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Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy
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| Zeitschriftentitel: | Proteins: Structure, Function, and Bioinformatics |
|---|---|
| Personen und Körperschaften: | , , |
| In: | Proteins: Structure, Function, and Bioinformatics, 83, 2015, 1, S. 169-177 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Wiley
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| Schlagwörter: |
| author_facet |
Nag, Moupriya Bera, Kallol Basak, Soumen Nag, Moupriya Bera, Kallol Basak, Soumen |
|---|---|
| author |
Nag, Moupriya Bera, Kallol Basak, Soumen |
| spellingShingle |
Nag, Moupriya Bera, Kallol Basak, Soumen Proteins: Structure, Function, and Bioinformatics Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy Molecular Biology Biochemistry Structural Biology |
| author_sort |
nag, moupriya |
| spelling |
Nag, Moupriya Bera, Kallol Basak, Soumen 0887-3585 1097-0134 Wiley Molecular Biology Biochemistry Structural Biology http://dx.doi.org/10.1002/prot.24715 <jats:title>ABSTRACT</jats:title><jats:p>Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (<jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub>) of ∼200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (<jats:italic>R</jats:italic><jats:sub><jats:italic>H</jats:italic></jats:sub>) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of <jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub> under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.</jats:p> Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy Proteins: Structure, Function, and Bioinformatics |
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Proteins: Structure, Function, and Bioinformatics |
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| title |
Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_unstemmed |
Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_full |
Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_fullStr |
Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_full_unstemmed |
Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_short |
Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_sort |
intermolecular disulfide bond formation promotes immunoglobulin aggregation: investigation by fluorescence correlation spectroscopy |
| topic |
Molecular Biology Biochemistry Structural Biology |
| url |
http://dx.doi.org/10.1002/prot.24715 |
| publishDate |
2015 |
| physical |
169-177 |
| description |
<jats:title>ABSTRACT</jats:title><jats:p>Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (<jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub>) of ∼200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (<jats:italic>R</jats:italic><jats:sub><jats:italic>H</jats:italic></jats:sub>) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of <jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub> under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.</jats:p> |
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| author | Nag, Moupriya, Bera, Kallol, Basak, Soumen |
| author_facet | Nag, Moupriya, Bera, Kallol, Basak, Soumen, Nag, Moupriya, Bera, Kallol, Basak, Soumen |
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| description | <jats:title>ABSTRACT</jats:title><jats:p>Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (<jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub>) of ∼200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (<jats:italic>R</jats:italic><jats:sub><jats:italic>H</jats:italic></jats:sub>) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of <jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub> under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.</jats:p> |
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| spelling | Nag, Moupriya Bera, Kallol Basak, Soumen 0887-3585 1097-0134 Wiley Molecular Biology Biochemistry Structural Biology http://dx.doi.org/10.1002/prot.24715 <jats:title>ABSTRACT</jats:title><jats:p>Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (<jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub>) of ∼200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (<jats:italic>R</jats:italic><jats:sub><jats:italic>H</jats:italic></jats:sub>) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of <jats:italic>τ</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub> under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.</jats:p> Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy Proteins: Structure, Function, and Bioinformatics |
| spellingShingle | Nag, Moupriya, Bera, Kallol, Basak, Soumen, Proteins: Structure, Function, and Bioinformatics, Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy, Molecular Biology, Biochemistry, Structural Biology |
| title | Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_full | Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_fullStr | Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_full_unstemmed | Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_short | Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| title_sort | intermolecular disulfide bond formation promotes immunoglobulin aggregation: investigation by fluorescence correlation spectroscopy |
| title_unstemmed | Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy |
| topic | Molecular Biology, Biochemistry, Structural Biology |
| url | http://dx.doi.org/10.1002/prot.24715 |