The Experts below are selected from a list of 1866 Experts worldwide ranked by ideXlab platform

Anna Tramontano - One of the best experts on this subject based on the ideXlab platform.

  • modelling Antibody antigen interactions Ferritin as a case study
    Molecular Immunology, 1995
    Co-Authors: Manuela Helmercitterich, Ermanna Rovida, Alessandra Luzzago, Anna Tramontano
    Abstract:

    In this work, we propose a model for the structure of the antigen-Antibody complex formed by human H-Ferritin and an Antibody that specifically recognizes it. We cloned and sequenced the Antibody gene, predicted the Antibody three-dimensional structure, and reconstructed the H-Ferritin-Antibody complex using an automated docking procedure previously validated on known complexes. This procedure allowed us to identify one putative complex which we carefully analysed, in order both to evaluate its likelihood, in light of a set of experimental results described in the literature, and to predict precisely which are the sites of interaction between the two molecules. Our model is compatible with the experimentally determined characteristics of the complex. Some of the residues that form the predicted antigenic site of Ferritin can be found in the amino acid sequence of peptides selected from a random peptide library because of their affinity for the Ferritin monoclonal Antibody. Furthermore, the structural difference between the antigenic site in human H-Ferritin and the corresponding region in other species permits us to rationalize the inability of the Antibody to recognize human L-Ferritin and rat, chicken and mouse H-Ferritin. Through the analysis of our model complex, we identify a number of other residues putatively involved in the interaction. This multidisciplinary approach shows that synergy between computational and experimental methods may bring further insight into the understanding of Antibody-antigen recognition rules.

Sergey P Martsev - One of the best experts on this subject based on the ideXlab platform.

  • folding of an Antibody variable domain in two functional conformations in vitro calorimetric and spectroscopic study of the anti Ferritin Antibody vl domain
    Protein Engineering Design & Selection, 2007
    Co-Authors: Yaroslav I Tsybovsky, Denis V Shubenok, Zinaida I Kravchuk, Sergey P Martsev
    Abstract:

    Understanding refolding pathways of recombinant Antibody fragments is essential for efficient production of these proteins of high biomedical significance. The recombinant VL domain of mouse anti-human Ferritin Antibody F11 formed two distinct functional conformations obtained by refolding from bacterial inclusion bodies using two different procedures. Involvement of a dialysis step at pH 2-3 resulted in the VL-1 conformation with fluorescence of the highly conserved Trp-35 residue quenched by the spatially proximal disulfide bond. This conformation was identical to the 'native' VL domain folded in host cells and purified from the cytoplasm. In the absence of the acidic dialysis step, the VL domain adopted a previously unreported conformation, VL-2, that demonstrated prominent fluorescence due to a local structural disorder around Trp-35. Furthermore, VL-2 showed changes in secondary structure and significantly lower stability as determined by differential scanning calorimetry and denaturant-induced unfolding. While more flexible VL-2 binds human Ferritin both in solution and after surface adsorption of the Antibody domain, the VL-1 conformer needs an adsorption-induced conformational change to allow the access of Ferritin to the antigen-binding site. Noteworthy, the two macroscopic conformations constitute kinetically trapped dimers and do not interconvert at elevated temperatures (3 weeks at 37 degrees C or 15 min at 60 degrees C), which indicates a high energetic barrier between them. As a major finding, this paper provides the first description for two stable and functional conformations of an Antibody domain.

  • partially structured state of the functional vh domain of the mouse anti Ferritin Antibody f11
    FEBS Letters, 2002
    Co-Authors: Sergey P Martsev, Yaroslav I Tsybovsky, Zinaida I Kravchuk, Anatoly P Dubnovitsky, Oleg A Stremovsky, Alexander A Chumanevich, Sergey M Deyev
    Abstract:

    Abstract An Antibody combining site generally involves the two variable domains, VH from the heavy and VL from the light chain. We expressed the individual VH domain of the mouse anti-human Ferritin monoclonal Antibody F11. The loss of affinity was not dramatic (Ka=4.0×107 M−1 versus 8.6×108 M−1 for the parent Antibody) and comparable to that previously observed for other VHs. However, the functional VH domain adopted a partially structured state with a significant amount of distorted secondary and compact yet greatly destabilized tertiary structures, as demonstrated by spectroscopic and calorimetric probes. These data provide the first description for a functional Antibody domain that meets all the criteria of a partially structured state.

Manuela Helmercitterich - One of the best experts on this subject based on the ideXlab platform.

  • modelling Antibody antigen interactions Ferritin as a case study
    Molecular Immunology, 1995
    Co-Authors: Manuela Helmercitterich, Ermanna Rovida, Alessandra Luzzago, Anna Tramontano
    Abstract:

    In this work, we propose a model for the structure of the antigen-Antibody complex formed by human H-Ferritin and an Antibody that specifically recognizes it. We cloned and sequenced the Antibody gene, predicted the Antibody three-dimensional structure, and reconstructed the H-Ferritin-Antibody complex using an automated docking procedure previously validated on known complexes. This procedure allowed us to identify one putative complex which we carefully analysed, in order both to evaluate its likelihood, in light of a set of experimental results described in the literature, and to predict precisely which are the sites of interaction between the two molecules. Our model is compatible with the experimentally determined characteristics of the complex. Some of the residues that form the predicted antigenic site of Ferritin can be found in the amino acid sequence of peptides selected from a random peptide library because of their affinity for the Ferritin monoclonal Antibody. Furthermore, the structural difference between the antigenic site in human H-Ferritin and the corresponding region in other species permits us to rationalize the inability of the Antibody to recognize human L-Ferritin and rat, chicken and mouse H-Ferritin. Through the analysis of our model complex, we identify a number of other residues putatively involved in the interaction. This multidisciplinary approach shows that synergy between computational and experimental methods may bring further insight into the understanding of Antibody-antigen recognition rules.

Yaroslav I Tsybovsky - One of the best experts on this subject based on the ideXlab platform.

  • folding of an Antibody variable domain in two functional conformations in vitro calorimetric and spectroscopic study of the anti Ferritin Antibody vl domain
    Protein Engineering Design & Selection, 2007
    Co-Authors: Yaroslav I Tsybovsky, Denis V Shubenok, Zinaida I Kravchuk, Sergey P Martsev
    Abstract:

    Understanding refolding pathways of recombinant Antibody fragments is essential for efficient production of these proteins of high biomedical significance. The recombinant VL domain of mouse anti-human Ferritin Antibody F11 formed two distinct functional conformations obtained by refolding from bacterial inclusion bodies using two different procedures. Involvement of a dialysis step at pH 2-3 resulted in the VL-1 conformation with fluorescence of the highly conserved Trp-35 residue quenched by the spatially proximal disulfide bond. This conformation was identical to the 'native' VL domain folded in host cells and purified from the cytoplasm. In the absence of the acidic dialysis step, the VL domain adopted a previously unreported conformation, VL-2, that demonstrated prominent fluorescence due to a local structural disorder around Trp-35. Furthermore, VL-2 showed changes in secondary structure and significantly lower stability as determined by differential scanning calorimetry and denaturant-induced unfolding. While more flexible VL-2 binds human Ferritin both in solution and after surface adsorption of the Antibody domain, the VL-1 conformer needs an adsorption-induced conformational change to allow the access of Ferritin to the antigen-binding site. Noteworthy, the two macroscopic conformations constitute kinetically trapped dimers and do not interconvert at elevated temperatures (3 weeks at 37 degrees C or 15 min at 60 degrees C), which indicates a high energetic barrier between them. As a major finding, this paper provides the first description for two stable and functional conformations of an Antibody domain.

  • partially structured state of the functional vh domain of the mouse anti Ferritin Antibody f11
    FEBS Letters, 2002
    Co-Authors: Sergey P Martsev, Yaroslav I Tsybovsky, Zinaida I Kravchuk, Anatoly P Dubnovitsky, Oleg A Stremovsky, Alexander A Chumanevich, Sergey M Deyev
    Abstract:

    Abstract An Antibody combining site generally involves the two variable domains, VH from the heavy and VL from the light chain. We expressed the individual VH domain of the mouse anti-human Ferritin monoclonal Antibody F11. The loss of affinity was not dramatic (Ka=4.0×107 M−1 versus 8.6×108 M−1 for the parent Antibody) and comparable to that previously observed for other VHs. However, the functional VH domain adopted a partially structured state with a significant amount of distorted secondary and compact yet greatly destabilized tertiary structures, as demonstrated by spectroscopic and calorimetric probes. These data provide the first description for a functional Antibody domain that meets all the criteria of a partially structured state.

Zinaida I Kravchuk - One of the best experts on this subject based on the ideXlab platform.

  • folding of an Antibody variable domain in two functional conformations in vitro calorimetric and spectroscopic study of the anti Ferritin Antibody vl domain
    Protein Engineering Design & Selection, 2007
    Co-Authors: Yaroslav I Tsybovsky, Denis V Shubenok, Zinaida I Kravchuk, Sergey P Martsev
    Abstract:

    Understanding refolding pathways of recombinant Antibody fragments is essential for efficient production of these proteins of high biomedical significance. The recombinant VL domain of mouse anti-human Ferritin Antibody F11 formed two distinct functional conformations obtained by refolding from bacterial inclusion bodies using two different procedures. Involvement of a dialysis step at pH 2-3 resulted in the VL-1 conformation with fluorescence of the highly conserved Trp-35 residue quenched by the spatially proximal disulfide bond. This conformation was identical to the 'native' VL domain folded in host cells and purified from the cytoplasm. In the absence of the acidic dialysis step, the VL domain adopted a previously unreported conformation, VL-2, that demonstrated prominent fluorescence due to a local structural disorder around Trp-35. Furthermore, VL-2 showed changes in secondary structure and significantly lower stability as determined by differential scanning calorimetry and denaturant-induced unfolding. While more flexible VL-2 binds human Ferritin both in solution and after surface adsorption of the Antibody domain, the VL-1 conformer needs an adsorption-induced conformational change to allow the access of Ferritin to the antigen-binding site. Noteworthy, the two macroscopic conformations constitute kinetically trapped dimers and do not interconvert at elevated temperatures (3 weeks at 37 degrees C or 15 min at 60 degrees C), which indicates a high energetic barrier between them. As a major finding, this paper provides the first description for two stable and functional conformations of an Antibody domain.

  • partially structured state of the functional vh domain of the mouse anti Ferritin Antibody f11
    FEBS Letters, 2002
    Co-Authors: Sergey P Martsev, Yaroslav I Tsybovsky, Zinaida I Kravchuk, Anatoly P Dubnovitsky, Oleg A Stremovsky, Alexander A Chumanevich, Sergey M Deyev
    Abstract:

    Abstract An Antibody combining site generally involves the two variable domains, VH from the heavy and VL from the light chain. We expressed the individual VH domain of the mouse anti-human Ferritin monoclonal Antibody F11. The loss of affinity was not dramatic (Ka=4.0×107 M−1 versus 8.6×108 M−1 for the parent Antibody) and comparable to that previously observed for other VHs. However, the functional VH domain adopted a partially structured state with a significant amount of distorted secondary and compact yet greatly destabilized tertiary structures, as demonstrated by spectroscopic and calorimetric probes. These data provide the first description for a functional Antibody domain that meets all the criteria of a partially structured state.