous to a family of bacterial repressor proteins including LacI. This implies that the structure of the repressor consists of a two‐domain binding protein portion attached to a DNA‐binding domain having the four‐helix structure of the LacI headpiece. The implications of these relationships to the mechanism of this class of repressors are discussed.
Identification of the guanine binding domain peptide of the GTP‐binding site of glucagon
Authors: Shoemaker Mike , Lin Paul C. , Haley Boyd
Published: February 2009

Glucagon, a peptide hormone synthesized and secreted by α islet cells, regulates glucose homeostasis by several mechanisms. Using [γ32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half‐maximal saturation of photoinsertion into the polypeptide hormone was at 8–12 μM with either [α32P]8N3GTP or [γ32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 μM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 μM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg2+ at 150 μM enhanced photoinsertion (twofold), whereas at 2–10 mM, it inhibited photoinsertion. Both Ca2+ and Zn2+ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse‐phase high performance liquid chromatography (HPLC) revealed that the N‐terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [32P]8N3GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N‐terminal region is suggestive of a physiological role for a glucagon‐GTP complex in the mechanism of action of this hormone.
Cis proline mutants of ribonuclease A. I. thermal stability
Authors: Schultz David A. , Baldwin Robert L.
Published: February 2009

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60–89). The expressed protein, which contains an additional N‐terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site‐directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give ΔTm ≅ 10 °C and ΔΔG0 (apparent) = 2–3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis ⟺ trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild‐type conformation, that allows the substituted amino acid to form a trans peptide bond.
83‐Kilodalton heat shock proteins of trypanosomes are potent peptide‐stimulated ATPases
Authors: Nadeau Kari , Walsh Christopher T. , Bradley Mark , Sullivan Mary Anne , Engman David M.
Published: February 2009

A Crithidia fasciculata 83‐kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min−1 and a Km of 60 μM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 × 10−3 min−1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min−1, Km = 80 μM, kautophosphorylation = 6.3 × 10−3 min−1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6–24 amino acids.
Chimeric prion protein expression in cultured cells and transgenic mice
Authors: Scott Michael R. , Köhler Ruth , Foster Dallas , Prusiner Stanley B.
Published: February 2009

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558–1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847–857; Prusiner, S.B., et al., 1990, Cell 63, 673–686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease‐resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa‐specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.