bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thioredoxin reductase from Escherichia coli: Evidence of restriction to a single conformation upon formation of a crosslink between engineered cysteines
Authors: Veine Donna M. , Ohnishi Kenji , Williams Charles H. 
Published: July 2010

AbstractThioredoxin reductase is a flavoprotein which catalyzes the reduction of the small protein thioredoxin by NADPH. It contains a redox active disulfide and an FAD in each subunit of its dimeric structure. Each subunit is further divided into two domains, the FAD and the pyridine nucleotide binding domains. The orientation of the two domains determined from the crystal structure and the flow of electrons determined from mechanistic studies suggest that thioredoxin reductase requires a large conformational change to carry out catalysis (Williams CH Jr, 1995, FASEB J 9:1267‐1276). The constituent amino acids of an ion pair, E48/R130, between the FAD and pyridine nucleotide binding domains, were mutagenized to cysteines to form E48C, R130C (CC mutant). Formation of a stable bridge between these cysteines was expected to restrict the enzyme largely in the conformation observed in the crystal structure. Crosslinking with the bifunctional reagent N, N,1,2 phenylenedimaleimide, spanning 4‐9 Å, resulted in a <95 % decrease in thioredoxin reductase and transhydrogenase activity. SDS‐PAGE confirmed that the crosslink in the CC‐mutant was intramolecular. Dithionite titration showed an uptake of electrons as in wild‐type enzyme, but anaerobic reduction of the flavin with NADPH was found to be impaired. This indicates that the crosslinked enzyme is in the conformation where the flavin and the active site disulfide are in close proximity but the flavin and pyridinium rings are too far apart for effective electron transfer. The evidence is consistent with the hypothesis that thioredoxin reductase requires a conformational change to complete catalysis.
Engineering protein for X‐ray crystallography: The murine major histocompatibility complex class II molecule I‐Ad
Authors: Scott Christoper A. , Christoper Garcia K. , Stura Enrico A. , Peterson Per A. , Wilson Ian A. , Tetton Luc 
Published: July 2010

AbstractClass II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I‐Ad, with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced inDrosophila melanogasterSC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six‐residue linker to the amino terminus of the I‐Adβ chain. This modified I‐Admolecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X‐ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I‐A and other class II MHC‐peptide complexes.
Expansion of the genetic code: Site‐directed p‐fluoro‐phenylalanine incorporation in Escherichia coli
Authors: Furter Rolf 
Published: July 2010

AbstractSite‐directed incorporation of the amino acid analogue p‐fluoro‐phenylalanine (p‐F‐Phe) was achieved in Escherichia coli. A yeast suppressor tRNAPheamber/phenylalanyl‐tRNA synthetase pair was expressed in an analogue‐resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64‐75% as p‐F‐Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p‐F‐Phe incorporation was 11‐21‐fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8‐12 mg/L of culture, corresponding to about two thirds of the expression level of the wild‐type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F‐NMR spectroscopy and other sample‐intensive methods. The use of a nonessential “21 st” tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.
A conserved deamidation site at asn 2 in the catalytic subunit of mammalian cAMP‐dependent protein kinase detected by capillary LC‐MS and tandem mass spectrometry
Authors: Jedrzejewski Paul T. , Girod Andreas , Tholey Andreas , König Norbert , Thullner Sandra , Kinzel Volker , Bossemeyer Dirk 
Published: July 2010

AbstractThe N‐terminal sequence myr‐Gly‐Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes C, Cβ, and O. By capillary LC‐MS and tandem MS, we show that, in approximately one third of the C and Cβ enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C‐subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from C and Cβ. Asn 2 deamidation does not occur during C‐subunit preparation and is absent in recombinant myristoylated Cα (rCα) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N‐terminal glycine, verified by the myristoylation negative phenotype of an rCa(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr‐Gly‐Asp sequence. Asp 2 seems to be required for the well‐characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr‐Gly‐Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.
Genfold: A genetic algorithm for folding protein structures using NMR restraints
Authors: Bayley Martin J. , Jones Gareth , Willett Peter , Williamson Michael P. 
Published: July 2010

AbstractWe report the development and validation of the program GENFOLD, a genetic algorithm that calculates protein structures using restraints obtained from NMR, such as distances derived from nuclear Overhauser effects, and dihedral angles derived from coupling constants. The program has been tested on three proteins: the POU domain (a small three‐helix DNA‐binding protein), bovine pancreatic trypsin inhibitor (BPTI), and the starch‐binding domain from Aspergillus niger glucoamylase I, a 108‐residue β‐sheet protein. Structures were calculated for each protein using published NMR restraints. In addition, structures were calculated for BPTI using artificial restraints generated from a high‐resolution crystal structure. In all cases the fittest calculated structures were close to the target structure, and could be refined to structures indistinguishable from the target structures by means of a low‐temperature simulated annealing refinement. The effectiveness of the program is similar to that of distance geometry and simulated annealing methods, and it is capable of using a very wide range of restraints as input. It can thus be readily extended to the calculation of structures of large proteins, for which few NOE restraints may be available.
Subunit asymmetry in the three‐dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis
Authors: Eisenberg David , Hart P. John , Liu Hongbin , Pellegrini Matteo , Nersissian Aram M. , Gralla Edith B. , Valentine Joan S. 
Published: July 2010

AbstractThe X‐ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig's disease (FALS) has been determined to 1.9 Å resolution. The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites. One subunit (subunitintact) shows a four‐coordinate ligand geometry of the copper ion, whereas the other subunit (subunitbroken) shows a three‐coordinate geometry of the copper ion. Also, subunitintact, displays higher atomic displacement parameters for backbone atoms (<B> = 30 ± 10 Å2) than subunitbroken (<B> = 24 ± 11 Å2). This structure is the first CuZnSOD to show large differences between the two subunits. Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested.
Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: Molecular basis of isozyme‐drug discrimination
Authors: Stams Travis , Chen Yun , Christianson David W. , Boriack‐Sjodin P. Ann , Hurt Jonathan D. , Laipis Philip , Silverman David N. , Liao John , May Jesse A. , Dean Tom 
Published: July 2010

AbstractCarbonic anhydrase IV (CAIV) is a membrane‐associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8‐Å resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt™). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the “130′s segment” and the residue‐63 loop (these may affect the nearby catalytic proton shuttle, His‐64). Similar to human CAIV, the C‐terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.
Cadmium‐induced crystallization of proteins: II. Crystallization of the Salmonella typhimuri histidine‐binding protein in complex with L‐histidine, L‐arginine, or L‐lysine
Authors: Ames Giovanna Ferro‐Luzzi , Kreimer David I. , Trakhanov Sergei , Parkin Sean , Rupp Bernhard 
Published: July 2010

AbstractTo further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine‐binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine‐binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 Å resolution and belong to the orthorhombic space group P212121. From our results and from the analysis of cadmium sites in well‐refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.
Anion binding to the ubiquitin molecule
Authors: Makhatadze George I. , Lopez Marin M. , Richardson John M. , Thmos Susan T. 
Published: July 2010

AbstractEffects of different salts (NaCl, MgCl2, CaCl2, GdmCl, NaBr, NaC1O4, NaH2PO4, Na2SO4) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for Cl− show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of ‐15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01‐2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of Cl− ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with Cl− binding effects.
Tautomeric state and pKa of the phosphorylated active site histidine in the N‐terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system
Authors: Garrett Daniel S. , Clore G.Marius , Gronenborn Angela M. , Seok Yeong‐Jae , Peterkfsky Alan 
Published: July 2010

AbstractThe phosphorylated form of the N‐terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one‐bond and long‐range 1H ‐ 1.5N correlation spectroscopy. The active site His 189 is phosphorylated at the Nϵ2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Nδ1‐H tautomer, and its Nϵ2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side‐chain conformation of His 189, specifically from a X2 angle in the g+ conformer in the unphosphorylated state to the g− conformer in the phosphorylated state.
S100B(ββ) inhibits the protein kinase C‐dependent phosphorylation of a peptide derived from p53 in a Ca2+‐dependent manner
Authors: Wilder Paul T. , Rustandi Richard R. , Drohat Alexander C. , Weber David J. 
Published: July 2010

AbstractS100B(ββ) is a dimeric Ca2+‐binding protein that is known to inhibit the protein kinase C (PKC)‐dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367‐388), neuromodulin (residues 37‐53), and the regulatory domain of PKC (residues 19‐31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full‐length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627‐11631), S100B(ββ) binds the p53 peptide and inhibits its PKC‐dependent phosphorylation (IC50 = 10 ± 7μM) in a Ca2+‐dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(ββ) in the presence of Ca2+ (IC50 = 17 ± 5 μM; IC50 = 1 ± 0.5 μM, respectively). At a minimum, the C‐terminal EF‐hand Ca2+‐binding domain (residues 61‐72) of each S100β subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition (CaIC50 = 29.3 ± 17.6 μM) to the dissociation of Ca2+ from the C‐terminal EF‐hand Ca2+‐ binding domain of S100B(ββ).
Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system
Authors: Chu Vano , Stayton Partrick S. , Freitag Stefanie , Le Trong Isolde , Stenkamp Ronald E. 
Published: July 2010

AbstractA circularly permuted streptavidin (CP51/46) has been designed to remove the flexible polypeptide loop that undergoes an open to closed conformational change when biotin is bound. The original termini have been joined by a tetrapeptide linker, and four loop residues have been removed, resulting in the creation of new N‐ and C‐termini. Isothermal titration calorimetric studies show that the association constant has been reduced approximately six orders of magnitude below that of wild‐type streptavidin to 107 M−1. The δH° of biotin association for CP51/46 is reduced by 11.1 kcal/mol. Crystal structures of CP51/46 and its biotin complex show no significant alterations in the binding site upon removal of the loop. A hydrogen bond between Ser45 and Ser52 found in the absence of biotin is broken in the closed conformation as the side‐chain hydroxyl of Ser45 moves to hydrogen bond to a ureido nitrogen of biotin. This is true in both the wild‐type and CP51/46 forms of the protein, and the hydrogen bonding interaction might thus help nucleate closure of the loop. The reduced entropic cost of binding biotin to CP51/46 is consistent with the removal of this loop and a reduction in entropic costs associated with loop closure and immobilization. The reduced enthalpic contribution to the free energy of binding is not readily explainable in terms of the molecular structure, as the binding contacts are nearly entirely conserved, and only small differences in solvent accessible surfaces are observed relative to wild‐type streptavidin.
Mutagenesis of histidine 26 demonstrates the importance of loop‐loop and loop‐protein interactions for the function of iso‐1‐cytochrome c
Authors: Fetrow Jacquelyn S. , Dreher Ulrike , Wiland Debra J. , Schaak Diane L. , Boose Terry L. 
Published: July 2010

AbstractIn yeast iso‐1‐cytochrome c, the side chain of histidine 26 (His26) attaches omega loop A to the main body of the protein by forming a hydrogen bond to the backbone atom carbonyl of glutamic acid 44. The His26 side chain also forms a stabilizing intra‐loop interaction through a hydrogen bond to the backbone amide of asparagine 31. To investigate the importance of loop‐protein attachment and intra‐loop interactions to the structure and function of this protein, a series of site‐directed and random‐directed mutations were produced at His26. Yeast strains expressing these variant proteins were analyzed for their ability to grow on non‐fermentable carbon sources and for their intracellular production of cytochrome c. While the data show that mutations at His26 lead toslightly decreased intracellular amounts of cytochrome c, the level of cytochrome c function is decreased more. The data suggest that cytochrome c reductase binding is affected more than cytochrome c oxidase or lactate dehydrogenase binding. We propose that mutations at this residue increase loop mobility, which, in turn, decreases the protein's ability to bind redox partners.
Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)
Authors: Woo Sang B. , Neet Kenneth E. , Whalen Christopher 
Published: July 2010

AbstractNerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and pl40TrkA. NGF binding to pl40TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA‐RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA‐RED is a 70 kDa acidic glycoprotein with a p1 of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA‐RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA‐RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA‐RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA‐RED)2–(NGF dimer) complex. Association and dissociation rates of 5.6 × 105 M−1 s−1 and 1.6 × lO−3 s−1 respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF‐induced receptor dimerization concomitant with a small conformational change.
Treatment of prostatic cancer: The eortc experience—preliminary results of prostatic carcinoma trials
Authors: Schroeder Fritz H. 
Published: July 2010

AbstractSix prospective studies in the field of prostatic carcinoma have been carried out to date by the EORTC Urological Group. In three phase II studies, adriamycin, procarbazine, vinde‐sine, and mytomycin C have been studied. Two of the three protocols have been completed. In three phase III studies, 3 mg of diethylstilbestrol (DES) is compared to cyproterone acetate (CPA), medroxyprogesterone acetate (MPA), and Estramustin phosphate (Estracyt). These two protocols have been closed to entry. The current protocol compares DES, 1 mg, to castration and to cyproterone acetate plus castration.From the phase II studies, no drug has emerged that is recommended for treatment of hormone‐resistant prostatic cancer. The endocrine protocols, designed for the primary treatment of T3, T4, and Ml carcinoma of the prostate, have resulted in several important observations. Responses to DES, 3 mg/day, and to Estracyt were very similar and amounted to 25‐30%. There was somewhat less objective response in the CPA and significantly less (P = 0.005) in the MPA group. It has become evident that DES at a dosage of 3 mg/day carries a significantly higher risk of overall cardiovascular toxicity than does cyproterone acetate, but severe cardiovascular complications did not differ between treatment groups. Up to now, no differences in survival were observed within the different treatment groups. Grade, local tumor extension, and performance were found to have a significant impact on survival.
Effects of Panax ginseng on glial cell‐derived neurotrophic factor (GDNF) expression and spermatogenesis in rats
Authors: Yang Woong Mo , Park Soo Yeon , Kim Hyung‐Min , Park Eun Hwa , Park Seong Kyu , Chang Mun Seog 
Published: July 2010

AbstractPanax ginseng (PG) is a medicinal herb which has been used to improve male reproduction in traditional Korean medicine. This study investigated the effect of PG on spermatogenesis and the regulation of glial cell‐derived neurotrophic factor (GDNF), which has a crucial role in spermatogonial stem cell maintenance. PG was administered to 8‐week‐old male Wistar rats (1.0 g/kg/day, p.o.) for 56 consecutive days, the sperm formation period of the rat. Sperm analysis, RT‐PCR and western blot assays were then carried out. The PG‐treated group had significantly enhanced sperm counts, GDNF mRNA level and protein level. These results suggest that PG induces spermatogenesis and GDNF activation in rat testes. Copyright © 2010 John Wiley & Sons, Ltd.