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Ralph Kratzner - One of the best experts on this subject based on the ideXlab platform.

  • structure of Ecballium elaterium trypsin inhibitor ii eeti ii a rigid molecular scaffold
    Acta Crystallographica Section D-biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

  • Structure of Ecballium elaterium Trypsin Inhibitor II (Eeti-II): A Rigid Molecular Scaffold
    Acta Crystallographica Section D Biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold. © 2005 International Union of Crystallography - all rights reserved.This work was supported by the Deutsche Forschungsgemeinschaft (SFB416) and by the Fonds der Chemischen Industrie. IU thanks the Spanish MEC for grant BIO2003-06653Peer Reviewe

  • strukturelle untersuchungen an varianten des Ecballium elaterium trypsin inhibitors ii eeti ii
    2001
    Co-Authors: Ralph Kratzner
    Abstract:

    The inhibitor cystine-knot family is composed of a large number of small, around 30 amino acids long proteins, that show a diversity of inhibitory functions. The characteristic structural feature of these microproteins is the so called cystine-knot motif, which fixes the polypeptide chain by 3 cystine bridges and compensates for the lack of a hydrophobic core region present in larger proteins. Typically the first cysteine binds to the fourth, the second to the fifth and the third to the sixth cysteine residue in the polypeptide chain. The 30 amino acids long Ecballium elaterium Trypsin Inhibitor (II), EETI-II, is a typical member of this family. It was first isolated from squash seeds and inhibits trypsin with a nanomolar dissociation constant. Given the very high tolerance of its fold for non-cysteine amino acid substitutions, EETI-II is an ideal framework for the generation of a constrained peptide library, from which variants of new properties can be isolated by repertoire techniques. In this work crystals of EETI-II were grown and X-ray diffraction data measured. To study the native inhibitory function the complex of EETI-II wildtype with porcine trypsin was crystallized and the X-ray structure was determined at 1.6 A resolution. The crystal structures of three folding variants of this inhibitor, carrying mutations in the c-terminal loop, were also studied in complex with porcine trypsin. Their X-ray structures were compared to the wildtype and related structures of other inhibitors.

  • sequence requirements of the gpng β turn of the Ecballium elaterium trypsin inhibitor ii explored by combinatorial library screening
    Journal of Biological Chemistry, 1999
    Co-Authors: Alexander Wentzel, Ralph Kratzner, Andreas Christmann, Harald Kolmar
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22–25 comprising a β-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22–25 tetrapeptide sequences and presented these proteins on the outer membrane ofEscherichia coli cells via fusion to the Igaβautotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1–5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in β-turns. The folding pathway of two selected variants, EETI-βNEDE and EETI-βTNNK, was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Isabel Uson - One of the best experts on this subject based on the ideXlab platform.

  • structure of Ecballium elaterium trypsin inhibitor ii eeti ii a rigid molecular scaffold
    Acta Crystallographica Section D-biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

  • Structure of Ecballium elaterium Trypsin Inhibitor II (Eeti-II): A Rigid Molecular Scaffold
    Acta Crystallographica Section D Biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold. © 2005 International Union of Crystallography - all rights reserved.This work was supported by the Deutsche Forschungsgemeinschaft (SFB416) and by the Fonds der Chemischen Industrie. IU thanks the Spanish MEC for grant BIO2003-06653Peer Reviewe

Harald Kolmar - One of the best experts on this subject based on the ideXlab platform.

  • structure of Ecballium elaterium trypsin inhibitor ii eeti ii a rigid molecular scaffold
    Acta Crystallographica Section D-biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

  • Structure of Ecballium elaterium Trypsin Inhibitor II (Eeti-II): A Rigid Molecular Scaffold
    Acta Crystallographica Section D Biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold. © 2005 International Union of Crystallography - all rights reserved.This work was supported by the Deutsche Forschungsgemeinschaft (SFB416) and by the Fonds der Chemischen Industrie. IU thanks the Spanish MEC for grant BIO2003-06653Peer Reviewe

  • sequence requirements of the gpng β turn of the Ecballium elaterium trypsin inhibitor ii explored by combinatorial library screening
    Journal of Biological Chemistry, 1999
    Co-Authors: Alexander Wentzel, Ralph Kratzner, Andreas Christmann, Harald Kolmar
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22–25 comprising a β-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22–25 tetrapeptide sequences and presented these proteins on the outer membrane ofEscherichia coli cells via fusion to the Igaβautotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1–5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in β-turns. The folding pathway of two selected variants, EETI-βNEDE and EETI-βTNNK, was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Alexander Wentzel - One of the best experts on this subject based on the ideXlab platform.

  • structure of Ecballium elaterium trypsin inhibitor ii eeti ii a rigid molecular scaffold
    Acta Crystallographica Section D-biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

  • Structure of Ecballium elaterium Trypsin Inhibitor II (Eeti-II): A Rigid Molecular Scaffold
    Acta Crystallographica Section D Biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold. © 2005 International Union of Crystallography - all rights reserved.This work was supported by the Deutsche Forschungsgemeinschaft (SFB416) and by the Fonds der Chemischen Industrie. IU thanks the Spanish MEC for grant BIO2003-06653Peer Reviewe

  • sequence requirements of the gpng β turn of the Ecballium elaterium trypsin inhibitor ii explored by combinatorial library screening
    Journal of Biological Chemistry, 1999
    Co-Authors: Alexander Wentzel, Ralph Kratzner, Andreas Christmann, Harald Kolmar
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22–25 comprising a β-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22–25 tetrapeptide sequences and presented these proteins on the outer membrane ofEscherichia coli cells via fusion to the Igaβautotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1–5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in β-turns. The folding pathway of two selected variants, EETI-βNEDE and EETI-βTNNK, was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Judit E Debreczeni - One of the best experts on this subject based on the ideXlab platform.

  • structure of Ecballium elaterium trypsin inhibitor ii eeti ii a rigid molecular scaffold
    Acta Crystallographica Section D-biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold.

  • Structure of Ecballium elaterium Trypsin Inhibitor II (Eeti-II): A Rigid Molecular Scaffold
    Acta Crystallographica Section D Biological Crystallography, 2005
    Co-Authors: Ralph Kratzner, Judit E Debreczeni, Thomas Pape, Thomas R Schneider, Alexander Wentzel, Harald Kolmar, George M Sheldrick, Isabel Uson
    Abstract:

    The Ecballium elaterium trypsin inhibitor II (EETI-II) belongs to the family of squash inhibitors and is one of the strongest inhibitors known for trypsin. The eight independent molecules of EETI-II in the crystal structure reported here provide a good opportunity to test the hypothesis that this small cystine-knot protein (knottin) is sufficiently rigid to be used as a molecular scaffold for protein-engineering purposes. To extend this test, the structures of two complexes of EETI-II with trypsin have also been determined, one carrying a four-amino-acid mutation of EETI-II. The remarkable similarity of these structures confirms the rigidity of the molecular framework and hence its suitability as a molecular scaffold. © 2005 International Union of Crystallography - all rights reserved.This work was supported by the Deutsche Forschungsgemeinschaft (SFB416) and by the Fonds der Chemischen Industrie. IU thanks the Spanish MEC for grant BIO2003-06653Peer Reviewe