bluuebunny/crossref_metadata_2025_split huggingface dataset:
Subunit interaction in extracellular superoxide dismutase: Effects of mutations in the N‐terminal domain
Authors: Stenlund Peter , Tibell Lena A. E. , Andersson Dick 
Published: February 2009

AbstractHuman extracellular superoxide dismutase (hEC‐SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC‐SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X‐ray structural determination have failed, the three‐dimensional structure of hEC‐SOD is unknown. This means that alternative strategies for structural studies are needed. The N‐terminal domain of EC‐SOD has already been studied using the fusion protein FusNN, comprised of the 49 N‐terminal amino acids from hEC‐SOD fused to human carbonic anhydrase (HCAII). The N‐terminal domain in this fusion protein forms a well‐defined three‐dimensional structure, which probably contains α‐helical elements and is responsible for the tetramerization of the protein.In this work, we have extended the studies, using site‐directed mutagenesis in combination with size‐exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic a‐helix (formed by residues 14‐32) in the N‐terminal domain is essential for the subunit interaction.
Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR
Authors: Huth Jeffrey R. , Bewley Carole A. , Clore G. Marius , Gronenborn Angela M. , Jackson Belinda M. , Hinnebusch Alan G. 
Published: February 2009

AbstractTwo protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin‐binding domain of streptococcal protein G (GB1 domain) linked to the N‐terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H‐15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15‐labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand‐receptor interactions, using 15N‐labeled GB1‐peptide fusions and unlabeled target.
Characterization of a helix‐loop‐helix (EF hand) motif of silver hake parvalbumin isoform B
Authors: Revett Sandra P. , Nelson Donald J. , King Gina , Shabanowitz Jeffrey , Hunt Donald F. , Laue Thomas M. , Hartman Kari L. 
Published: February 2009

AbstractParvalbumins are a class of calcium‐binding proteins characterized by the presence of several helix‐loop‐helix (EF‐hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF‐hand. Silver hake parvalbumin (SHPV) consists of three EF‐type helix‐loop‐helix regions, two of which have the ability to bind calcium. The three helix‐loop‐helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV‐B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a β‐lineage parvalbumin. SHPV‐B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33‐amino acid EF fragment (residues 76‐108), containing one of the calcium ion binding sites in native SHPV‐B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion‐induced self‐association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge‐density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer‐dimer equilibrium when complexed with La3+.
Alteration of T4 lysozyme structure by second‐site reversion of deleterious mutations
Authors: Poteete Anthony R. , Rennell Dale , Bouvier Suzanne E. , Hardy Larry W. 
Published: February 2009

AbstractMutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second‐site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures.
Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry
Authors: Neville David C. A. , Townsend R. Reid , Rozanas Christine R. , Verkman A. S. , Price Elmer M. , Gruis Darren B. 
Published: February 2009

AbstractThe cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane C1− channel regulated by protein phosphorylation. To identify cAMP‐dependent protein kinase (PKA)‐phosphorylated residues in full‐length CFTR, immobilized metal‐ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron‐loaded (Fe3+) nitrilotriacetic (NTA) Sepharose compared to iminodiacetic acid (IDA) metal‐chelating matrices was demonstrated using a PKA‐phosphorylated recombinant NBD1‐R protein from CFTR. Fe3+‐loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly‐His‐containing peptides were co‐purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA‐phosphorylated full‐length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix‐assisted laser desorption mass spectrometry (MALDI/MS) with post‐source decay (PSD) analysis and collision‐induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full‐length CFTR.
The proteasome 11S regulator subunit REGα (PA28α) is a heptamer
Authors: Johnston Steven C. , Whitby Frank G. , Realini Claudio , Rechsteiner Martin , Hill Christopher P. 
Published: February 2009

AbstractActivity of the 20s proteasome, which performs much of the cytosolic and nuclear proteolysis in eukaryotic cells, is controlled by regulatory complexes that bind to one or both ends of the cylindrical proteasome. One of these complexes, the 11S regulator (REG), is a complex of 28 kDa subunits that is thought to activate proteasomes toward the production of antigenic peptides. REG, purified from red blood cells, is a complex of REGα and REGβ subunits. We have crystallized recombinant REGα (rREGα) and collected diffraction data to 3.0 Å resolution. The self‐rotation function indicates that rREGα forms a heptameric ring in the crystal. Equilibrium sedimentation demonstrates that rREGα is a heptamer in solution also.
Crystal structures of a marginally active thymidylate synthase mutant, Arg 126 → Glu
Authors: Strop Pavel , Montfort William R. , Changchien Liming , Maley Frank 
Published: February 2009

AbstractThymidylate synthase (TS) is a long‐standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co‐enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon‐carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000‐fold and Km reduced 600‐fold. The crystal structure of R126E was determined under two conditions—in the absence of bound ligand (2.4 Å resolution), and with dUMP and the antifolate CB3717 (2.2 Å resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a ∼ 1 Å shift in the phosphate group. Interestingly, the protein displayed the typical ligand‐induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.
A flavodoxin that is required for enzyme activation: The structure of oxidized flavodoxin from Escherichia coli at 1.8 Å resolution
Authors: Hoover David M. , Ludwig Martha L. 
Published: February 2009

AbstractIn Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate‐lyase, anaerobic ribonucleotide reductase, and B12‐dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P212121, a = 126.4, b = 41.10, c = 69.15 Å, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 Å, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8‐Å resolution were collected at 140 K and the structure was refined to an Rworkof 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non‐hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis‐Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 Å,) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 Å (R = 0.191; Rfree = 0.249). The sequence Tyr 58‐Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.
Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin
Authors: Lazaridis Themis , Lee Irwin , Karplus Martin 
Published: February 2009

AbstractMolecular dynamics simulations in solution are performed for a rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus (RdPf) and one from the mesophilic organism Desulfovibrio vulgaris (RdDv). The two proteins are simulated at four temperatures: 300 K, 373 K, 473 K (two sets), and 500 K; the various simulations extended from 200 ps to 1,020 ps. At room temperature, the two proteins are stable, remain close to the crystal structure, and exhibit similar dynamic behavior; the RMS residue fluctuations are slightly smaller in the hyperthermophilic protein. An analysis of the average energy contributions in the two proteins is made; the results suggest that the intraprotein energy stabilizes RdPf relative to RdDv. At 373 K, the mesophilic protein unfolds rapidly (it begins to unfold at 300 ps), whereas the hyperthermophilic does not unfold over the simulation of 600 ps. This is in accord with the expected stability of the two proteins. At 473 K, where both proteins are expected to be unstable, unfolding behavior is observed within 200 ps and the mesophilic protein unfolds faster than the hyperthermophilic one. At 500 K, both proteins unfold; the hyperthermophilic protein does so faster than the mesophilic protein. The unfolding behavior for the two proteins is found to be very similar. Although the exact order of events differs from one trajectory to another, both proteins unfold first by opening of the loop region to expose the hydrophobic core. This is followed by unzipping of the β‐sheet. The results obtained in the simulation are discussed in terms of the factors involved in flexibility and thermostability.
Crystallization and preliminary X‐ray diffraction analysis of arginyl‐tRNA synthetase from Escherichia coli
Authors: Lin Sheng‐Xiang , Zhou Ming , Campbell Robert L. , Wang En‐Duo , Wang Ying‐Lai 
Published: February 2009

AbstractArginyl‐tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 Å with a rotating anode X‐ray source and R‐axis IIc image plate detector. They have an ortho‐rhombic space group P21212 with unit cell parameters of a = 251.51 Å, b = 53.12 Å, and c = 52.35 Å. A complete native data set has been collected at 3.1 Å resolution for these crystals.
Crystal structure of a non‐toxic mutant of heat‐labile enterotoxin, which is a potent mucosal adjuvant
Authors: Van Den Akker Focco , Pizza Mariagrazia , Rappuoli Rino , Hol Wim G. J. 
Published: February 2009

AbstractTwo closely related bacterial toxins, heat‐labile enterotoxin (LT‐I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single‐site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT‐I is determined at 2.0 Å resolution. Its structure appears to be essentially the same as the wild‐type LT‐I structure. The substitution Ser63Lys was designed, based on the wild‐type LT‐I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT‐I. The fact that the Ser63Lys mutant of LT‐I does not disrupt the wild‐type LT‐I structure makes the non‐toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild‐type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.
Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold
Authors: Buss Kathryn A. , Sanders David A. , Hasson Miriam S. , Ingram‐Smith Cheryl , Ferry James G. 
Published: February 2009

AbstractThe unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme‐catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 Å resolution. Analysis of X‐ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 Å, b = 67 Å, c = 83 Å, β = 103°. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic twofold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.
Crystallization of the first three domains of the human insulin‐like growth factor‐1 receptor
Authors: Mckern Neil M. , Frenkel Maurice J. , Verkuylen Amanda , Bentley John D. , Lovrecz George O. , Ivancic Neva , Elleman Thomas C. , Cosgrove Leah J. , Lou Meizhen , Garrett THOMAS P. J. , Ward Colin W. 
Published: February 2009

AbstractThe insulin‐like growth factor‐1 receptor (IGF‐1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1‐462) comprising the L1‐cysteine rich‐L2 domains of the human IGF‐1R ectodomain has been overexpressed in glycosylation‐deficient Lec8 cells and has been affinity‐purified via a c‐myc tag followed by gel filtration. The fragment was recognized by two anti‐IGF‐1R monoclonal antibodies, 24‐31 and 24‐60, but showed no detectable binding of IGF‐1 or IGF‐2. Isocratic elution of IGF‐1R/462 on anion‐exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 Å resolution with cell dimensions a = 77.0 A, Å = 99.5 Å, c = 120.1 Å, and space group P212121.
Crystallization and preliminary X‐ray studies of I‐PpoI: A nuclear, intron‐encoded homing endonuclease from Physarum polycephalum
Authors: Flick Karen E. , Mchugh Diane , Stoddard Barry L. , Heath Joe Don , Stephens Kathryn M. , Monnat Raymond J. 
Published: February 2009

AbstractThe homing endonuclease I‐PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26s rRNA gene. This endonuclease promotes the lateral transfer or “homing” of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26s rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I‐PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombi‐nant I‐PpoI has been co‐crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3121, with unit cell dimensions a = b = 114 Å, c = 89 Å. The results of initial X‐ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 Å3/dalton. These experiments also provide strong evidence that I‐PpoI contains several bound zinc ions as part of its structure.
Calculated electrostatic gradients in recombinant human H‐chain ferritin
Authors: Douglas Trevor , Ripoll Daniel R. 
Published: February 2009

AbstractCalculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H‐chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three‐fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic potential. However, the outer entrance to the three‐fold channels are surrounded by regions of positive potential, creating an electrostatic field directed toward the interior cavity. This electrostatic gradient provides a guidance mechanism for cations entering the protein cavity, indicating the three‐fold channel as the major entrance to the protein.Pathways from the three‐fold channels, indicated by electrostatic gradients on the inner surface, lead to the ferroxidase center, the nucleation center and to the interior entrance to the four‐fold channel. Six glutamic acid residues at the nucleation site give rise to a region of very negative potential, surrounding a small positively charged center due to the presence of two conserved arginine residues, R63, in close proximity (4.9 Å), suggesting that electrostatic fields could also play a role in the nucleation process.A large gradient in the electrostatic potential at the 4‐fold channel gives rise to a field directed outward from the internal cavity, indicating the possibility that this channel functions to expel cations from inside the protein. The 4‐fold channel could therefore provide an exit pathway for protons during mineralization, or iron leaving the protein cavity during de‐mineralization.
Synthesis, physicochemical characterization, and crystallization of a putative retro‐coiled coil
Authors: Liu Niankun , Deillon Christine , Klauser Stephan , Gutte Bernd , Thomas Richard M. 
Published: February 2009

AbstractAn artificial HIV enhancer‐binding polypeptide has recently been dimerized by covalently linking it to the leucine zipper motif of the yeast transcriptional activator GCN4 (Liu N et al., 1997, Eur Biophys J 25:399‐403). Although it seemed that the dimerization of this peptide could be best achieved by the use of the retro sequence of the leucine zipper, this approach was not implemented in the original construct. As the first step toward the synthesis of a basic region‐retro leucine zipper HIV enhancer‐binding fusion protein, we have now prepared the retro version of the leucine zipper (r‐LZ35) and performed initial physicochemical characterization. Circular dichroism and sedimentation equilibrium studies showed that, at concentrations <100 μ, the retro peptide was an unstructured monomer. At higher concentrations, however, the monomer was in equilibrium with a tetramer and, at 1 mM, the retro peptide was almost fully helical. N‐terminal extension of the retro peptide by the tripeptide Cys‐Gly‐Gly resulted in a 38‐residue polypeptide that could be covalently dimerized by forming a disulfide bond between two chains to give the peptide (r‐LZ38)2. Even in the low micromolar concentration range peptide (r‐LZ38)2 formed a stable, noncovalent, helical dimer as revealed by circular dichroism and sedimentation equilibrium in the presence and absence of guanidinium chloride. (r‐LZ38)2 has been crystallized and X‐ray structural analysis is under way. The disulfide‐crosslinked retro‐leucine zipper may lend itself to interesting protein structural studies, including protein design. The present work also highlights the structural and functional potential of retro proteins in general.