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  1. Article: Structural characterisation of the hepatitis C envelope glycoprotein E1 ectodomain derived from a mammalian and a yeast expression system.

    Lorent, Eric / Bierau, Horst / Engelborghs, Yves / Verheyden, Gert / Bosman, Fons

    Vaccine

    2008  Volume 26, Issue 3, Page(s) 399–410

    Abstract: The structure of the ectodomain of the hepatitis C envelope glycoprotein E1 (E1s) was characterised by spectroscopic methods. Monomeric E1s was purified from a mammalian and from a Hansenula polymorpha cell lysate, and cysteine-blocked monomers were ... ...

    Abstract The structure of the ectodomain of the hepatitis C envelope glycoprotein E1 (E1s) was characterised by spectroscopic methods. Monomeric E1s was purified from a mammalian and from a Hansenula polymorpha cell lysate, and cysteine-blocked monomers were reconstituted into stable particles. Particles from yeast E1s and mammalian E1s showed a comparable reactivity in ELISA with sera from human chronic HCV carriers, similar antibody titers in the sera of immunised mice as well as a comparable structure as analyzed by spectroscopic methods (tryptophan fluorescence, circular dichroism, and Fourier transform infrared spectroscopy). The overall secondary structure of E1s was neither influenced by the degree of glycosylation nor by the nature of cysteine modification used during purification. The structural comparability of mammalian- and H. polymorpha-expressed E1s opens new perspectives for further development of E1s-based therapeutics as yeast systems generally allow a more easy scaling up.
    MeSH term(s) Animals ; Cercopithecus aethiops ; Circular Dichroism ; Hepacivirus/immunology ; Hepatitis C, Chronic/diagnosis ; Hepatitis C, Chronic/immunology ; Hepatitis C, Chronic/virology ; Humans ; Kidney/cytology ; Kidney/virology ; Mice ; Mice, Inbred BALB C ; Pichia/genetics ; Pichia/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Spectroscopy, Fourier Transform Infrared ; Vaccinia virus/genetics ; Vaccinia virus/metabolism ; Vero Cells ; Viral Envelope Proteins/chemistry ; Viral Envelope Proteins/genetics ; Viral Envelope Proteins/immunology ; Viral Envelope Proteins/metabolism ; Virion/metabolism ; Virion/ultrastructure
    Chemical Substances E1 protein, Hepatitis C virus ; Recombinant Proteins ; Viral Envelope Proteins
    Language English
    Publishing date 2008-01-17
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 605674-x
    ISSN 1873-2518 ; 0264-410X
    ISSN (online) 1873-2518
    ISSN 0264-410X
    DOI 10.1016/j.vaccine.2007.11.004
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1930: Show issues Location:
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    Zs.MO 458: Show issues

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  2. Article: Simulation of the activation of alpha-chymotrypsin: analysis of the pathway and role of the propeptide.

    Mátrai, Janka / Verheyden, Gert / Krüger, Peter / Engelborghs, Yves

    Protein science : a publication of the Protein Society

    2004  Volume 13, Issue 12, Page(s) 3139–3150

    Abstract: Alpha-chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular ... ...

    Abstract Alpha-chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular dynamics and targeted molecular dynamics simulations in aqueous environment on the activation and inactivation processes of three different types of chymotrypsin. These are the wild-type bovine chymotrypsin containing the propeptide and the bovine and rat chymotrypsin lacking the propeptide. From these simulations, the importance of the propeptide and of the sequence differences between the rat and bovine variants from the viewpoint of activation could be evaluated and compared with previous fluorescence stopped flow results. The obtained results show the unambiguous influence of the propeptide on the explored conformational space, whereas the sequence differences between bovine and rat chymotrypsin play a minor role. The main features of activation are present in both the wild type and the variant lacking the propeptide, despite the fact that different parts of the conformational space were explored. The comparison of all trajectories shows that particular amino acid residues, such as 17, 18, 19, 187, 217, 218, and 223, undergo large dihedral transitions during the activation process, suggesting a role as hinge residues during the conformational change.
    MeSH term(s) Amino Acid Sequence ; Animals ; Cattle ; Chymotrypsin/metabolism ; Computer Simulation ; Enzyme Activation ; Enzyme Precursors/physiology ; Hydrogen-Ion Concentration ; Models, Molecular ; Molecular Sequence Data ; Protein Conformation ; Rats ; Sequence Alignment ; Signal Transduction ; Time Factors
    Chemical Substances Enzyme Precursors ; Chymotrypsin (EC 3.4.21.1) ; alpha-chymotrypsin (EC 3.4.21.1)
    Language English
    Publishing date 2004-12
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1106283-6
    ISSN 1469-896X ; 0961-8368
    ISSN (online) 1469-896X
    ISSN 0961-8368
    DOI 10.1110/ps.04825004
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article: A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants.

    Verheyden, Gert / Matrai, Janka / Volckaert, Guido / Engelborghs, Yves

    Protein science : a publication of the Protein Society

    2004  Volume 13, Issue 9, Page(s) 2533–2540

    Abstract: The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from ... ...

    Abstract The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.
    MeSH term(s) Animals ; Cattle ; Chymotrypsin/chemistry ; Chymotrypsin/genetics ; Chymotrypsin/metabolism ; Enzyme Activation ; Fluorescence ; Hydrogen-Ion Concentration ; Kinetics ; Models, Molecular ; Mutation ; Protein Conformation ; Rats ; Structure-Activity Relationship ; Tryptophan/chemistry
    Chemical Substances Tryptophan (8DUH1N11BX) ; Chymotrypsin (EC 3.4.21.1) ; alpha-chymotrypsin (EC 3.4.21.1)
    Language English
    Publishing date 2004-09
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1106283-6
    ISSN 1469-896X ; 0961-8368
    ISSN (online) 1469-896X
    ISSN 0961-8368
    DOI 10.1110/ps.04709604
    Database MEDical Literature Analysis and Retrieval System OnLINE

    Full text online

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 3407: Show issues Location:
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    Zs.MO 1: Show issues

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