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  • 101
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 219-225 
    ISSN: 0887-3585
    Schlagwort(e): enolase ; fluoride ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Enolase in the presence of its physiological cofactor Mg2+ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase-Mg2+-F--Pi has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-lo9, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate-enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg2+ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg2+. The synergism of inhibition by F- and Pi most probably is due to the requirement of Pi, for the closed conformation. It is possible that other Mg2+-dependent enzymes that have OH- ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 102
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 268-277 
    ISSN: 0887-3585
    Schlagwort(e): trp-repressor ; TIP3P water ; hydrophobic shell ; hydrogen bond ; AMBER ; diffusion coefficient ; radial distribution ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The solvent structure and behavior around a protein were examined by analyzing a trajectory of molecular dynamics simulation of thetrp-holorepressor in a periodic box of water. The calculated selfdiffusion coefficient indicated that the solvent within 10 Å of the protein had lower mobility. Examination of the solvent diffusion around different atoms of different kinds of residues showed no general tendency. Thisfact suggested that the solvent mobility is not influenced significantly bythe kind of the atom or residue they solvated. Distribution analysis aroundthe protein revealed two peaks of water oxygen: a sharp one at 2.8 Å around polar and charged atoms and a broad one at ∼3.4 Å aroundapolar atoms. The former was stabilized by water-protein hydrogen bonds, and the latter was stabilized by water-lwater hydrogen bonds, suggesting the existence of a hydrophobic shell. An analysis of protein atom-water radial distribution functions confirmed these shell structures around polar or charged atoms and apolar ones. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 103
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 226-245 
    ISSN: 0887-3585
    Schlagwort(e): molecular dynamics ; biotin ; avidin ; free energy calculations ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We present calculations of the absolute and relative binding free energies of complexation of streptavidin with biotin and its analogsby means of a thermodynamic free energy perturbation method implemented with molecular dynamics. Using the recently solved crystal structure of the streptavidin-biotin complex, biotin was mutated into a dummy molecule as well as thiobiotin and iminobiotin both in the protein and insolution. The calculated absolute binding free energy was dependent on the simulation model used. Encouragingly, the “best models” provided a reasonable semiquantitative reproduction (-20 to -22 kcal/mol) of the experimental free energy (-18.3 kcal/ mol). Furthermore, the calculated results give clear insights into the binding nature of the protein-ligand complex, showing that the van der Waals energy dominates the electrostatic and hydrogen bonding energies in thebinding of biotin by streptavidin. Specifically, the mutation of biotin into a dummy molecule in solution has a ΔG (van der Waals) ∼ -4 kcal/mol, due to the cancellation of dispersion and repulsion “cavity” effects. On the other hand, in the protein, a very small free energy price must be paid to create a cavity since one already exists and the mutation of biotin into a dummy molecule has a ΔG (van der Waals) ∼ 15 kcal/mol. These results are also consistent with the interpretation that the entropy increase to be expected from hydrophobic interactions from desolvation of biotin is counterbalanced by a decrease in entropy accompanying the formationof buried hydrogen bonds, which have been derived from the apparentlyconflicting experimental data. They provide an alternative interpretationofthe reason for the extremely high affinity of the biotin-streptavidin interaction than that recently proposed by Weber et al. (J. Am. Chem. Soc. 114:3197, 1992). In the case of the relative binding freeenergies, the calculated values of 3.8 ± 0.6 and 7.2 ± 0.6 kcal/mol compare well with the experimental values of 3.6 and 6.2 kcal/mol for the perturbation of biotin to thiobiotin and iminobiotin, respectively in the related protein avidin. The calculations indicate that desolvation of the ligand is important in understanding the relative affinity of the ligands with the protein. The above successful simulations suggestthat the molecular dynamics/free energy perturbation method is useful for understanding the energetic features affecting the binding between proteins and ligands, since it is generally difficult to determine these factors unambiguously by experiment. This set of studies provide a textbook example of the key elements of protein-ligand recognition: the electrostatic free energy dominates the relative affinities, the van der Waals free energy dominates the absolute free energy; the free energy of desolvation is a key to why iminobiotin is so much more weakly bound than biotin and the free energy of binding explains why thiobiotin is so weakly bound relative to biotin. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 104
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 246-267 
    ISSN: 0887-3585
    Schlagwort(e): generic binding cavity ; ligand design ; site-filling peptides ; effects of pH on binding D- and L-enantiomers ; immunoglobulin light chain dimer ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: An immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X-ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallo-graphically refined structures of 14 peptide-protein complexes at 2.7 Å resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change inconformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program anend-to-end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligandswere diffused into preexisting crystals. In sucha large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 105
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 278-292 
    ISSN: 0887-3585
    Schlagwort(e): protein structural comparison ; 3-D protein motifs ; surface motifs ; docking ; computer vision ; geometric hashing ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We describe the application of a method geared toward structural and surface comparison of proteins. The method is based on the Geometric Hashing Paradigm adapted from Computer Vision. It allows for comparison of any two sets of 3-D coordinates, such as protein backbones, protein core or protein surface motifs, and small molecules such as drugs. Here we apply our method to 4 types of comparisons between pairs of molecules: (1) comparison of the backbones of two protein domains; (2) search for a predefined 3-D Cα motif within the full backbone of a domain; and in particular, (3) comparison of the surfaces of two receptor proteins; and (4) comparison of the surface of a receptor to the surface of a ligand. These aspects complement each other and can contribute toward a better understandingof protein structure and biomolecular recognition. Searches for 3-D surface motifs can be carried out on either receptors or on ligands. The latter may result in the detection of pharmacophoric patterns. If the surfaces of the binding sites of either the receptors or of the ligands are relatively similar, surface superpositioning may aid significantly in the docking problem. Currently, only distance invariants are used in the matching, although additional geometric surface invariants are considered. The speed of our Geometric Hashing algorithm is encouraging, with a typical surface comparison taking only seconds or minutes of CPU time on a SUN 4 SPARC workstation. The direct application of this method to the docking problem is also discussed. We demonstrate the success of this methodin its application to two members of the globin family and to two dehydrogenases. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 106
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 301-305 
    ISSN: 0887-3585
    Schlagwort(e): molecular replacement ; rubredoxin ; phase error ; intensity distribution ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Crystals of racemic rubredoxin, prepared by independent chemical synthesis of the two enantiomers, have been grown and characterized. The unit cell contains two molecules, one of each enantiomer. Examination of the intensity distribution in the diffraction pattern revealed that the crystals are centrosymmetric. This was confirmed by solution of thestructure to 2 Å resolution via molecular replacement methods. The electron density maps are of very high quality due to the fact that the phaseof each reflection must be exactly 0° or exactly 180°. These results demonstrate the feasibility of using synthetic racemic proteins to yield centrosymmetric protein crystals with electron density maps that have very low phase error and model bias. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 107
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 306-308 
    ISSN: 0887-3585
    Schlagwort(e): rubredoxin ; lgG antibody ; lgM antibody ; immunogen ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The immunogenicity of a folded, all D-amino acid protein- rubredoxin, has been compared with that for the corresponding L-protein enantiomer. Following multiple administrations with alum adjuvant, the L-protein induced a strong, specific lgG antibody response, whereas the D-protein did not. This relative lack of responsiveness to the D-protein cannot be attributed to rapid excretion, since it is retained at least 4 times longer than the natural L-protein. These observations provide the first direct evidence that a folded D-amino acid protein has low immunogenicity and is long lived in vivo. Proteins with such properties may be useful as molecular platforms in a variety of chemical and pharmaco-logical applications. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 108
    ISSN: 0887-3585
    Schlagwort(e): ribonuclease ; synthesized peptide ; protein folding ; protein conformation ; molecular evolution ; exon shuffling ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Proteins consist of structural units such as globular domains, secondary structures, and modules. Modules were originally defined by partitioning a globular domain into compact regions, each of which is a contiguous polypeptide segment having a compact conformation. Since modules show close correlations with the intron positions of genes, they are regarded as primordial polypeptide pieces encoded by exons and shuffled, leading to yield new combination of them in early biological evolution. Do modules maintain their native conformations in solution when they are excised at their boundaries? In order to find answers to this question, we have synthesized modules of barnase, one of the bacterial RNases, and studied the solution structures of modules M2 (amino acid residues 24-52) and M3 (52-73) by 2D NMR studies. Some local secondary structures, α-helix, and β-turns in M2 and β-turns in M3, were observed in the modules at the similar positions to those in the intact barnase but the overall state seems to be in a mixture of random and native conformations. The present result shows that the excised modules have propensity to form similar secondary structures to those of the intact barnase. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
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  • 109
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 357-363 
    ISSN: 0887-3585
    Schlagwort(e): hydrophobic core ; protein structure ; ribonuclease ; protein folding ; exon shuffling ; molecular evolution ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Proteins in eukaryotes are composed of structural units, each encoded by discrete exons. The protein module is one such structural unit; it has been defined as the least extended or the most compact contiguous segment in a globular domain. To elucidate roles of modules in protein evolution and folding, we examined roles of hydrogen bonds and hydrophobic cores, as related to the stability of these modules. For this purpose we studied barnase, a bacterial Rnase from Bacillus amylolique-faciens. Barnase is decomposed into at least six modules, M1-M6; the module boundaries are identified at amino acid residues 24, 52, 73, 88, and 98. Hydrogen bonds are localized mainly within each of the modules, with only a few between them, thereby indicating that their locations are designed to primarily stabilize each individual module. To obtain support for this notion, an analysis was made of hypothetical modules defined as segments starting at a center of one module and ending at the center of the following one. We found that the hydrogen bonds did not localize in each hypothetical module and that many formed between the hypothetical modules. The native conformations of modules of barnase may be specified predominantly by interactions within the modules. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 110
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 437-439 
    ISSN: 0887-3585
    Schlagwort(e): peptide antigen ; high affinity complex ; crystallization ; preliminary X-ray data ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The Fab fragment of CB 4-1, a monoclonal murine antibody against HIV protein p24, has been produced. It forms a complex with a synthetic antigen, an epitope of p24 made up of 11 amino acids, with the binding constant kd = 3.6 × 10-9 M. Crystals of hexagonal and orthorhombic space group has been obtained by cocrystallization of the Fab with the epitope and crystallization without the epitope, respectively. In either case, the crystals are suitable for X-ray structural analysis. Crystals of the Fab fragment cocrystallized with the peptide have the space group P 6322 with cell dimensions of a = b = 105 Å, c = 297 Å. Fab crystals without the epitope are in space group C 222 with cell dimensions a = 110.1 Å, b = 110.2 c Å = 150.1 Å. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 111
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 408-422 
    ISSN: 0887-3585
    Schlagwort(e): heme ; flavin ; electron transfer proteins ; crystal packing ; molecular modeling ; energy minimization ; electrostatic interactions ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Flavocytochrome b2 and cytochrome c are physiological electron transfer partners in yeast mitochondria. The formation of a stable complex between them has been demonstrated both in solution and in the crystalline state. On the basis of the three-dimensional structures, using molecular modeling and energy minimization, we have generated a hypothetical model for the interaction of these redox partners in the crystal lattice. General criteria such as good charge and surface complementarity, plausible orientation, and separation distance of the prosthetic groups, as well as more specific criteria such as the stoichiometry determined in the crystal, and the involvement of both domains and of more than one subunit of flavocytochrome b2 led us to discriminate between several possible interaction sites. In the hypothetical model we present, four cytochrome c molecules interact with a tetramer of flavocytochrome b2. The b2 and c hemes are coplanar, with an edge-to-edge distance of 14 Å. the contact surface area is ca. 800 Å2. Several electrostatic interactions involving the flavin and the heme domains of flavocytochrome b2 stabilize the binding of cytochrome c. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 112
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 438-443 
    ISSN: 0887-3585
    Schlagwort(e): crystallization ; antitumor ; antibody ; primary structure ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The Fab′ of CC49, a murine monoclonal antibody directed against the human tumor-associated antigen TAG-72 has been crystallized. The crystals are monoclinic, space group P21 with cell parameters a = 115.6 Å, b = 116.4 Å, and c = 70.3 Å; β = 97.8°. The size of the unit cell is compatible with four Fab′ molecules in the asymmetric unit. The Fab molecules are related by two approximately perpendicular pseudo-2-fold axes. One pseudo-2-fold axis is parallel to the crystallographic 2-fold axis and was found by inspection of the Harker section of the native Patterson map; the other was found by a self rotation function. The primary structures of the variable regions of the CC49 antibody light and heavy chains have been determined and are compared with those of the related antitumor antibody B72.3. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 113
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 205-208 
    ISSN: 0887-3585
    Schlagwort(e): lectin ; stinging nettle ; Urtica dioica ; crystallization ; X-ray diffraction ; protein-carbohydrate interactions ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN′N″ -triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P212121 with unit cell dimensions a = 54.3 (1) Å, b = 62.2 (1) Å, and c = 92.4 (2) Å, and diffracts to 3.0 Å resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P212121 with cell constants a = 37.69 (4) Å, b = 48.97 (6) Å, and c = 57.32 (4) Å. These crystals diffract to at least 2.0 Å resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 114
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
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  • 115
    ISSN: 0887-3585
    Schlagwort(e): crystallization ; antilysozyme system ; epitope ; antigen-antibody complex ; cross-reactivity ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The complex formed between the Fab fragment of a murine monoclonal anti-hen egg lysozyme antibody F9.13.7 and the het-erologous antigen Guinea-fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X-rays to 3 Å resolution, belong to the monoclinic space group P21, with a = 83.7 Å, b = 195.5 Å, c = 50.2 Å, β = 108.5° and have two molecules of the complex in the asymmetric unit The three-dimensional structure has been determined from a preliminary data set to 4 Å using molecular replacement techniques. The lysozyme-Fab complexes are arranged with their long molecular axes approximately parallel to the crystallo-graphic unique axis. Fab F9.13.7 binds an anti-genie determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 116
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 213-215 
    ISSN: 0887-3585
    Schlagwort(e): crystals ; bacterial esterase ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm3 within a month. The crystals belong to the trigonal space group P31 (or P32), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM) of 2.53 Å3/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 117
    ISSN: 0887-3585
    Schlagwort(e): peroxidase ; protein crystals ; X-ray crystallography ; protein structure ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Peroxidase (donor: H2O2 oxi-doreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P42212, with unit cell dimensions of a = b = 74.5 Å, c = 117.6 Å. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 Å resolution and are stable to X-rays. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 118
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 223-234 
    ISSN: 0887-3585
    Schlagwort(e): leucine zipper ; protein folding ; molecular dynamics ; skip residue ; hemaglutinin ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Two complementary methods for measuring local pitch based on heptad position in α-helical coiled coils are described and applied to six crystal structures. The results reveal a diversity of pitch values: two-stranded coiled coils appear to have pitch values near 150 Å the values for three- and four-stranded coiled coils range closer to 200 Å. The methods also provide a rapid and sensitive gauge of local coiled-coil conformation. Polar or charged residues in the apolar interface between coiled-coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled-coil stability. Moreover, the identification of a skip residue (heptad frame shift) in the hemaglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes. These results on relatively short coiled-coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established. We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high-resolution crystal structure - a result not readily discerned by direct comparison of atomic coordinates. Taken together, these methods reveal specific aspects of coiled-coil structure which may escape detection by global analyses of pitch. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 119
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 252-265 
    ISSN: 0887-3585
    Schlagwort(e): titration curves ; electrostatics ; ionization ; ion pairs ; salt bridges ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: This paper describes a general method to calculate the pKas of ionizable groups in proteins. Electrostatic calculations are carried out using the finite difference Poisson-Boltzmann (FDPB) method. A formal treatment of the calculation of pKas within the framework of the FDPB method is presented. The major change with respect to previous work is the specific incorporation of the complete charge distribution of both the neutral and charged forms of each ionizable group into the formalism. This is extremely important for the treatment of salt bridges. A hybrid statistical mechanical/Tanford-Roxby method, which is found to be significantly faster than previous treatments, is also introduced. This simplifies the problem of summing over the large number of possible ionization states for a complex polyion. Applications to BPTI and serine proteases suggest that the calculations can be quite reliable. However, the necessity of including bound waters in the treatment of the Asp-70…His-31 salt bridge in T4 lysozyme and experience with other proteins suggest that additional factors ultimately need to be considered in a comprehensive treatment of pKas in proteins. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 120
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 235-251 
    ISSN: 0887-3585
    Schlagwort(e): conformational analysis ; 2D NMR spectroscopy ; restrained molecular dynamics ; RGD peptides ; proline ; cis/trans isomerism ; peptide templates ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: To investigate the role of proline in defining β turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L- and D-proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L-proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D-proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala-), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo(-Pro-Ala-Ala-Ala-Ala--), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon βVI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 121
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 266-282 
    ISSN: 0887-3585
    Schlagwort(e): electrostatics ; conformational energies ; solvation ; ionization ; force fields ; energy functions ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Computer models of proteins frequently treat the energies and forces associated with ionizable groups as if they were purely electrostatic. This paper examines the validity of the purely electrostatic approach, and concludes that significant errors in energies can result from the neglect of ionization changes. However, a complete treatment of ionizable groups presents substantial computational obstacles, because of the large number of ionization states which must be examined in systems having multiple interacting titratable groups. In order to address this problem, two novel methods for treating the energetics and forces associated with ionizable groups with a minimum of computer time have been developed. The most rapid method yields approximate energies by computing the free energy of a single highly occupied ionization state. The second method separates ionizable groups into clusters, and treats intracluster interactions exactly, but intercluster interactions approximately. This method yields both accurate energies and fractional charges. Good results are obtained in tests of both methods on proteins having has many as 123 ionizable groups. The more rapid method requires computer times of 0.01 to 0.34 sec, while the more accurate method requires 0.7 to 15 sec. These methods may be fast enough to permit the incorporation of ionization effects in iterative computations, such as energy minimizations and conformational searches. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 122
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 283-289 
    ISSN: 0887-3585
    Schlagwort(e): phosphoglycerate kinase ; Bacillus stearothermophilus ; crystallographic structure ; thermal stability ; increased hydrophobicity ; helix stability ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 Å, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 Å for both bonds and planes.The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53°C, respectively. Differential scanning calorimetry indicates that the energy difference (ΔΔG) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol-1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better α-helix stability resulting from the removal of helix destablising residues and extra helix-dipole/helix side chain ionic interactions. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 123
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 312-321 
    ISSN: 0887-3585
    Schlagwort(e): RGD sequence ; snake venom disintegrin ; GP IIb-IIIa antagonist ; platelet aggregation inhibitor ; protein structure/conformation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and 〉200-fold, respectively. The conservative D51E mutant was still 〉100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 124
    ISSN: 0887-3585
    Schlagwort(e): correlation time ; crystallographic B-factor ; hydrogen exchange rate ; NMR relaxation ; order parameter ; protein motion ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody VL domain has been characterized by measurements of 15N T1 and T2 relaxation times, 1H-15N NOE values, and 1H-2H exchange rates. These data were acquired with 2D inverse detected heteronuclear 1H-15N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R2 (= 1/T2). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R2 exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R2 exchange contributions. The measured 1H-2H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the VL domain, including the three hypervariable loops, undergo dynamic changes upon VL:VH association and/ or complexation with digoxin. Overall, the 26-10 VL domain exhibits relatively low flexibility on the ps-ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 125
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 126
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 330-337 
    ISSN: 0887-3585
    Schlagwort(e): DNA-binding proteins ; DNA-protein interactions ; nonhistone protein ; site-specific proteolytic cleavage ; nuclear autoantibody ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Ku protein, a relatively abundant nuclear protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule. The functional role played by this protein in the cell, however, remains to be elucidated. We have observed here that Ku protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact. This cleavage releases an 18 kDa polypeptide and converts Ku protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit. The proteolyzed form of Ku protein, denoted Ku′, has DNA binding properties similar to those of Ku protein. The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0. In addition, cleavage occurs only when Ku protein is bound to DNA, not free in solution. We suggest that in vivo, such proteolysis might be necessary for Ku protein function at some stage of the cell cycle. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 127
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 5-9 
    ISSN: 0887-3585
    Schlagwort(e): protein simulations ; molecular dynamics ; serine protease ; enzyme-substrate interactions ; hydrogen bonding analysis ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The binding free energy difference for the Gly-169 → Ala-169 (G169A) mutation in subtilisin BPN′ complexed with a tripeptide substrate analogue is explored using the thermodynamic integration approach. The structure of the mutant enzyme-substrate complex obtained from free energy simulation is in good agreement with experimental X-ray refinement. The near perfect reversibility is obtained in the present work for ensuring the correctness of the free energy calculations. The results of the binding free energy difference are close to similar experimental data. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 128
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 176-192 
    ISSN: 0887-3585
    Schlagwort(e): transforming growth factor ; X-ray structure ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The crystal structure of TGF-β2 has been refined using data collected with synchrotron radiation (CHESS) to 1.8 Å resolution with a residual R (= ∑ | |Fo| - |Fc| | /∑ |Fo|) factor of 17.3%. The model consists of 890 protein atoms from all 112 residues and 59 water molecules. The monomer of TGF-β2 assumes a rather extended conformation and lacks a well-defined hydrophobic core. Surface accessibility calculations show only 44% of the nonpolar surface is buried in the monomer. In contrast, 55.8% of the nonpolar surface area is buried when the two monomers from a dimer, a typical value for globular proteins. This includes a 1300 Å2 buried interface area that is largely hydrophobic. Sequence comparisons using a profile derived from the refined TGF-β2 structure suggest that the cluster of four disulfides (three intramonomeric disulfide bonds 15-78, 44-109, 48-111 forming a disulfide knot, and one intermonomeric disulfide 77-77) together with the extended β strand region constitutes the conserved structural motif for the TGF-β superfamily. This structural motif, without the 77-77 disulfide bond, defines also the common fold for a general family of growth factors, including the nerve growth factor and platelet-derived growth factor families. The fold is conserved only at the monomer level, while the active forms are dimers, suggesting that dimerization plays an important role in regulating the binding of these cytokines to their receptors and in modulating the biological responses. © 1993 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
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  • 129
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 217-218 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 130
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 219-219 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 131
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 193-202 
    ISSN: 0887-3585
    Schlagwort(e): molecular surface ; molecular volume ; molecular similarity ; pharmacophore ; drug design ; elastase ; protease inhibitors ; serine protease ; turkey ovomucoid inhibitor ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A novel analytical method for comparing molecular shapes by optimizing the intersection of molecular “SKINS” has been developed. This method provides a quantitative measure of the shape similarity by maximizing the intersection volume of molecular surfaces with a finite thickness; a molecular skin. We report shape matching of a small tripeptide inhibitor (DFKi) of elastase class proteins with the 56 residue turkey ovomucoid inhibitor (TOMI). To match a large elastase inhibitor such as TOMI with a small inhibitor or drug, we found that it is necessary to use a skin match rather than molecular volume. Skin based comparisons of TOMI protein with DFKi successfully found the alignment expected from comparison of their respective crystallographic complexes with elastase (i.e. HLE/TOMI complex and PPE/tripeptide complex). In the skin comparison of the tripeptide with the TOMI protein, blind searching for skin matches involved optimization of the skin intersection from 172 starting positions randomly selected from a set of 500 points on the TOMI van der Waals surface [within 9.5 Å of the Leu-18 on the TOMI binding loop (1 point/Å2)]. The tripeptide center of mass was placed at these points and its orientation was randomized before optimization was initiated. The best skin intersection, 86.4 Å3, was found thre times and corresponds to the experimental alignment. The next best skin intersection was 78.1 Å3 giving a discrimination factor in this case of 10%. Searches over the entire surface of the TOMI protein did not identify any new matches with skin intersection greater than 78.1 Å3. Matching the DFKi with a TOMI structure relaxed from its crystal conformation by molecular dynamics gives similar results. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 132
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 203-216 
    ISSN: 0887-3585
    Schlagwort(e): carbonic anhydrase ; anion binding ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Computer simulation techniques are used to address the question of how cyanide and related ions interact with human carbonic anhydrase II (HCAII). Spectroscopic results have suggested that cyanide is coordinated with the zinc ion, while recent X-ray results suggest that the cyanide ion is noncovalently associated with the zinc-water or zinchydroxide form of the enzyme. We have carried out simulations on three models in an attempt to shed light on why the spectroscopic and X-ray results differ. The first model we studied (Model I) has cyanide directly coordinated to the zinc ion, the second has it noncovalently interacting with the zinc-hydroxide (high pH) form of the enzyme (Model II), and the third has cyanide noncovalently interacting with the zinc-water (low pH) form of the enzyme (Model III). None of these models is satisfactory in explaining the available structural data obtained from X-ray crystallography. This leads us to propose an alternative model, in which HCAII hydrates HCN to form an OH-/HCN complex coordinated to the Zn ion. Ab initio calculations are consistent with this model. Based on these results we are able to explain the observed crystallographic behavior of cyanate and, by inference, thiocyanate. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 133
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 220-220 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 134
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 135
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 223-231 
    ISSN: 0887-3585
    Schlagwort(e): isometric virus ; architecture ; decapsidation ; neutron small angle scattering ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The organization of turnip yellow mosaic virus has been investigated by neutron small angle scattering at 300 K and 80 K in buffers containing various amounts of D2O. We confirm that in native virions, no substantial part of the RNA is located at a radius larger than ca. 100-110 Å, i.e., that there is very little interpretation of the RNA into the capsid. At 80 K, scattering curves do not depend much upon contrast, from 40% D2O to 100% D2O buffers, but are strongly affected by interparticle interference. We could, however, show that it is not the case for the subsidiary intensity maximum at q ∼ 0.06 Å-1. From the position of this maximum, we conclude that upon freezing, the radius of the capsid expands by c.a. 3.5% and the RNA penetrates deeply into the protein shell. Biological implications of this conformational change immediately preceding decapsidation are dicussed. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 136
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 232-251 
    ISSN: 0887-3585
    Schlagwort(e): protein structure ; NMR ; conformation ; φ, Ψ distribution ; χ1 distribution ; heterogeneity ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A study is presented of the conformational characteristics of NMR-derived protein structures in the Protein Data Bank compared to X-ray structures. Both ensemble and energy-minimized average structures are analyzed. We have addressed the problem using the methods developed for crystal structures by examining the distribution of φ, Ψ, and χ angles as indicators of global conformational irregularity. All these features in NMR structures occur to varying degrees in multiple conformational states. Some measures of local geometry are very tightly constrained by the methods used to generate the structure, e.g., proline φ angles, α-helix φ, Ψ angles, ω angles, and Cα chirality. The more lightly restrained torsion angles do show increasead clustering as the number of overall experimental observations increases. φ, Ψ, and χ1 angle conformational heterogeneity is strongly correlated with accessibility but shows additional differences which reflect the differing number of observations possible in NMR for the various side chains (e.g., many for Trp, few for Ser). In general, we find that the core is defined to a notional resolution of 2.0 to 2.3 Å. Of real interest is the behavior of surface residues and in particular the side chains where multiple rotameric states in different structures can vary from 10% to 88%. Later generation structures show a much tighter definition which correlates with increasing use of J-coupling information, stereospecific assignments, and heteronumclear techniques. A suite of programs is being developed to address the special needs of NMR-derived structures which will take into account the existence of increased mobility in solution. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 16 Ill.
    Materialart: Digitale Medien
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  • 137
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 266-278 
    ISSN: 0887-3585
    Schlagwort(e): molecular recognition ; ligand binding ; complementarity ; interaction energy ; automated prediction ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The biological activities of proteins depends on specific molecular recognition and binding. Computational methods for predicting binding modes can facilitate the discovery and design of ligands and yeild information on the factors governing complementarity. The DOCK suite of programs has been applied to several systems; here, the degree of orientational sampling required to reproduce and identify known binding modes, with and without rigid-body energy minimization, is investigated for four complexes. There is a tradeoff between sampling and minimization. The known binding modes can be identified with intensive sampling alone (10,000 to 20,000 orientations generated per system) or with moderate sampling combined with minimization. Optimization improves energies significantly, particularly when steric clashes are present, and brings many orientations closer to the experimentally observed position. Whether or not minimization is performed, however, sampling must be sufficient to find at least one structure in the vicinity of the presumed true binding mode. Hybrid approaches combining docking and minimization are promising and will become more viable with the use of faster algorithms and the judicious selection of fewer orientations for minimization. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 138
    ISSN: 0887-3585
    Schlagwort(e): thrombin ; bifunctional inhibitor ; crystal structure ; hirutonis ; drugdesing ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The crystal structure of the complexes of hirutonin-2 and hirutonin-6 with human α-thrombin have been solved and refined to R-factors of 0.169 (2.0 Å resolution) and 0.162 (201Å), respectively. Hirutonins belong to a family of bifunctional inhibitors bearing a noncleavable moiety mimicking the scissile bond. Hirutonin-2 is an analog of (D)Phe-Pro-Arg-Gly-hirudin49-65; hirutonin-6 has the same N-terminal tripeptide connected to a shortened fibrinogen exosite-binding part by a short, non-peptidyl linker. The hirutonin-6 molecule is well defined in the electron density with the exception of the C-terminal Leu-h61. The linker follows near the bottom of the canyon connecting the active site with the exosite, forms a short antiparallel β-sheet-like arrangement with Leu-40-Leu41 and makes van der Waals contacts with Glu39-Leu40-Leu41 of thrombin. In the thrombin-hirutonin-2 complex, the N- and C-terminal parts of the inhibitor are well or dered (except the C-terminal Gln-h65) while the central portion of the linker is partially disordered. The glycine analog in the P1′ position of hirutonin-2 assumes a conformation similar to that of the canonical form (Bode and Huber (1992) Eur. J. Biochem. 204 : 433-451) and supports the identification of the S1′ site as restricted by His57, Trp60D, Lys60F, and the Cys42-Cys58 disulfide bridge. The carbonyl oxygen of the P1 arginine residue is located in the oxyanion hole formed by the NH groups of Gly193 and Ser195, while the carbonyl carbon is positioned within a short distance, 2.8 Å, from the Oγ of Ser195. This resembles the conformation of the substrate-like inhibitors bound to other serine proteases. The N-terminal (D)Phe-pro-Arg fragment common to both inhibitors binds to thrombin in a fashion very similar to that of other inhibitors having this motif. The binding of the C-terminus of hirutonins to the fibrinogen-binding exosite is similar to that observed in hirudin and hirulog complexes. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 139
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 297-309 
    ISSN: 0887-3585
    Schlagwort(e): nuclear magnetic resonance ; nuclear overhauser effect ; dimer ; solution structure ; symmetry ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The structure determination of symmetric dimers by NMR is impeded by the ambiguity of inter- and intramonomer NOE crosspeaks. In this paper, a calculation strategy is presented that allows the calculation of dimer structures without resolving ther ambuguity by additional experiments (like asymmetric labeling). The strategy employs a molecular dynamic-based simulated annealing approach to minimize a traget function. The experimental part of the target function contains distance restraints that correctly describe the ambiguity of the NOE peaks, and a novel term that restrains the symmetry of the dimer without requiring the knowledge of the symmetry axis. The use of the method is illustrated by three examples, using experimentally obtained data and model data derived from a known structure. For the purpose of testing the method, it is assumed that every NOE crosspeak is ambiguous in all three cases. It is shown that the structure of a homologous protein is known and in ab intio structure determination. The method can be extended to higher order symmetric multimers. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 140
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 279-296 
    ISSN: 0887-3585
    Schlagwort(e): hemoglobin ; cooperativity ; single-site modifications ; half-ligated tetramers ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The patterns of energetic response elicited by single-site hemoglobin mutations and chemical mocdifications have been determined in order to probe the dimer-dimer interface of the half-ligated tetramer (species[21]) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules1. In this study the free energies of quaternary assembly(dimers to tetramers) were determined for aseries of 24 tetrameric species in which one dimeric half-molecule is ligated while the adjacent αβ dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half-ligated tetramers and the unligated tetramers over the entire spatial distrubution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer-dimer interface of the half-ligated tetramer(species[21]) has the same quaternary sturcture as that of the unligated molecule, i.e, “quaternary T.” This quaternary structure assignment of species [21] strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T → R quaternary switchpoints2. Analysis of the observed Symmetry Rule behaviour in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between α and β subunits of the dimeric half-tetramer within quaternary T. The mutational results indicate that these interactions are not “paid for” by breaking or making noncovalent bonds at the dimer-dimer interface (α1β2). They arise from structural and energetic changes that are “internal” to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of “tertiary constraint” is thus generated by the first binding step and is propagated to the second hemesite while the dimer-dimer interface α1β2serves as a constraint. The “sequential” cooperativity that occurs within the half-molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers. When the constraint is released functionally by T to R dimer rearrangement (at each switch-point specified by the a Symmetry Rule) the alterations of interfacial bonds then dominate the energetics of cooperativity. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 141
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 310-324 
    ISSN: 0887-3585
    Schlagwort(e): NMR restraint analysis ; molecular dynamics ; simulated annealing ; restraint satisfaction ; low-resolution modeling ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A reduced representation of paroteins has been developed for use in restraint satisfaction calculations with dynamic simulated annealing. Each amino acid residue is represented by up to four spherical virtual atoms. The virtual bonds and excluded volume of these atoms has ben parameterized by analysis of 83 protein structures determined at high resolution by X-ray crystallography. The use of the new representation in NOE distance restraint satisfaction has been compared with the standard all-atom represntation for the determination of the structures of crambin, eshistatin, and protein G. Using the reduced representation, there is a 30-fold decrease in the computer time needed for generatin a single structure, and up to a 20-fold decrease in the time taken to produce an acceptable structure compared to using the all-atom representation. The root mean square deviation between the mean structure obtain with all-atom and reduced representation si between 1.5 and 1.7 Å for Cα atoms. The new representation is adequate for describing the “low-resolution” features of protein structure such as the general fold and the positions of the secondary structure for more detailed refinement with the full all-atom representation. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 142
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 325-328 
    ISSN: 0887-3585
    Schlagwort(e): crystallization ; ribonuclease ; inhibitor ; amyloliquefaciens ; protein-protein complex ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Crystals of barstar, the intracellular inhibitor of the extracellular ribonuclease produced by Bacillus amyloliquefaciens (barnase), were obtained through vapor phase equilibration using the hanging drop technique. Three crystal forms have been characterized. Forms I and II, crystallized eithr in potetragonal; they exhibit a superstructure along the c-axis. Form III crystals, suitable for a high resolution structure determination, were grown from 55-65% ammomnium sulfate. This crystal form is hexagonal and diffracts to at least 2 Å resolution at a synchrotron radiation source. It belongs to the hexagonal space group P6, with unit cell dimensions a = b = 143.6 Å, c = 35.6 Å. There are four molecules of barstar in the asymmetric unit. X-ray data have been collected to 2.2 Å Bragg sapcing. The structure determination is underway in order to analyze conformational changes of barstar upon complexation with barnas. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 143
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 144
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 329-334 
    ISSN: 0887-3585
    Schlagwort(e): bacterial toxins ; superantigens ; X-ray crystallography ; crystallization ; Staphylococcus aureus ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Native toxic shock syndrom toxin 1 (TSST-1) purified from Staphylococcus aurius has been crystallized in four different forms. The highest resolution data (2.05 Å) was collected from orthorhombic crystals belonging to the space group C2221. The unit cell dimension are a = 108.7 Å, b = 177.5 Å, c = 97.6 Å. Rotation function analysis of this from indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 Å. The space group is P21 with unit cell parameters of a = 44.4 Å, b = 34.0 Å, c = 55.2 Å, β = 93.0°. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 145
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 347-354 
    ISSN: 0887-3585
    Schlagwort(e): EPR ; structure ; spectroscopy ; contraction ; polymer ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin-labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL-fibers). The EPR spectrum of unoriented PHSL-labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T‖′) of 42.8 ± 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated halfcone angle of 76°. When the PHSL-fiber bundle was oriented parallel to the magnetic field, the splitting between the zero-crossing points (2T′) was 42.7 ± 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T′ decreased to 34.5 ± 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL-fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL-actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5°, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1°. These results rule out large changes in the overall geometry of the actin filament and in the local conformation of actin near the phalloidin binding site during the generation of isometric tension in muscle fibers. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 146
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 29-42 
    ISSN: 0887-3585
    Schlagwort(e): crystallography ; molecular replacement ; refinement ; structure ; muscle ; carbonic anhydrase ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 147
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 48-56 
    ISSN: 0887-3585
    Schlagwort(e): pattern recognition ; structure-activity data base ; receptor binding ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A novel computer modeling approach suitable for the structure analysis of small bioactive peptides has been developed. This approach involves identification of conformational patterns in protein structure data bank based on the sequence homology with the bioactive peptide. The models built on the basis of this homology and having common conformational patterns are analyzed under the structural constraints derived from the activity data of various synthetic analogs of the peptide. Application of this procedure to the gonadotropin releasing hormone (GnRH) resulted in a library of possible structures for GnRH, 9 among which shared a common β-turn. Further analysis of the structures containing the β-turn motif, in the context of the structure-activity data, led to a model for the active conformation of GnRH. The topology of the putative receptor binding site of the hormone is defined by a contiguous surface formed through an appropriate juxtaposition of the N-terminal pGlu1 the guanidyl group of Arg8, aromatic side chain of Trp3, and the Gly10-NH2 at the C-terminal end. ©Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 148
    ISSN: 0887-3585
    Schlagwort(e): X-ray crystallography ; antibody domain ; recombinant DNA ; binding affinity ; antigen-antibody complex ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P4212, with unit cell dimensions a = b = 141 Å, c = 218 Å. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 149
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 64-78 
    ISSN: 0887-3585
    Schlagwort(e): hydrolase ; mechanism ; catalytic triad ; inhibitor binding ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 150
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 151
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 79-91 
    ISSN: 0887-3585
    Schlagwort(e): protein structure prediction ; neural networks ; amino acid composition ; protein folding classes ; 4α-helical bundles ; parallel (α/β)8 barrels ; nucleotide binding fold ; immunoglobulin fold ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: An empirical relation between the amino acid composition and three-dimensional folding pattern of several classes of proteins has been determined. Computer simulated neural networks have been used to assign proteins to one of the following classes based on their amino acid composition and size: (1) 4α-helical bundles, (2) parallel (α/β)8 barrels, (3) nucleotide binding fold, (4) immunoglobulin fold, or (5) none of these. Networks trained on the known crystal structures as well as sequences of closely related proteins are shown to correctly predict folding classes of proteins not represented in the training set with an average accuracy of 87%. Other folding motifs can easily be added to the prediction scheme once larger databases become available. Analysis of the neural network weights reveals that amino acids favoring prediction of a folding class are usually over represented in that class and amino acids with unfavorable weights are underrepresented in composition. The neural networks utilize combinations of these multiple small variations in amino acid composition in order to make a prediction. The favorably weighted amino acids in a given class also form the most intramolecular interactions with other residues in proteins of that class. A detailed examination of the contacts of these amino acids reveals some general patterns that may help stabilize each folding class. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
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  • 152
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 364-383 
    ISSN: 0887-3585
    Schlagwort(e): X-ray structure ; TEM1 ; β-lactamase ; antibiotics ; bacterial resistance ; serine hydrolase ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The X-ray structure of Escherichia coli TEM1β-lactamase has been refined to a crystallorgphic R-factor of 16.4% for 22,510 reflections between 5.0 and 1.8 Å resolution; 199 water molecules and 1 sulphate ion were included in refinement. Except for the tips of a few solvent-exposed side chains, all protein atoms have clear electron density and refined to an average atomic temperature factor of 11 Å2. The estimated coordinates error is 0.17 Å. The substrate binding site is located at the interface of the two domains of the protein and contains 4 water molecules and the sulphate anion. One of these solvent molecules is found at hydrogen bond distance from S70 and E166. S70 and S130 are hydrogen bonded to K73 and K234, respectively. It was found that the E. coli TEM1 and Staphylococcus aureus PC1 β-lactamases crystal structures differ in the relative orientations of the two domains composing the enzymes, which result in a narrowed substrate binding cavity in the TEM1 enzyme. Local but significant differences in the vicinity of this site may explain the occurrence of TEM1 natural mutants with extended substrate specificities. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 21 Ill.
    Materialart: Digitale Medien
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  • 153
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 154
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 412-425 
    ISSN: 0887-3585
    Schlagwort(e): normal modes ; constraint dynamics ; molecular dynamics ; lysozyme ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Analysis of extended molecular dynamics (MD) simulations of lysozyme in vacuo and in aqueous solution reveals that it is possible to separate the configurational space into two subspaces: (1) an “essential” subspace containing only a few degrees of freedom in which anharmonic motion occurs that comprises most of the positional fluctuations; and (2) the remaining space in which the motion has a narrow Gaussian distribution and which can be considered as “physically constrained.” If overall translation and rotation are eliminated, the two spaces can be constructed by a simple linear transformation in Cartesian coordinate space, which remains valid over several hundred picoseconds. The transformation follows from the covariance matrix of the positional deviations. The essential degrees of freedom seem to describe motions which are relevant for the function of the protein, while the physically constrained subspace merely describes irrelevant local fluctuations. The near-constraint behavior of the latter subspace allows the separation of equations of motion and promises the possibility of investigating independently the essential space and performing dynamic simulations only in this reduced space. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 155
    ISSN: 0887-3585
    Schlagwort(e): transcription factors ; zinc finger ; generalized order parameter ; effective correlation time ; internal protein motions ; Lipari-Szabo model ; “model-free” approach ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The rapid motions of the backbone of the DNA-binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton-detected heteronuclear NMR experiments on 15N-labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S2) and an effective correlation time (τe) for the internal motion of each amide bond. A back calculation, using the same model, yielded the {1H}-15N nuclear Overhauser effects (NOEs) and the 15N spin-lattice relaxation times (T1) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major α-helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S2-values was as good as quantitative for most of the residues, except for some residues that were subject to a more large-scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile-487 between the charged oxygens of the side chain of Asp-485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental τe-values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (〈0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower τe-values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile-487. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 156
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 426-434 
    ISSN: 0887-3585
    Schlagwort(e): enolase ; mutagenesis ; activity ; enzyme ; active site ; charge shuttle ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Yeast has two enolase isozymes (called 1 and 2), either of which suffices for growth. We cloned DNA encoding the enolase 1 protein coding and promoter regions flanked by BamHI termini using the PCR. The DNA, which contained no nucleotide base changes altering the protein sequence, was cloned into the multicopy shuttle vector pRS314 and transformed into a yeast strain with a deletion in its enolase 1 gene. The resulting plasmid-containing strain makes enolase 1 in quantities which depend on cell growth. A ‘charge shuttle’ mechanism of action of enolase based on X-ray crystallographic evidence (Lebioda and Stec, Biochemistry 30 : 2817, 1991) involves Glu-168 accepting a proton from a water molecule that in turn accepts a proton from a carbon-2 of the substrate. We prepared the E168Q mutant of enolase 1 by oligonucleotide-directed site-directed mutagenesis. Its identity was confirmed by N-terminal sequence analysis, HPLC on Superose 12, SDS-gel electrophoresis, and the sequence of the mutated DNA protein-coding region. The E168Q mutant has approximately 0.01% of the activity of native enolase. It binds substrate/product, AEP (3-aminoenolpyruvate-2-phosphate, the 3-amino analogue of the product phosphoenolpyruvate) and TSP (D-tartronate semialdehyde-2-phosphate, the aldehyde analogue of the substrate 2-phosphoglycerate), the latter two at least with affinities similar to those of the native enzyme. The E168Q enolase also produces absorbance changes in the analogues. The reaction with AEP is consistent with the ‘charge shuttle’ mechanism; the reaction with TSP, which presumably requires proton removal from carbon-2, is complex but shows a very slow phase consistent with expectations. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 157
    ISSN: 0887-3585
    Schlagwort(e): metalloproteinase ; inhibitor ; X-ray diffraction ; tumor invasion ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A nonglycosylated (N30QN78Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P21, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
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  • 158
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 159
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 160
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 360-373 
    ISSN: 0887-3585
    Schlagwort(e): conformational substates ; protein conformations ; molecular dynamics ; nonbanded relaxations ; ergodic measures ; s-peptide ; RNase A ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The dynamic properties of the RNase A/3′-UMP enzyme/product complex and the S-peptide of RNase A have been investigated by molecular dynamics simulations using suitable generalization of ideas introduced to probe the energy landscape in structural glasses. We introduce two measures, namely, the kinetic energy fluctuation metric and the force metric, both of which are used to calculate the time needed for sampling the conformation space of the molecules. The calculation of the fluctuation metric requires a single trajectory whereas the force metric is computed using two independent trajectories. The vacuum MD simulations show that for both systems the time required for kinetic energy equipartitioning is surprisingly long even at high temperatures. We show that the force metric is a powerful means of probing the nature and relative importance of conformational substates which determine the dynamics at low temperatures. In particular the time dependence of the non-bonded force metric is used to demonstrate that at low temperatures the system is predominantly localized hi a single cluster of conformational substates. The force metric is used to show that relaxation of long range (in sequence space) interactions must be mediated by a sequence of local dihedral angle transitions. We also argue that the time needed for compact structure formation is intimately related to the time needed for the relaxation of the dihedral angle degrees of freedom. The tame for non-bonded interactions, which drive protein molecules to fold under appropriate conditions, to relax becomes extremely long as the temperature is lowered suggesting that the formation of maximally compact structure hi proteins must be a very slow process. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 15 Ill.
    Materialart: Digitale Medien
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  • 161
    ISSN: 0887-3585
    Schlagwort(e): crambin ; terative relaxation matrix approach ; ensemble averaging ; solution structure ; restrained molecular dynamics ; stereospecific assignments ; 2D NMR ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The structure in solution of crambin, a small protein of 46 residues, has been determined from 2D NMR data using an iterative relaxation matrix approach (IRMA) together with distance geometry, distance bound driven dynamics, molecular dynamics, and energy minimization. A new protocol based on an “ensemble” approach is proposed and compared to the more standard initial rate analysis approach and a “single structure” relaxation matrix approach. The effects of fast local motions are included and R-factor calculations are performed on NOE build-ups to describe the quality of agreement between theory and experiment. A new method for stereospecific assignment of prochiral groups, based on a comparison of theoretical and experimental NOE intensities, has been applied. The solution structure of crambin could be determined with a precision (rmsd from the average structure) of 0.7 Å on backbone atoms and 1.1 Å on all heavy atoms and is largely similar to the crystal structure with a small difference observed in the position of the side chain of Tyr-29 which is determined in solution by both J-coupling and NOE data. Regions of higher structural variability (suggesting higher mobility) are found hi the solution structure, in particular for the loop between the two helices (Gly-20 to Pro-22). © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 162
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 138-151 
    ISSN: 0887-3585
    Schlagwort(e): structure prediction ; computer modeling ; structure comparison ; sequence identity ; protein homology ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Protein structure prediction is based mainly on the modeling of proteins by homology to known structures; this knowledgebased approach is the most promising method to date. Although it is used in the whole area of protein research, no general rules concerning the quality and applicability of concepts and procedures used in homology modeling have been put forward yet. Therefore, the main goal of the present work is to provide tools for the assessment of accuracy of modeling at a given level of sequence homology. A large set of known structures from different conformational and functional classes, but various degrees of homology was selected. Pairwise structure superpositions were performed. Starting with the definition of the structurally conserved regions and determination of topologically correct sequence alignments, we correlated geometrical properties with sequence homology (defined by the 250 PAM Dayhoff Matrix) and identity. It is shown that both the topological differences of the protein backbones and the relative positions of corresponding side chains diverge with decreasing sequence identity. Below 50% identity, the deviation in regions that are structurally not conserved continually increases, thus implying that with decreasing sequence identity modeling has to take into account more and more structurally diverging loop regions that are difficult to predict. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 163
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 152-160 
    ISSN: 0887-3585
    Schlagwort(e): rubredoxin ; redox potential ; ironsulfur protein ; protein relaxation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A theoretical investigation of the protein contribution to the redox potential of the iron-sulfur protein rubredoxin is presented. Structures of the oxidized and reduced forms of the protein were obtained by energy minimizing the oxidized crystal structure of Clostridium pasteurianum rubredoxin with appropriate charges and parameters. By including 102 crystal waters, structures close to the original crystal structure were obtained (rms difference of 1.16 Å), even with extensive minimization, thus allowing accurate calculations of comparative energies. Our calculations indicate an energy change of about -60 kcal/mol (2.58 eV) in the protein alone upon reduction. This energy change was due to both the change in charge of the redox site and the subsequent relaxation of the protein. An energy minimization procedure for the relaxation gives rms differences between the oxidized and reduced states of about 0.2 Å. The changes were small and occurred in both the backbone and sidechain mainly near the Fe-S center but contributed about - 16 kcal/mol (0.69 eV) to the total protein contribution. Although the neglect of certain effects such as electronic polarization may make the relaxation energies calculated an upper limit, the results indicate that protein relaxation contributes substantially to the redox potential. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 164
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 80-87 
    ISSN: 0887-3585
    Schlagwort(e): refined structures ; mutant ; substrate binding ; zinc coordination ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A complex of carbonic anhydrase (CA) with one of its substrates, bicarbonate, has been studied crystallographically. Human isoenzyme II was mutated at position 200 from threonine to histidine, which results in higher affinity for bicarbonate. The HCO3- ion binds in the active site to the zinc ion as a pseudo-bidentate ligand which gives the metal a coordination geometry between tetrahedral and trigonal bipyramide. The water/hydroxide normally bound with tetrahedral coordination to the zinc is probably replaced by the OH group of the bicarbonate ion. The importance of residues Thr-199 and Glu-106 in controlling the binding orientation of HCO3- is discussed as well as the catalytic mechanism. Both the complex as well as the uncomplexed mutant were studied at 1.9 Å resolution. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 165
    ISSN: 0887-3585
    Schlagwort(e): α-zeins ; α-kafirins ; α-coixins ; structure prediction ; gene duplication ; helix packing ; quaternary structure ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: α-Prolamins are the major seed storage proteins of species of the grass tribe Andropogonea. They are unusually rich in glutamine, proline, alanine, and leucine residues and their sequences show a series of tandem repeats presumed to be the result of multiple intragenic duplication. Two new sequences of α-prolamin clones from Coix (pBCX25.12 and pBCX25.10) are compared with similar clones from maize and Sorghum in order to investigate evolutionary relationships between the repeat motifs and to propose a schematic model for their three-dimensional structure based on hydrophobic membrane-helix propensities and helical “wheels.” A scheme is proposed for the most recent events in the evolution of the central part of the molecule (repeats 3 to 8) which involves two partial intragenic duplications and in which contemporary odd-numbered and even-numbered repeats arise from common ancestors, respectively. Each pair of repeats is proposed to form an antiparallel α-helical hairpin and that the helices of the molecule as a whole are arranged on a hexagonal net. The majority of helices show six faces of alternating hydrophobic and polar residues, which give rise to intersticial holes around each helix which alternate in chemical character. The model is consistent with proteins which contain different numbers of repeats, with oligomerization and with the dense packaging of α-prolamins within the protein body of the seed endosperm. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 166
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 103-107 
    ISSN: 0887-3585
    Schlagwort(e): aequorin ; photoprotein ; crystallization ; bioluminescence ; X-ray diffraction ; metal-binding ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 Å resolution. The crystals are orthorhombic, space group P21212 1; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) Å. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 167
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 168
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 100-102 
    ISSN: 0887-3585
    Schlagwort(e): dogfish C-reactive protein ; vapor phase equilibration ; ammonium sulfate ; sitting drop technique ; hexamers ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Crystals of dogfish (Mustelus canis) C-reactive protein were obtained through vapor phase equilibration using the sitting drop rod technique with ammonium sulfate as the precipitating agent. The space group was determined to be P1 (triclinic lattice) with unit cell dimensions of a = 82.91, b = 92.25 and c = 103.40 Å; α = 83.36°, β = 89.76°, and γ = 81.30°. These crystals diffract to about 2.6 Å resolution and contain two hexamers in the asymmetric unit. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 169
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 108-111 
    ISSN: 0887-3585
    Schlagwort(e): ribonuclease H ; Thermus thermophilus HB8 ; DNA/RNA hybrid ; crystallization ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6 122 (or P6 522), with unit cell parameters a = b = 44.7 Å, c = 314.7 Å. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 Å resolution. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 170
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. i 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 171
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 113-120 
    ISSN: 0887-3585
    Schlagwort(e): thermodynamics ; calorimetry ; protein-hormone interaction ; drug design ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The ability to predict the strength of the association of peptide hormones or other ligands with their protein receptors is of fundamental importance in the fields of protein engineering and rational drug design. To form a tight complex between a flexible peptide hormone and its receptor, the large loss of configurational entropy must be overcome. Recently, the crystallographic structure of the complex between angiotensin II and the Fab fragment of a high affinity monoclonal antibody has been determined (Garcia, K. C., Ronco, P. M., Verroust, P. J., Brünger, A. T., Amzel, L. M. Three-dimensional structure of an angiotensin II-Fab complex at 3 Å: Hormone recognition by an anti-idiotypic antibody. Science 257:502-507, 1992). In this paper we present a study of the thermodynamics of the association by high sensitivity isothermal titration calorimetry. The results of the experiments indicate that at 30°C the binding is characterized by (1) a ΔH of -8.9 ± 0.7 kcal mol-1, (2) a ΔCp of -240 ± 20 cal K-1 mol-1, and (3) the release of 1.1 ± 0.1 protons per binding site in the pH range 6.0-7.3. Using these values and the previously determined binding constant in phosphate buffer, ΔG at 30°C is estimated as -11 kcal mol-1 and ΔS as 6.9 cal K-1 mol-1. The calorimetric data indicate that binding is favored both enthalpically and entropically. These results have been complemented by structural thermodynamic calculations. The calculated and experimentally determined thermodynamic quantities are in good agreement. Entropically, the loss of configurational entropy is more than compensated by the entropy gain from solvent release associated with the hydrophobic effect. Enthalpically, binding is favored by polar interactions (hydrogen bonding). Consequently, the problem of binding flexible hormones is solved in much the same way as the folding of an unstructured polypeptide chain into a globular protein. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 172
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 133-146 
    ISSN: 0887-3585
    Schlagwort(e): coiled coil ; GCN4 ; leucine zipper ; model building ; structure prediction ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The recently solved X-ray structure of the dimerization region (“leucine zipper”) of the yeast transcriptional activator GCN4 (O'Shea, E. K., Klemm, J. D., Kim, P. S., Alber, T. Science 254:539-544, 1991) is compared to previously predicted models which had been obtained by a conformational search procedure employing simulated annealing without any knowledge of the crystal coordinates (Nilges, M., Brünger, A. T. Protein Eng. 4:649-659,1991). During the course of the simulated annealing procedure, the models converged towards the X-ray structure. The averaged root mean square difference between the models and the X-ray structure is 1.26 and 1.75 Å for backbone atoms and all nonhydrogen atoms at the dimerization interface, respectively. The local helix-helix crossing angle of the X-ray structure falls within the range predicted by the models; a slight unwinding of the coiled coil toward the N-terminal DNA-binding end of the dimerization region has been correctly predicted. Distance maps between the helices are largely identical. The region around asparagine 20 is asymmetric in the X-structure and in the models. Surface side chain dihedrals showed a large variation in the models although the χ1, χ2, χ3, χ4 3-fold dihedrals were correctly predicted in 69, 42, 43, and 44% of the cases, respectively. Phenomenological free energies of dimerization of the models show little correlation with the root mean square difference between the models and the X-ray structure. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 173
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 121-132 
    ISSN: 0887-3585
    Schlagwort(e): epitope ; neuraminidase ; critical contacts ; antigen-antibody complex ; monoclonal antibody ; site-directed mutagenesis ; influenza virus ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab1 shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 174
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 205-213 
    ISSN: 0887-3585
    Schlagwort(e): calcium binding ; crystal structure ; protein stability ; site-directed mutagenesis ; subtilisin ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A version of subtilisin BPN′ lacking the high affinity calcium site (site A) has been produced through genetic engineering methods, and its crystal structure refined at 1.8 Å resolution. This protein and the corresponding version containing the calcium A site are describedand compared. The deletion of residues 75-83 was made in the context of four site-specific replacements previously shown to stabilize subtilisin. The helix that in wild type is interrupted by the calcium binding loop, is continuous in the deletion mutant, with normal geometry. A few residues adjacent to the loop, principally those that were involved in calcium coordination, are repositioned and/or destabilized by the deletion. Because refolding is greatly facilitated by the absence of the Caloop, this proteinoffers a new vehicle for analysis and dissection of the folding reaction. This is among the largest internal changes to a protein to be described at atomic resolution. © Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 175
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 195-204 
    ISSN: 0887-3585
    Schlagwort(e): HIV-1 protease ; HIV-2 protease specificity ; 8-D space ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A vector projection method is proposed to predict the cleavability of oligopeptides by extended-specificity site proteases. For an enzyme with eight specificity subsites the substrate octapeptide can be uniquely expressed as a vector in an 8-dimensional space, whose eight bases correspond to the amino acids at the eight subsites, P1, P1′, P2′, P3′ and P4′, respectively. The component of such a characteristic vector on each of the eight bases is defined as the frequency of an amino acid occurring at a given site. These frequencies were derived from a set of octapeptides known to be cleaved by HIV protease. The cleavability of an octapeptide can then be estimated from the projection of its characteristic vector on an idealized, optimally cleavable vector. The high ratio of correct prediction vs. total prediction for the data in both the training and the testing sets indicates that the new method is self-consistent and efficient. It provides a rapid and accurate algorithm for analyzing the specificity of any multisubsite enzyme for which there is no coupling between subsites. In particular, it is useful for predicting the cleavability of an oligopeptide by either HIV-1 or HIV-2 protease, and hence offers a supplementary means for finding effective inhibitors of HIV protease as potential drugs against AIDS. © Wiley-Liss, Inc.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 176
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 214-216 
    ISSN: 0887-3585
    Schlagwort(e): ROP ; 4-α-helix bundles ; protein stability ; protein crystallization ; calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.
    Zusätzliches Material: 2 Tab.
    Materialart: Digitale Medien
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  • 177
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 178
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 293-300 
    ISSN: 0887-3585
    Schlagwort(e): domains ; compactness ; area calculations ; volume calculations ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A new method for calculating compactness (Z) that uses look-up table-based algorithms for area and volume computations is introduced. These algorithms can be used in any iterative area orvolume calculation, are more than 1000 times faster than the originalalgorithms, and have equal or better precision. With the faster algorithms it is now possible to calculate the compactness of all continuous units in a protein, and to precisely locate the optimal compact units without the screening functions and limited resolution used previously. These methods have been incorporated into a fully automatic domain finding algorithm, and this method has been applied to the 21 proteins originally analyzed as well as 12 additional proteins. This method is robust, and yields similar units even when applied to coordinates of protein crystals grown under different experimental conditions. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 179
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 172-194 
    ISSN: 0887-3585
    Schlagwort(e): serine protease ; active site solvation ; stochastic simulation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The motions of water molecules, the acyl moiety, the catalytic triad, and the oxyanion binding site of acyl-chymotrypsin were studied by means of a stochastic boundary molecular dynamics simulation. A water molecule that could provide the nucleophilic OH- for the deacylation stage of the catalysis was found to be trapped between the imidazole ring of His-57 and the carbonyl carbon of the acyl group. It makes a hydrogen bond with the Nε2 of His-57 and is heldin place through a network of hydrogen-bonded water molecules in theactive site. The water molecule was found as close as 2.8 Å to the carbonyl carbon. This appears to be due to the constraints imposed by nonbonded interaction in the active site. Configurations were found in which one hydrogen of the trapped water shared a bifurcated hydrogen bond with His-57-Nε2 and Ser-195-0γ with the water oxygen very close to the carbonyl carbon. The existence of such a water molecule suggests that large movement of the His-57 imidazole ring between positions suitable for providing general-base catalyzed assistance and for providing general-acid catalyzed assistance may notbe required during the reaction. The simulation indicates that the side chains of residues involved in catalysis (i.e., His-57, Ser-195, and Asp-102) are significantly less flexible than other side chains in the protein. The 40% reduction in rms fluctuations is consistent with a comparable reduction calculated from the temperature factors obtained in the X-ray crystal-lographic data of γ-chymotrypsin. The greater rigidity of active site residues seems to result from interconnected hydrogen bonding networks among the residues and between the residues and the solvent water in the active site. © Wiley-Liss, Inc.
    Zusätzliches Material: 15 Ill.
    Materialart: Digitale Medien
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  • 180
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 337-346 
    ISSN: 0887-3585
    Schlagwort(e): RNase H ; Mg2+-binding site ; catalytic mechanism ; molecular replacement ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: To obtain more precise insight into the Mg2+-binding site essential for RNase HI catalytic activity, we have determined the crystal structure of E. coli RNase HI in complex with Mg2+. The analyzed cocrystal, which is not isomorphous with the Mg2+-free crystal previously refined at 1.48 Å resolution, was grown at a high MgSO4 concentration more than 100 mM so that even weakly bound Mg2+ sites could be identified. The structure was solved by the molecular replacement method, using the Mg2+-free crystal structure as a search model, and was refined to give a final R-value of 0.190 for intensity data from 10 to 2.8 Å, using the XPLOR and PROLSQ programs. The backbone structures are in their entirety very similar to each other between the Mg2+-bound and the metal-free crystals, except for minor regions in the enzyme interface with the DNA/RNA hybrid. The active center clearly revealed a single Mg2+ atom located at a position almost identical to that previously found by the soaking method. Although the two metal-ion mechanism had been suggested by another group (Yang, W., Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990) and partially supported by the crystallographic study of inactive HIV-1 RT RNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan, S.R., Matthews, D. Science 252:88-95, 1991), the present result excludes the possibility that RNase HI requires two metal-binding sites for activity. In contrast to the features in the metal-free enzyme, the side chains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg2+ in the metal-bound crystal. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 181
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 355-362 
    ISSN: 0887-3585
    Schlagwort(e): protein folding ; protein modeling ; molecular force fields ; protein structure determination ; Boltzmann's principle ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A major problem in the determination of the three-dimensional structure of proteins concerns the quality of the structural models obtained from the interpretation of experimental data. New developments in X-ray crystallography and nuclear magnetic resonance spectroscopy have acceleratedd the process of structure determination and the biological community is confronted with a steadily increasing number of experimentally determined protein folds. However, in the recent past several experimentally determined protein structures have been proven to contain major errors, indicating that in some cases the interpretation of experimental data is difficult and may yield incorrect models. Such problems can be avoided when computational methods are employed which complement experimental structure determinations. A prerequisite of such computational tools is that they are independent of the parameters obtained from a particular experiment. In addition such techniques are able to support and accelerate experimental structure determinations. Here we present techniques based on knowledge based mean fields which can be used to judge the quality of protein folds. The methods can be used to identify misfolded structures as well as faulty parts of structural models. The techniques are even applicable in cases where only the Cα trace of a protein conformation is available. The capabilities of the technique are demonstrated using correct and incorrect protein folds. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 182
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 363-374 
    ISSN: 0887-3585
    Schlagwort(e): sequence analysis ; homology search ; ANK repeat ; horizontal gene transfer ; cell cycle proteins ; transcription factor NF-κB ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Based on pattern searches and systematic database screening, almost 650 different ankyrin-like (ANK) repeats from nearly all phyla have been identified; more than 150 of them are reported here for the first time. Their presence in functionally diverse proteins such as enzymes, toxins, and transcription factors strongly suggests domain shuffling, but their occurrence in prokaryotes and yeast excludes exon shuffling. The spreading mechanism remains unknown, but in at least three cases horizontal gene transfer appears to be involved. ANK repeats occur in at least four consecutive copies. The terminal repeats are more variable in sequence. One feature of the internal repeats is a predicted central hydrophobic α-helix, which is likely to interact with other repeats. The functions of the ankyrin-like repeats are compatible with a role in protein-protein interactions. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 183
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 391-411 
    ISSN: 0887-3585
    Schlagwort(e): protein sequence ; sequence repeats ; sequence alignments ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: An automated algorithm is presented that delineates protein sequence fragments which display similarity. The method incorporates a selection of a number of local nonoverlapping sequence alignments with the highest similarity scores and a graphtheoretical approach to elucidate the consistent start and end points of the fragments comprising one or more ensembles of related subsequences. The procedure allows the simultaneous identification of different types of repeats within one sequence. A multiple alignment of the resulting fragments is performed and a consensus sequence derived from the ensemble(s). Finally, a profile is constructed form the multiple alignment to detect possible and more distant members within the sequence. The method tolerates mutations in the repeats as well as insertions and deletions. The sequence spans between the various repeats or repeat clusters may be of different lengths. The technique has been applied to a number of proteins where the repeating fragments have been derived from information additional to the protein sequences. © 1993 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 184
    ISSN: 1860-1499
    Schlagwort(e): Hepatocellular carcinoma ; Tumor grade ; Ultrastructure ; Morphometry ; Cell organelles
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract In 31 cases of hepatocellular carcinoma, electron microscopic observation and morphometry on the cell organelles were carried out to evaluate the usefulness of electron microscopy for the diagnosis of well differentiated hepatocellular carcinoma. The cell organelles in well differentiated tumor cells were very similar to those in normal hepatocytes or hepatocytes with liver cirrhosis (LC). We found that in poorly differentiated tumor cells, the nuclear area, N/C ratio, nucleolar area, the amount of dispersed chromatin, and the number of free ribosomes had increased, but the cellular area, degree of nuclear roundness, and mitochondrial area had decreased. These results seem to indicate that electron microscopy is not as useful as light microscopy in the diagnosis of well differentiated hepatocellular carcinoma, but is useful in the study of poorly differentiated tumor cells, which indicated that the cell proliferation through mitoses was activated.
    Materialart: Digitale Medien
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  • 185
    Digitale Medien
    Digitale Medien
    Springer
    Medical molecular morphology 26 (1993), S. 65-75 
    ISSN: 1860-1499
    Schlagwort(e): Ultrastructure ; Rats ; Muscle spindles ; Immobilization
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract The ultrastructure of rat muscle spindles was examined after the anterior tibial muscles had been immobilized in a plaster cast. There was an increase in the number of collagen fibrils and external laminae around the outer capsules and in the intracapsular space 2 weeks after immobilization. The changes in the intrafusal muscle fibers within 4 weeks included disorientation of myofilaments. After 6 weeks, Z bands had become disarranged, and there was vacuolar degeneration of the sarcoplasmic reticulum in some fibers. Myelin sheaths of many of the myelinated nerve fibers (especially the thick ones, which were probably sensory nerve fibers) had degenerated within 2 weeks. These results indicate that immobilization of skeletal muscles affects not only extrafusal muscle fibers but also the structure of the muscle spindle.
    Materialart: Digitale Medien
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  • 186
    ISSN: 1860-1499
    Schlagwort(e): Human megakaryocytic leukemia cell line ; Adhesion ; Glycoprotein Ib ; Ultrastructure
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract To characterize the CMK11-5 subclone of a human megakaryocytic leukemia cell line (CMK), an ultrastructural study on adhesion was performed. The CMK11-5 cells showed irreversible attachment to rabbit aortic subendothelium accompanied by several morphological changes such as flattening and the spreading out of pseudopodia. These morphological changes are similar to those observed in normal platelets during attachment. The attachment of CMK11-5 cells to rabbit aortic subendothelium indicates that human megakaryocytes may possess an adhesion function like that of platelets. The CMK cell line and its CMK11-5 subclone with megakaryocytic properties appear to be useful for studying the function of megakaryocytes.
    Materialart: Digitale Medien
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  • 187
    ISSN: 1860-1499
    Schlagwort(e): Endotoxemia ; Enzyme histochemistry ; Ca++-ATPase ; Ultrastructure ; Myocardium
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract Enzyme-cytochemical studies were performed to clarify the pathogenesis of myocardial damage in endotoxemia. The reaction product of Ca++-ATPase was localized on the pinocytotic vesicles and plasma membrane of endothelial cells of capillaries and sarcolemma, sarcoplasmic reticulum and mitochondria of cardiomyocytes in control rats. However, the enzyme reaction product was reduced in amount and irregularly distributed on the plasma membrane, and on the increased number of pinocytotic vesicles of the endothelial cells of capillaries and the sarcolemma and sarcoplasmic reticulum of edematous cardiomyocytes after endotoxin injection. It is concluded that endotoxin induces structural changes and decreased activity in Ca++-ATPase in the vascular endothelium and myocytes of the heart.
    Materialart: Digitale Medien
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  • 188
    Digitale Medien
    Digitale Medien
    Springer
    Medical molecular morphology 26 (1993), S. 215-217 
    ISSN: 1860-1499
    Schlagwort(e): Ultrastructure ; Esophagus ; Candida albicans ; Candidial lesion
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract Although the esophagus is the most frequent site ofCandida infections in the gastrointestinal tract, and many clinical studies about it have been reported, little attention has been directed toward experimental candidiasis of the esophagus, especially with regard to its ultrastructure. Using transmission electron microscopy, this study was performed to clarify the ultrastructure of experimental lesions, obtained from five New Zealand white male rabbits which were given a suspension ofCandida albicans cells (107/ml) for 13 days. The results showed that the lesions consisted of exfoliating, squamous epithelial cells with mycelial elements ofCandida albicans cells penetrating through them, and that a widened intercellular space between individual cells in the area of candidial invasion seems to be a characteristic finding of candidial infection.
    Materialart: Digitale Medien
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  • 189
    Digitale Medien
    Digitale Medien
    Springer
    Brain tumor pathology 20 (1993), S. 89-92 
    ISSN: 1861-387X
    Schlagwort(e): Amyloid ; Magnetic resonance imaging ; Pituitary adenoma ; Prolactinoma ; Ultrastructure
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract An unusual case of prolactin-producing adenoma with extensive amyloid deposition is reported to clarify its radiological, intraoperative, and light- and electron-microscopic findings. A 41-year-old female patient complained of amenorrhea persisting for 20 years. Magnetic resonance imaging (MRI) revealed a pituitary adenoma, which included low-intensity spots on T1- and T2-weighted images. Intraoperative examination found multiple small, yellowish, spherical masses resembling salmon roe within the adenoma. Light and electron microscopy revealed the presence of immunoreactive cells for prolactin intermingled with concentric lamellar bodies of radially arranged amyloid fibrils originating from the endoplasmic reticulum in prolactinoma cells. The extracellular lamellar amyloid deposits were apparently due to degradation of prolactin-producing cells, but the reason for the production and radially arranged accumulation of amyloid remains to be identified.
    Materialart: Digitale Medien
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  • 190
    ISSN: 1861-387X
    Schlagwort(e): Atypical teratoid/rhabdoid tumor ; Fourth ventricle ; Immunohistochemistry ; Ultrastructure ; Basal lamina
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract Atypical teratoid/rhabdoid tumor (AT/RT), a recently established central nervous system tumor entity, occurs in children and is more malignant than medulloblastoma/primitive neuroectodermal tumors (PNET). We report here a case of AT/RT in a male infant who was 9 months old at the time of diagnosis. Magnetic resonance imaging revealed that the tumor occupied the fourth ventricle, and at surgery it was found to adhere to the floor of the fourth ventricle. After subtotal removal of the tumor mass, chemotherapy and radiotherapy were performed, but the patient died about 8 months after the diagnosis following rapid regrowth of the residual tumor. Light-microscopically, the tumor was composed mainly of nests of rhabdoid cells with fields of PNET. Occasional mesenchymal and epithelial fields were also evident. Immunohistochemically, these rhabdoid cells were positive for vimentin, epithelial membrane antigen, smooth-muscle actin, cytokeratin, and S-100 protein, and less frequently for glial fibrillary acidic protein. Electron-microscopically, the typical rhabdoid cells contained whorled bundles of intermediate filaments in their cytoplasm. Occasionally, such rhabdoid cells were covered partially by basal lamina at their stromal interface. These findings are typical of AT/RT. Although it is well known that AT/RT often arises in the posterior fossa, detailed reports of cases affecting the fourth ventricle are rare. In this case, the ultrastructural relationship between rhabdoid cells and the basal lamina, which has not so far been described in AT/RT, was of great interest when the nature of the rhabdoid cells was considered.
    Materialart: Digitale Medien
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  • 191
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 1-1 
    ISSN: 0006-3592
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Materialart: Digitale Medien
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  • 192
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 35-42 
    ISSN: 0006-3592
    Schlagwort(e): pattern recognition ; machine vision ; tissue cultures ; Betula pendula Roth ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: This article deals with the automation of the process of somatic embryogenesis for the propagation of plants. An important problem is the monitoring of the embryo production process in order to decide the time to start harvesting embryos for further processing. The classification algorithm development for somatic embryos of birch (Betula pendula Roth) showed that automated recognition of embryos at different developmental stages is possible. No globular stage embryos were classified to be heart or torpedo stage and no heart or torpedo stage embryos were classified to be at globular stage. Heart and torpedo stage embryos were classified into three developmental classes by a new index that describes the relation of embryo breadth to the length of the root. The probability of classifying a nonembryo as an embryo was less than 1%, and 14% of the object classified as embryos by a human expert were discarded by the algorithm. A computer vision system suitable for automated monitoring of samples from the bioreactor was constructed. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 193
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 43-54 
    ISSN: 0006-3592
    Schlagwort(e): acetic acid ; alkaline protease ; Bacilus firmus ; continuous culture ; extracellular enzymes ; carbon/nitrogen/phosphorus limitation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 194
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 95-103 
    ISSN: 0006-3592
    Schlagwort(e): enzymatic esterification ; equilibrium ; log P ; organic solvent choice ; lipase ; two-phase system ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: The effect of organic solvents on the equilibrium position of lipase-catalyzed esterification of glycerol and decanoic acid has been investigated. The reaction is carried out in an aqueous-organic two-phase system. In polar solvents, high mole fractions of monoacylglycerol and low mole fractions of triacylglycerol and measured, while in nonpolar solvents, the measured differences in the mole fractions of monodi-, and triacylglycerols are less. There is a good correlation between the ester mole fractions at equilibrium and the log P of the solvent (partition coefficient in n-octanolwater), however, only if the group of tertiary alcohols is excluded. In the plot of the easter mole fractions as a function of the logarithm of hte solubility of water in the organic solvent, the tertiary alcohols can be included; however, in this case other deviations appear.For the prediction of the effect of organic solvents on the ester mole fractions at reaction equilibrium in nondilute reaction systems with a water activity below 1, the program TREP (Two-phase Reaction Equilibrium Prediction) is developed, which is based on the UNIFAC group contribution method. With this model the equilibrium data are essentially predicted from basic thermodynamic data. The required equilibrium constants are estimated from experiments without an organic solvent in the reaction medium. The mole fractions calculated by TREP show the same trends as the experimentally measured mole fractions; however, some variation is observed in the absolute values. These deviations may be due to inaccuracies in the UNIFAC group contribution method. TREP is found to be a correct method to predict within some limits the ester mole fractions at equilibrium for all mixtures of solvents, substrates, and products. The production of monoester can be enhanced in reaction system with a sufficient high concentration of a polar solvent. In experiments with a triglymeto-decanoic acid ratio of 5, almost no di-and triesters can be detected at equilibrium. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 195
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 134-147 
    ISSN: 0006-3592
    Schlagwort(e): displacement ; elution ; optimization ; preparative chromatography ; production rate ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: The results of a study of the optimization of the experimental conditions for maximum production rate in overloaded elution and displacement chromatography are discussed. This study is based on the use of the equilibrium-dispersive model of chromatography and the competitive Langmuir isotherms to calculate individual band profiles in the elution and displacement modes, and of a simplex algorithm to optimize the production rate. The operating parameters (sample size, mobile phase velocity, and the displacer concentration in the displacement model) and the column design parameters (column length and average particle diameter) are optimized simultaneously. Binary mixtures having relative concentrations 3:1 and 1:3, and separation factors of 1.2 to 1.8 are investigated. One of our major results is that, in both modes of chromatography, the maximum production rate is achieved at very low values of the retention factors, k′, much lower than those used in current practice. In all cases, unless k′ exceeds greatly that optimum value, the production rate is higher in overloaded elution than in displacement chromatography. This is particularly true for the extraction of a minor component, which is eluted second. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 196
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 316-324 
    ISSN: 0006-3592
    Schlagwort(e): Escherichia coli ; acetic acid ; inhibition ; glycine ; methionine ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more β-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h-1, the methionine addition enhanced β-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h -1, the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 197
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 330-340 
    ISSN: 0006-3592
    Schlagwort(e): hybridoma ; Immobilization ; monoclonal antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Immobilization offers several intrinsic advantages over free suspension cultures for the production of monoclonal antibodies. An important advantage of immobilization is the improved specific monoclonal antibody (MAb) productivity (qMAb) that can be obtained. However, there are conflicting reports in the literature on the enhancement of the qMAb with immobilization. The discrepancies between these reports can be attributed to the different to either the cultivation methods used for immobilized cell or to difference between the cell lines used in the various studies. We show that these differences may be attributed to the different cultivation methods used for one model hybridoma cell line. S3H5/ϒ2bA2 hybridoma cells entrapped in different sizes of calcium alginate beads were cultivated in both T- and spinner flasks in order to determine whether cultivation methods (T- and spinner flasks) and bead size influence the qMAb Free-suspended cell cultures inoculated with cells recovered from alginate beads were also carried out in order to determine whether changes in the qMab of the entrapped cells are reversible.The cultivation methods was found to influence significantly the qMAb of the entrapped cells. When the entrapped cells in 1-mn diameter beads were cultivated in T-flasks, the qMAb was not increased by 200% as previously observed in an entrapped cell culture using 1-mm-diameter alginate beads in spinner flasks. The qMAb of the entrapped cell was approximately 58% higher than that of the free-suspended cells in a control experiment. Unlike the cultivation method, the bead size in the range of 1- to 3-mm diameter did not significantly influence the qMAb, regardless of cultivations methods. The changes in qMAb of an entrapped cells were reversible. When the free-suspended cells recovered from the T- and spinner flasks were sub-cultured in T- and spinner flasks enhanced qMAb of the entrapped cells in both cases decreased to the level of the free-suspended cell in a control experiments. Taken together, these results shows that the method of cultivation of hybridoma cells immobilized in alginate beads determines the extent of enhancement of the qMAb. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 198
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 380-389 
    ISSN: 0006-3592
    Schlagwort(e): density-dependent growth ; anchorage-dependent cells ; image analysis ; CHO cells ; model simulation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: The density-dependent growth of Chinese hamster ovary (CHO) cells was monitored on-line by using an inverted microscope. A flow system was employed for cell cultivation so that nutrient concentration could be maintained and metabolic wastes were removed. With the help of video image analysis, local cells density could be accurately calculated and cell motility and exposed cell surface area could be estimated. A computer program which accounted for change of sell size and translocation of cells was developed to stimulate dell growth. The stimulated results of the population dynamics and the variations in cell size showed good agreement with our experimental observations, Cell motility and initial cell distribution on the substratum were found to have strong effect on cell growth. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 15 Ill.
    Materialart: Digitale Medien
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  • 199
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 390-393 
    ISSN: 0006-3592
    Schlagwort(e): organic solvents ; enzyme catalysis ; immiscibility with water ; hydrophobicity of solvents ; dipole moment dielectric constant ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: The question of whether the solvent's water-immiscibility is relevant to enzymatic activity was addressed by assaying four different hydrolases (three lipases and one protease) in nine anhydrous solvents of similar hydrophobicities of which four were infinitely miscible with water and five were not. For no enzyme was a jump in activity observed upon a transition from water-miscible to water-immiscible solvent. The relevance of solvent apolarity to enzymatic efficiency was also examined. To this end, three groups of isomeric anhydrous solvents were selected where within each group of isomeric anhydrous solvents were selected where within each group one solvent was apolar (i.e., lacked a permanent dipole moment). For none of the four enzymes studied was activity significantly higher in apolar solvents than in their polar counterparts. Thus we conclude that often-cited solvent's immiscibility with water and apolarity by themselves are irrelevant to enzymatic activity. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 200
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 422-428 
    ISSN: 0006-3592
    Schlagwort(e): entropy of growth ; Escherichia coli K-12 ; entropy of anabolism ; entropy change ; entropy of formation ; entropy of formation of cells ; cellular entropy ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: The ΔSf′ of one unit carbon formula weight of Escherichia coli K-12 cells, when grown on succinic acid, was calculated to be -80.13 J/deg. This value could then be used to calculate the entropy change accompanying the anabolism and metabolism of succinic acid to be 30.82 J/deg and 32.40 J/mol deg, respectively. The entropy of one unit carbon formula weight of dried E. Coli K-12 cells is calculated to be 94.40 J/deg, which when divided by the mass of these cells becomes 3.90 J/g deg. The corresponding entropy of succinic acid is 2.77 J/g deg, making it apparent that the entropy per unit mass of the cells is greater than that of the substrate. It might be thought that because the cells appear to be so much more complex than the substrate, the cells should have a lesser entropy per unit mass than the substrate. That this does not appear to be true leads to the conclusion that the macromolecular organization (informational content?) of the cells contributes only in a very minor way to the total physical entropy of cells. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 5 Tab.
    Materialart: Digitale Medien
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