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  • 1
    Electronic Resource
    Electronic Resource
    Hydrogen bonding and biological specificity analysed by protein engineering (1985)
    [s.l.] : Nature Publishing Group
    Nature 314 (1985), S. 235-238 
    Details
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/314235a0
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  • 2
    Electronic Resource
    Electronic Resource
    Isolation and characterization of CHO cell mutants with altered asparagine synthetase (1979)
    Springer
    Somatic cell and molecular genetics 5 (1979), S. 625-639 
    Details
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat− strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10−6 per cell per generation. Three particular revenants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01542699
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  • 3
    Electronic Resource
    Electronic Resource
    Analyse von Struktur-Aktivitäts-Beziehungen bei Enzymen durch Protein-EngineeringNach einem Vortrag beim Dritten Europäischen Symposium über Organische Chemie (ESOC III, 5.-9. September 1983). Diese Arbeit wurde vom Medical Research Council (Großbritannien) unterstützt. (1984)
    Weinheim : Wiley-Blackwell
    Zeitschrift für die chemische Industrie 96 (1984), S. 455-462 
    Details
    ISSN: 0044-8249
    Keywords: Chemistry ; General Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Die Beziehung zwischen Struktur und Aktivität von Enzymen kann heute erstmals durch systematische Veränderung der Proteinstruktur analysiert werden. Die rapide Entwicklung der DNA-Rekombinations-Techniken einerseits und die Erarbeitung leistungsfähiger Methoden zur chemischen Synthese von DNA-Fragmenten andererseits ermöglichen es, auf einfache Weise Proteine durch spezifische Mutation der entsprechenden Gene gezielt zu verändern. Die kinetische Analyse von Mutanten-Enzymen und die durch hochauflösende Röntgen-Kristallographie erhaltenen Befunde lassen direkt Rückschlüsse auf die Beziehung zwischen Struktur und Funktion zu. Insbesondere können jetzt Enzym-Substrat-Wechselwirkungen und deren Bedeutung für Katalyse und Spezifität detailliert untersucht werden. Den gezielten Austausch einer oder mehrerer Aminosäuren eines Proteins - eine „ortsspezifische Mutagenese“ („site-directed mutagenesis“) führten wir exemplarisch zur Analyse der Struktur-Funktions-Beziehung an der Tyrosyl-tRNA-Synthetase aus Bacillus stearothermophilus durch; dabei konzentrierten wir uns auf das Studium der Rolle der Wasserstoffbrückenbindungen für Substratspezifität und Katalyse. Die Bindung von Tyrosin und ATP kann nur unter Berücksichtigung der Austauschreaktion mit den Wassermolekülen der Hydrathülle verstanden werden. Diese Tatsache und die Kenntnis der detaillierten Struktur ermöglichten es uns, ein Enzym mit erhöhter Substrataffinität maßzuschneidern. Durch ein solches „Protein-Engineering“ können Enzyme mit neuen Spezifitäten, Aktivitäten und Struktureigenschaften gewonnen und direkte Einblicke in die Art und Weise der Enzymkatalyse erhalten werden. Wir fanden beispielsweise, daß die Katalyse der Bildung von Tyr-AMP aus Tyr und ATP hauptsächlich auf elektrostatischen Kräften und Wasserstoffbrückenbindungen beruht, die im Übergangszustand stärker als im Grundzustand sind - ein „strain“-Mechanismus also und weniger eine Säure-Base-Katalyse oder eine kovalente Katalyse.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/ange.19840960704
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  • 4
    Electronic Resource
    Electronic Resource
    Isolation of cDNA clones from an osteosarcoma-ROS17/2.8 library by differential hybridization (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 54 (1994), S. 273-280 
    Details
    ISSN: 0730-2312
    Keywords: differential hybridization ; osteoblastic cDNA ; ROS17/2.8 ; chondrocytes ; calvaria ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have used differential hybridization to isolate and characterize two novel cDNAs expressed in chondrocytes and some osteoblastic cells. A rat osteosarcoma ROS17/2.8 cDNA library was screened and cDNA clones hybridizing strongly to radiolabeled porcine calvaria cDNA but weakly to a control radiolabeled cDNA were isolated. Two clones were obtained - p.6.1 and p.10.15. A radiolabeled probe of p10.15 was shown to hybridize specifically to a 2.3 Kb message RNA from a chondrogenic clonal cell population from rat calvaria-RCJ 3.1C5.18, and the mRNA was downregulated by 1,25 (OH)2D3, which inhibits chondrogenesis in these cells. The other clone, p6.1, was found to hybridize to a 0.95 Kb message that is expressed in rat liver, kidney, lung, muscle, and brain, but not expressed in spleen and expressed only in low levels in thymus.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240540303
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  • 5
    Electronic Resource
    Electronic Resource
    Analysis of Enzyme Structure and Activity by Protein EngineeringThe review article is based on a lecture presented at The Third European Symposium on Organic Chemistry (ESOC III) in September, 1983. This work was supported by the Medical Research Council of the UK. (1984)
    Weinheim : Wiley-Blackwell
    Angewandte Chemie International Edition in English 23 (1984), S. 467-473 
    Details
    ISSN: 0570-0833
    Keywords: Analytical methods ; Enzymes ; Protein engineering ; Enzyme activity ; Chemistry ; General Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Structure-activity relationships of enzymes can now be analyzed for the first time by the systematic alteration of protein structure. Recent developments in the chemical synthesis of DNA fragments and recombinant DNA technology enable the facile modification of proteins by highly specific mutagenesis of their genes. Kinetic analysis of the mutant enzymes combined with high-resolution structural data from protein X-ray crystallography allow direct measurements on the relationships between structure and function. In particular, the strength and nature of enzyme-substrate interactions and their detailed roles in catalysis and specificity can now be studied. We have developed such analysis of enzyme structure-function by site-directed mutagenesis of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus, concentrating so far on the subtle role of hydrogen bonding in both substrate specificity and catalysis. We find that the energetics of tyrosine and ATP binding must be analyzed in terms of an exchange reaction with solvent water. Based on this idea and structural data, we have engineered an enzyme of improved enzyme-substrate affinity, and there thus appear to be real prospects of engineering proteins of new specificities, activities, and structural properties. We are also using protein engineering to gather direct information on the nature of enzyme catalysis. For example, we find the catalysis of formation of Tyr-AMP from Tyr and ATP is due largely to electrostatic and hydrogen bonding interactions that are stronger in the transition state than in the ground state - a “strain” mechanism rather than acid-base or covalent catalysis.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/anie.198404673
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