bluuebunny/crossref_metadata_2025_split huggingface dataset:
The DNA replication inhibitor microcin B17 is a forty‐three‐amino‐acid protein containing sixty percent glycine
Authors: Davagnino Juan , Herrero Marta , Furlong Dierdre , Moreno Felipe , Kolter Roberto 
Published: May 2005

AbstractMicrocin B17 is a low‐molecular‐weight protein that inhibits DNA replication in a number of enteric bacteria. It is produced by bacterial strains which harbor a 70‐kilobase plasmid called pMccB17. Four plasmid genes (named mcbABCD) are required for its production. The product of the mcbA gene was identified by labelling minicells. The mcbA gene product was slightly larger when a mutation in any of the other three production genes was present. This indicates that these genes are involved in processing the primary mcbA product to yield the active molecule. The mcbA gene product predicted from the nucleotide sequence has 69 amino acids including 28 glycine residues. Microcin B17 was extracted from the cells by boiling in 100 mM acetic acid, 1 mM EDTA, and purified to homogeneity in a single step by high‐performance liquid chromatography through a C18 column. The N‐terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17. The active molecule is a processed product lacking the first 26 N‐terminal residues. The 43 remaining residues include 26 glycines. While microcin B17 is an exported protein, the cleaved N‐terminal peptide does not have the characteristic properties of a “signal sequence,” which suggests that it is secreted by a mechanism different from that used by most secreted proteins of E. coli.
Primary structure of the reaction center from Rhodopseudomonas sphaeroides
Authors: Williams J. C. , Steiner L. A. , Feher G. 
Published: May 2005

AbstractThe reaction center is a pigmentprotein complex that mediates the initial photochemical steps of photosynthesis. The amino‐terminai sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single‐stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that hte H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly bviased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch opf hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was contructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.
Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells
Authors: Schneider William P. , Oi Vernon T. , Yanofsky Charles 
Published: May 2005

AbstractWe have produced a series of hybrid IgG1‐IgG2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differnces between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse γ1 and γ2a gene segments. Crossovers occured through out these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H2L2 tetramers and properly glycosylated. In addition, all 11 immunoglbulins have identical antigen combining sites specific for the fluorescent hapten ε‐dansyll‐L‐lysine. Protein A binding was used as probe of the structural integrity of the Fc portion of the variant antibodies. Differeneces in protein A binding between IgG1 and IgG2a appear to be due to amino acid differances at postions 252 (Thr→Met) and 254 (Thr→Ser) of the heavy chain (EU numbering).
Prediction of the tertiary structure of the α‐subunit of tryptophan synthase
Authors: Hurle Mark R. , Matthews C. Robert , Cohen Fred E. , Kuntz I.D. , Toumadje Arazdordi , Johnson W. Curtis 
Published: May 2005

AbstractThe tertiary structure of the α‐subunit of tryptophan synthase was proposed using a combination of experimental data and computational methods. The vacuum‐ultraviolet circular dichroism spectrum was used to assign the protein to the α/β‐class of supersecondary structures. The two‐domain structure of the α‐subunit (Miles et al.: Biochemistry 21:2586, 1982; Beasty and Matthews: Biochemistry 24:3547, 1985) eliminated consideration of a barrel structure and focused attention on a β‐sheet structure. An algorithm (Cohen et al.: Biochemistry 22:4894, 1983) was used to generate a secondary structure prediction that was consistent with the sequence data of the α‐subunit from five species. Three potential secondary structures were then packed into tertiary structures using other algorithms. The assumption of nearest neighbors from second‐site revertant data eliminated 97% of the possible tertiary structures; consideration of conserved hydrophobic packing regions on the β‐sheet eliminated all but one structure. The native structure is predicted to have a parallel β‐sheet flanked on both sides by α‐helices, and is consistent with the available data on chemical cross‐linking, chemical modification, and limited proteolysis. In addition, an active site region containing appropriate residues could be identified as well as an interface for β2‐subunit association. The ability of experimental data to facilitate the prediction of protein structure is discussed.
Lysine/fibrin binding sites of kringles modeled after the structure of kringle 1 of prothrombin
Authors: Tulinsky Alexander , Park Chang H. , Mao Boryeu , Llináas Miguel 
Published: May 2005

AbstractThe Lys binding site of kringle 1 and 4 (K1 and K4) of plasminogen (PG) has been modeled on the basis of the three‐dimensional structure of kringle 1 of prothrombin and 300‐ and 600‐MHz proton nuclear magnetic resonance observations. These structures were then compared to the corresponding regions of modeled kringle 1 and 2 of tissue plasminogen activator (PA). The coordinates of the modeled structures have been refined by energy minimization in the presence and absence of ϵ‐aminocaproic acid ligand in order basically to remove unacceptable van der Waals contacts. The binding site is characterized by an apparent dipolar surface, the polar parts of which are separated by a hydrophobic region of highly conserved aromatic residues. Zwitterionic ligands such as Lys and ϵ‐aminocaproic acid form ion pair interactions with Asp55 and Asp57 located on the dipolar surface; the latter are also conserved in all the Lys binding kringles. The cationic center of the dipolar surface is Arg71, in the case of PGK4, and is composed of Arg34 and Arg71 in PGK1. The doubly charged anionic/cationic interaction centers of the latter might account for the larger binding constants of PGK1 for like‐ligands but the modeling suggests that PGK4 might be kinetically faster in binding bulkier ligands. The binding site region of PAK2, which also binds Lys, resembles those of PGK1 and PGK4. Since PAK2 lacks both cationic center Arg residues, ligand carboxylate binding appears to be accomplished though an imidazolium ion of His64, which is located just below the outer surface of the kringle.
Model of a complex between the tetrahemic cytochrome c3 and the ferredoxin I from Desulfovibrio desulfuricans (Norway strain)
Authors: Cambillau C. , Frey M. , Mossé J. , Guerlesquin F. , Bruschi M. 
Published: May 2005

AbstractA three‐dimensional model of an electron‐transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfatereducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X‐ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X‐ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe‐4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the “best” structure in terms of charges, interactions, and complementary f the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 Å, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 Å2.
Crystallization and preliminary investigation of single crystals of deoxyuidine triphosphate nucleotidohydrolase from Escherichia coli
Authors: Cedergren‐Zeppezauer Eilia S. , Larsson Gunilla , Hoffmann Ingrid , Törnoos Karl W. , Al‐Karadaghi Salam , Nyman Per Olof 
Published: May 2005

AbstractDeoxyuridine triphosphate nucleotidohydrolase (dUTPase), an enzyme in the nucleotide metabolism that is a pyrophosphatase hydrolyzing dUTP, has been crystallized. The crystals belong to the trigonal space group R3 and diffract beyond 2 Å. The native dUTPase crystals and a mercury derivative are stable in the X‐ray beam and are suitable for a high resolution X‐ray structure analysis.
Homology of lysosomal enzymes and related proteins: Prediction of posttranslational modification sites including phosphorylation of mannose and potential epitopic and substrate binding sites in the α‐ and β‐subunits of hexosaminidases, α‐glucosidase, and rabbit and human isomaltase
Authors: Barnes Alison K. , Wynn Colin H. 
Published: May 2005

AbstractRecently developed computer programs, including secondary structure and epitopic site predictions, have been used to align lysosomal proteins for maximum homology, based on conservative interchanges, and the aligned sequences have been searched for potential sites for posttranslational modification, glycosyaltion, and binding and catalysis of substrate. The homology and prediction of the posttranslational modification of the α‐ and β‐subunits of hexosaminidase is in good agreement with previous observations, and an explanation of the differing substrate specificities of the two subunits is advanced. We shows that the striking homology between α‐glucosidase and isomaltase is reflected in that apparent conversation of the active site in both enzymes. Nonhomologous regions have been examined in detail in a search for binding sited for glycogen and maltose, and two such sites have been tentatively identified. A highly redundant consensus sequence for the Phosphorylation of mannose in lysosomal proteins, YXX(Y, W, or F), is suggested.
Second virial coefficient of α‐crystallin
Authors: Wang Xiaowei , Bettelheim Frederick A. 
Published: May 2005

AbstractLight scattering studies were performed on bovine α‐crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of α‐crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of α‐crystallin are positive. The Flory thetatemperature was found to be 271 K.
A protein structural motif that bends DNA
Authors: White Stephen W. , Appelt Krzysztof , Wilson Keith S. , Tanaka Isao 
Published: May 2005

AbstractThe prokaryotic protein HU, integration host factor (IHF) from Escherichia coli and transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules. Biochemical results suggest that the role of these proteins is to bind and bend DNA. From the high‐resolution structure of HU, we propose a model for thisinteraction with DNA. Crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.
Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV 1) protease purified from Escherichia coli
Authors: Strickler James E. , Gorniak Joselina , Dayton Brian , Meek Thomas , Moore Michael , Magaard Victoria , Malinowski John , Debouck Christine 
Published: May 2005

AbstractA recombinant plasmid encompassing the human immunodeficiency virus type 1 (HIV 1) protease coding sequence and flanking regions (Ala‐13 to Gly‐185 of the pol open reading frame) has been expressed in two distinct strains of Escherichia coli, AR58 and AR68. In the first strain, AR58, the primary translation product, a 25 kilodalton (kDa) precursor protein, is short‐lived and rapidly processes itself to the 11 kDa mature protease in vivo. In the second strain, AR68, the 25 kDa species isonly partially processed, and it, a 13 kDA intermediate, and the mature 11 kDA enzyme accumulate at a ratio of 3:4.5:2.5, respectively. The 11 kDa mature protease from AR58 and the 25 kDa precursor from AR68 have been purified to homogeneity. The yield of 11 kDa enzyme from AR58 is approximately 0.02 mg/g wet weight of E. coli cell pellet. The protease has both the expected NH2‐ and COOH‐terminal sequences. The yield of 25 kDa enzyme from AR68 is approximately 0.1 mg/g wet weight of E. coli cell pellet. In vitro, the 25 kDa precursor enzyme rapidly (t1/2≅ 9 min) processes itself into a species with a mass of ∼13kDa and a species with a mass of ∼11 kDa. Both of these latter species can be separated by RP‐HPLC, have the NH2‐terminal sequence expected for the mature protease, and are active. The 11 kDa enzyme from AR58 comigrates with the 11 kDa enzyme from AR68 on RP‐HPLC and SDS poly acrylamide gel electrophoresis. On extended incubation at 4°C at either neutral or acidic pH all species of the proteinexhibit further autodegradation at defined sequences. The availability of the mature, 11 kDa enzyme and the 25 kDa precursor will allow biochemical and physical studies on this critical viral enzyme.
Site‐directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation
Authors: Todd A. Paul , Cong Jianping , Levinthal Francoise , Levinthal Cyrus , Hubell Wayne L. 
Published: May 2005

AbstractColicin E1 is an E. coli plasmid‐lencoded water‐soluble protein that spontaneously inserts into lipid membranes to form a voltage‐gated ion channel. We have employed a novel approach is which site‐directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels. A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine. A hydrophilic sequence (398–406) within the C‐terminal domain of the water‐soluble form of the protein was investigated and exhibited an electron paramagnetic resonancc (EPR) spectral periodicity strongly suggesting an amphiphilic α‐helix. After removal of the Nterminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.
Three‐dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy
Authors: Widmer Hans , Billeter Martin , Wüthrich Kurt 
Published: May 2005

AbstractWith the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three‐dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and χ1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sapling of al conformations that are consistent with the local and medium‐range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of β‐methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOCE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 ± 0.3 Å. This core is formed by a four‐stranded β‐sheet connected by two well‐defined loops, and there is an additional flexible loop consisting of the eleven residues 8‐18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.
Protein–drug interactions: Characterization of inhibitor binding in complexes of DHFR with trimethoprim and related derivatives
Authors: Fleischmann Stephen H. , Brooks Charles L. 
Published: May 2005

AbstractStructural and thermodynamic interactions for the binding of trimethoprim and related congeners to the binary complex of diphydrofolate reductase (from chicken) and NADPH are explored using free energy simulation methods. Good agreement between structures from experimental X‐ray refinement and molecular dynamics simulations is found for the complexes. Agreement with thermodyanmic measurements is found as well. Our thermodynamic calculations suggest that entropic contributions and desolvation thermodynamics can play a crucial role in overall bindings, and that extreme care must be taken in the use of simple model building to rationalize or predict protein–drug binding.
Structure determination and refinment of Bacillus stearothermophilus lactate dehydrogenase
Authors: Piontek Klaus , Chakrabarti Pinakpani , Schär Hans‐Peter , Rossmann Michael G. , Zuber Herbert 
Published: May 2005

AbstractStructures have been determined of Bacillus stearothermophilus “apo” and holo lactate dehydrogenase. The holo‐enzyme had been co‐crystallized with the activator fructose 1,6‐biosphosphate. The “apo” lactate dehydrogenase structure was solved by use of the known apo‐M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 Å resolution by means of the noncrystallographic molecular 222 symmetry. The R‐factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least‐squares refnement in which the molecula rsymmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the “apo” enzyme.Further refinement proceeded with the isomorphous holo‐enzyume from Bacillus Stearothermophilus. After removing the noncrystallographic constraints, the R‐factor dropped from 30.3% to a final value of 26.0% with a 0.019 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively.Two sulfate ions per subunit were included in the final model of the “apo” ‐from‐one at the substrate binding site and one close to the molecular P ‐axis near the location of the fructose 1,6‐bisphosphate activator. The final model of the holo‐enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R‐axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6‐bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6‐bisphosphate are close to the sulfate ion near the P‐axis in the “apo” model.This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo‐enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with incotinamide adenine dinucleotide and oxamate than to apo‐M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose, 1,6‐bisphosphate were also analyzed.
Computer analysis of mutations that affect antibody specificity
Authors: Novotny Jiri , Bruccoleri Robert E. , Haber Eldgar 
Published: May 2005

AbstractThe mouse hybridoma cell line 40–150 scretes antibodies with high affinity towards the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40–150 A2.4, produces and antibody which carries a single residue mutation, Ser → Arg, in its heavy chain (H94) and has an altered specificity. A second order mutant 40–150 A2.4 P.10, produces two antibody molecules, one the same as 40–150 A2.4, the other lacking two residues at the N‐terminus of its H chain, and having a specificity profile approaching that of 40–150 antibody. 1 The N‐terminus and the position H94 are distant from the antigen‐binding site of the antibody; thus, the structural basic of the specificity changes was not immediately clear. Approximate structures of the 40–150 antibody and its mutants were constructed in the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. Using the program OCNGEN, the torsional space of the polypeptide backbone and side chains around position H94 was uniformly sampled, and the lowest energy conformations were analyzed in detail. The results indicate that when Arg‐H94 is substituted for Ser. Agr‐H94 can hydrogen bond to side chains of Asp‐H101, Arg‐L46, and Asp‐L55. The results in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N‐terminal residues increases solvent accessibility of Arg‐H94. The solvation may cause a hydrogen bond between Arg‐H94 and Asp‐H101 to be lost, restoring the structure to one similar to that of 40–150.