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Izumi Kumagai - One of the best experts on this subject based on the ideXlab platform.

  • contribution of asparagine residues to the stabilization of a proteinaceous antigen antibody complex hyhel 10 hen egg white lysozyme
    Journal of Biological Chemistry, 2010
    Co-Authors: Akiko Yokota, Kouhei Tsumoto, Mitsunori Shiroishi, Takeshi Nakanishi, Hidemasa Kondo, Izumi Kumagai
    Abstract:

    Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of Antigen-Antibody complexes, we examined the Interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of l-Asn-31, l-Asn-32, and l-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the Interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the Interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other Interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the Interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the Antigen-Antibody Interaction can be attributed to their limited flexibility and accessibility at the complex interface.

  • structural consequences of mutations in interfacial tyr residues of a protein antigen antibody complex the case of hyhel 10 hel
    Journal of Biological Chemistry, 2007
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Takeshi Nakanishi, Hidemasa Kondo, Yoshikazu Tanaka, Izumi Kumagai
    Abstract:

    Abstract Tyrosine is an important amino acid in protein-protein Interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein Interactions, we performed crystallographic studies and thermodynamic analyses of the Interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the Interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the Antigen-Antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the Antigen-Antibody Interaction through a diverse set of structural and thermodynamic Interactions.

  • structural evidence for entropic contribution of salt bridge formation to a protein antigen antibody Interaction the case of hen lysozyme hyhel 10 fv complex
    Journal of Biological Chemistry, 2001
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Hidemasa Kondo, Yoshiyuki Nishimiya, Katsunori Horii, Masaaki Matsushima, Kyoko Ogasahara, Katsuhide Yutani, Izumi Kumagai
    Abstract:

    A structural and thermodynamic study of the entropic contribution of salt bridge formation to the Interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the Interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the Antigen-Antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein Antigen-Antibody Interaction through reduction of entropy loss due to dehydration and structural changes.

  • crystal structure of anti hen egg white lysozyme antibody hyhel 10 fv antigen complex local structural changes in the protein antigen and water mediated Interactions of fv antigen and light chain heavy chain interfaces
    Journal of Biological Chemistry, 1999
    Co-Authors: Hidemasa Kondo, Kouhei Tsumoto, M Shiroishi, Masaaki Matsushima, Izumi Kumagai
    Abstract:

    In order to address the recognition mechanism of the fragments of antibody variable regions, termed Fv, toward their target antigen, an x-ray crystal structure of an anti-hen egg white lysozyme antibody (HyHEL-10) Fv fragment complexed with its cognate antigen, hen egg white lysozyme (HEL), was solved at 2.3 A. The overall structure of the complex is similar to that reported in a previous article dealing with the Fab fragment-HEL complex (PDB ID code,). However, the areas of Fv covered by HEL upon complex formation increased by about 100 A(2) in comparison with the Fab-HEL complex, and two local structural differences were observed in the heavy chain of the variable region (VH). In addition, small but significant local structural changes were observed in the antigen, HEL. The x-ray data permitted the identification of two water molecules between the VH and HEL and six water molecules retained in the interface between the antigen and the light chain complementarity determining regions (CDRs) 2 and 3 (CDR-L2 and CDR-L3). These water molecules bridge the Antigen-Antibody interface through hydrogen bond formation in the VL-HEL interface. Eleven water molecules were found to complete the imperfect VH-VL interface, suggesting that solvent molecules mediate the stabilization of Interaction between variable regions. These results suggest that the unfavorable effect of deletion of constant regions on the Antigen-Antibody Interaction is compensated by an increase in favorable Interactions, including structural changes in the Antigen-Antibody interface and solvent-mediated hydrogen bond formation upon complex formation, which may lead to a minimum decreased affinity of the antibody Fv fragment toward its antigen.

  • role of salt bridge formation in antigen antibody Interaction entropic contribution to the complex between hen egg white lysozyme and its monoclonal antibody hyhel10
    Journal of Biological Chemistry, 1996
    Co-Authors: Kouhei Tsumoto, Kyoko Ogasahara, Katsuhide Yutani, Yoshitaka Ueda, Kimitsuna Watanabe, Izumi Kumagai
    Abstract:

    Abstract For elucidation of the role of salt bridge formation in the Antigen-Antibody complex, the Interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, the structure of which has been well characterized and forms one salt bridge (Lys97 of HEL and Asp32 of HyHEL10 heavy chain variable region (VH)), was investigated. Asp32 of VH was substituted with Ala, Asn, or Glu by site-directed mutagenesis, and the Interaction between HEL and the mutant fragments of the variable region of light chain was investigated by inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. Inhibition assay indicated that these mutations lowered the inhibition only slightly. Thermodynamic study indicated that the negative enthalpic change in the Interaction between each of the mutant variable regions of light chain and HEL was significantly increased, although the association constant was slightly decreased, suggesting that these mutations increased the entropy change upon Antigen-Antibody binding. These results indicate that the role of salt bridge formation in the HyHEL10-HEL Interaction is to lower the entropic loss due to binding. In the mutant proteins, the numbers of residues that were perturbed structurally on binding increased, suggesting that the salt bridge suppresses excess structural movement of the antibody upon binding.

Kouhei Tsumoto - One of the best experts on this subject based on the ideXlab platform.

  • contribution of asparagine residues to the stabilization of a proteinaceous antigen antibody complex hyhel 10 hen egg white lysozyme
    Journal of Biological Chemistry, 2010
    Co-Authors: Akiko Yokota, Kouhei Tsumoto, Mitsunori Shiroishi, Takeshi Nakanishi, Hidemasa Kondo, Izumi Kumagai
    Abstract:

    Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of Antigen-Antibody complexes, we examined the Interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of l-Asn-31, l-Asn-32, and l-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the Interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the Interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other Interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the Interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the Antigen-Antibody Interaction can be attributed to their limited flexibility and accessibility at the complex interface.

  • structural consequences of mutations in interfacial tyr residues of a protein antigen antibody complex the case of hyhel 10 hel
    Journal of Biological Chemistry, 2007
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Takeshi Nakanishi, Hidemasa Kondo, Yoshikazu Tanaka, Izumi Kumagai
    Abstract:

    Abstract Tyrosine is an important amino acid in protein-protein Interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein Interactions, we performed crystallographic studies and thermodynamic analyses of the Interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the Interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the Antigen-Antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the Antigen-Antibody Interaction through a diverse set of structural and thermodynamic Interactions.

  • structural evidence for entropic contribution of salt bridge formation to a protein antigen antibody Interaction the case of hen lysozyme hyhel 10 fv complex
    Journal of Biological Chemistry, 2001
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Hidemasa Kondo, Yoshiyuki Nishimiya, Katsunori Horii, Masaaki Matsushima, Kyoko Ogasahara, Katsuhide Yutani, Izumi Kumagai
    Abstract:

    A structural and thermodynamic study of the entropic contribution of salt bridge formation to the Interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the Interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the Antigen-Antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein Antigen-Antibody Interaction through reduction of entropy loss due to dehydration and structural changes.

  • crystal structure of anti hen egg white lysozyme antibody hyhel 10 fv antigen complex local structural changes in the protein antigen and water mediated Interactions of fv antigen and light chain heavy chain interfaces
    Journal of Biological Chemistry, 1999
    Co-Authors: Hidemasa Kondo, Kouhei Tsumoto, M Shiroishi, Masaaki Matsushima, Izumi Kumagai
    Abstract:

    In order to address the recognition mechanism of the fragments of antibody variable regions, termed Fv, toward their target antigen, an x-ray crystal structure of an anti-hen egg white lysozyme antibody (HyHEL-10) Fv fragment complexed with its cognate antigen, hen egg white lysozyme (HEL), was solved at 2.3 A. The overall structure of the complex is similar to that reported in a previous article dealing with the Fab fragment-HEL complex (PDB ID code,). However, the areas of Fv covered by HEL upon complex formation increased by about 100 A(2) in comparison with the Fab-HEL complex, and two local structural differences were observed in the heavy chain of the variable region (VH). In addition, small but significant local structural changes were observed in the antigen, HEL. The x-ray data permitted the identification of two water molecules between the VH and HEL and six water molecules retained in the interface between the antigen and the light chain complementarity determining regions (CDRs) 2 and 3 (CDR-L2 and CDR-L3). These water molecules bridge the Antigen-Antibody interface through hydrogen bond formation in the VL-HEL interface. Eleven water molecules were found to complete the imperfect VH-VL interface, suggesting that solvent molecules mediate the stabilization of Interaction between variable regions. These results suggest that the unfavorable effect of deletion of constant regions on the Antigen-Antibody Interaction is compensated by an increase in favorable Interactions, including structural changes in the Antigen-Antibody interface and solvent-mediated hydrogen bond formation upon complex formation, which may lead to a minimum decreased affinity of the antibody Fv fragment toward its antigen.

  • role of salt bridge formation in antigen antibody Interaction entropic contribution to the complex between hen egg white lysozyme and its monoclonal antibody hyhel10
    Journal of Biological Chemistry, 1996
    Co-Authors: Kouhei Tsumoto, Kyoko Ogasahara, Katsuhide Yutani, Yoshitaka Ueda, Kimitsuna Watanabe, Izumi Kumagai
    Abstract:

    Abstract For elucidation of the role of salt bridge formation in the Antigen-Antibody complex, the Interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, the structure of which has been well characterized and forms one salt bridge (Lys97 of HEL and Asp32 of HyHEL10 heavy chain variable region (VH)), was investigated. Asp32 of VH was substituted with Ala, Asn, or Glu by site-directed mutagenesis, and the Interaction between HEL and the mutant fragments of the variable region of light chain was investigated by inhibition of the enzymatic activity of HEL and isothermal titration calorimetry. Inhibition assay indicated that these mutations lowered the inhibition only slightly. Thermodynamic study indicated that the negative enthalpic change in the Interaction between each of the mutant variable regions of light chain and HEL was significantly increased, although the association constant was slightly decreased, suggesting that these mutations increased the entropy change upon Antigen-Antibody binding. These results indicate that the role of salt bridge formation in the HyHEL10-HEL Interaction is to lower the entropic loss due to binding. In the mutant proteins, the numbers of residues that were perturbed structurally on binding increased, suggesting that the salt bridge suppresses excess structural movement of the antibody upon binding.

Hidemasa Kondo - One of the best experts on this subject based on the ideXlab platform.

  • contribution of asparagine residues to the stabilization of a proteinaceous antigen antibody complex hyhel 10 hen egg white lysozyme
    Journal of Biological Chemistry, 2010
    Co-Authors: Akiko Yokota, Kouhei Tsumoto, Mitsunori Shiroishi, Takeshi Nakanishi, Hidemasa Kondo, Izumi Kumagai
    Abstract:

    Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of Antigen-Antibody complexes, we examined the Interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of l-Asn-31, l-Asn-32, and l-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the Interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the Interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other Interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the Interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the Antigen-Antibody Interaction can be attributed to their limited flexibility and accessibility at the complex interface.

  • structural consequences of mutations in interfacial tyr residues of a protein antigen antibody complex the case of hyhel 10 hel
    Journal of Biological Chemistry, 2007
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Takeshi Nakanishi, Hidemasa Kondo, Yoshikazu Tanaka, Izumi Kumagai
    Abstract:

    Abstract Tyrosine is an important amino acid in protein-protein Interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein Interactions, we performed crystallographic studies and thermodynamic analyses of the Interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the Interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the Antigen-Antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the Antigen-Antibody Interaction through a diverse set of structural and thermodynamic Interactions.

  • structural evidence for entropic contribution of salt bridge formation to a protein antigen antibody Interaction the case of hen lysozyme hyhel 10 fv complex
    Journal of Biological Chemistry, 2001
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Hidemasa Kondo, Yoshiyuki Nishimiya, Katsunori Horii, Masaaki Matsushima, Kyoko Ogasahara, Katsuhide Yutani, Izumi Kumagai
    Abstract:

    A structural and thermodynamic study of the entropic contribution of salt bridge formation to the Interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the Interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the Antigen-Antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein Antigen-Antibody Interaction through reduction of entropy loss due to dehydration and structural changes.

  • crystal structure of anti hen egg white lysozyme antibody hyhel 10 fv antigen complex local structural changes in the protein antigen and water mediated Interactions of fv antigen and light chain heavy chain interfaces
    Journal of Biological Chemistry, 1999
    Co-Authors: Hidemasa Kondo, Kouhei Tsumoto, M Shiroishi, Masaaki Matsushima, Izumi Kumagai
    Abstract:

    In order to address the recognition mechanism of the fragments of antibody variable regions, termed Fv, toward their target antigen, an x-ray crystal structure of an anti-hen egg white lysozyme antibody (HyHEL-10) Fv fragment complexed with its cognate antigen, hen egg white lysozyme (HEL), was solved at 2.3 A. The overall structure of the complex is similar to that reported in a previous article dealing with the Fab fragment-HEL complex (PDB ID code,). However, the areas of Fv covered by HEL upon complex formation increased by about 100 A(2) in comparison with the Fab-HEL complex, and two local structural differences were observed in the heavy chain of the variable region (VH). In addition, small but significant local structural changes were observed in the antigen, HEL. The x-ray data permitted the identification of two water molecules between the VH and HEL and six water molecules retained in the interface between the antigen and the light chain complementarity determining regions (CDRs) 2 and 3 (CDR-L2 and CDR-L3). These water molecules bridge the Antigen-Antibody interface through hydrogen bond formation in the VL-HEL interface. Eleven water molecules were found to complete the imperfect VH-VL interface, suggesting that solvent molecules mediate the stabilization of Interaction between variable regions. These results suggest that the unfavorable effect of deletion of constant regions on the Antigen-Antibody Interaction is compensated by an increase in favorable Interactions, including structural changes in the Antigen-Antibody interface and solvent-mediated hydrogen bond formation upon complex formation, which may lead to a minimum decreased affinity of the antibody Fv fragment toward its antigen.

Akiko Yokota - One of the best experts on this subject based on the ideXlab platform.

  • contribution of asparagine residues to the stabilization of a proteinaceous antigen antibody complex hyhel 10 hen egg white lysozyme
    Journal of Biological Chemistry, 2010
    Co-Authors: Akiko Yokota, Kouhei Tsumoto, Mitsunori Shiroishi, Takeshi Nakanishi, Hidemasa Kondo, Izumi Kumagai
    Abstract:

    Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of Antigen-Antibody complexes, we examined the Interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of l-Asn-31, l-Asn-32, and l-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the Interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the Interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other Interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the Interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the Antigen-Antibody Interaction can be attributed to their limited flexibility and accessibility at the complex interface.

  • structural consequences of mutations in interfacial tyr residues of a protein antigen antibody complex the case of hyhel 10 hel
    Journal of Biological Chemistry, 2007
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Takeshi Nakanishi, Hidemasa Kondo, Yoshikazu Tanaka, Izumi Kumagai
    Abstract:

    Abstract Tyrosine is an important amino acid in protein-protein Interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein Interactions, we performed crystallographic studies and thermodynamic analyses of the Interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the Interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the Antigen-Antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the Antigen-Antibody Interaction through a diverse set of structural and thermodynamic Interactions.

  • structural evidence for entropic contribution of salt bridge formation to a protein antigen antibody Interaction the case of hen lysozyme hyhel 10 fv complex
    Journal of Biological Chemistry, 2001
    Co-Authors: M Shiroishi, Akiko Yokota, Kouhei Tsumoto, Hidemasa Kondo, Yoshiyuki Nishimiya, Katsunori Horii, Masaaki Matsushima, Kyoko Ogasahara, Katsuhide Yutani, Izumi Kumagai
    Abstract:

    A structural and thermodynamic study of the entropic contribution of salt bridge formation to the Interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the Interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the Antigen-Antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein Antigen-Antibody Interaction through reduction of entropy loss due to dehydration and structural changes.

Matthew R Scholfield - One of the best experts on this subject based on the ideXlab platform.

  • versatile photosensitizers for photodynamic therapy at infrared excitation
    Journal of the American Chemical Society, 2007
    Co-Authors: Peng Zhang, Wim F A Steelant, Manoj Kumar, Matthew R Scholfield
    Abstract:

    A new type of photosensitizers used in photodynamic therapy, which is based on photon upconverting nanoparticles, is reported. These photosensitizers are excitable with infrared irradiation, which has several times larger tissue penetration depth than the currently available ones. They are brought close to the target cancer cells through Antigen-Antibody Interaction with good specificity and versatility. The design is also flexible in that various photosensitive molecules can be potentially adopted into the design. Results from in vitro experiments demonstrate their promise of becoming the next generation photodynamic therapy drugs.

  • versatile photosensitizers for photodynamic therapy at infrared excitation
    Journal of the American Chemical Society, 2007
    Co-Authors: Peng Zhang, Wim F A Steelant, Manoj Kumar, Matthew R Scholfield
    Abstract:

    A new type of photosensitizers used in photodynamic therapy, which is based on photon upconverting nanoparticles, is reported. These photosensitizers are excitable with infrared irradiation and have several times larger tissue penetration depth than the currently available ones. They are brought close to the target cancer cells through antigen−antibody Interaction with good specificity and versatility. The design is also flexible in that various photosensitive molecules can be potentially adopted into the design. Results from in vitro experiments demonstrate their promise of becoming the next generation photodynamic therapy drugs.