دورية أكاديمية
Effects of substitutions in the binding surface of an antibody on antigen affinity.
| العنوان: | Effects of substitutions in the binding surface of an antibody on antigen affinity. |
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| المؤلفون: | Dougan DA; CSIRO Molecular Science and CRC for Diagnostic Technologies, Parkville, Victoria, Australia., Malby RL, Gruen LC, Kortt AA, Hudson PJ |
| المصدر: | Protein engineering [Protein Eng] 1998 Jan; Vol. 11 (1), pp. 65-74. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Oxford University Press Country of Publication: England NLM ID: 8801484 Publication Model: Print Cited Medium: Print ISSN: 0269-2139 (Print) Linking ISSN: 02692139 NLM ISO Abbreviation: Protein Eng Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Oxford : Oxford University Press Original Publication: Oxford [Oxfordshire] ; Washington, DC : IRL Press, [c1986-c2003] |
| مواضيع طبية MeSH: | Antigen-Antibody Reactions*, Immunoglobulin Fab Fragments/*metabolism , Neuraminidase/*metabolism, Base Sequence ; DNA Primers ; Hydrogen Bonding ; Immunoglobulin Fab Fragments/chemistry ; Immunoglobulin Fab Fragments/isolation & purification ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Orthomyxoviridae/enzymology ; Recombinant Proteins/chemistry ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/metabolism ; Thermodynamics |
| مستخلص: | The interactions between the Fab and single-chain Fv (scFv) fragments of an antibody (NC10) and its antigen, influenza virus neuraminidase, were analysed in the crystal structures of the Fab-neuraminidase and scFv-neuraminidase complexes. To investigate the contribution to binding made by cavities, salt links and hydrogen bonds in the antibody-antigen interface, 14 single amino acid replacements were made at six contact residues in the scFv fragment by site-directed mutagenesis. The binding affinity of each mutant scFv antibody for neuraminidase was determined with a BIAcore optical biosensor. Four of the mutations resulted in large changes in the free energy of binding to neuraminidase (deltadeltaG > 1 kcal/mol) and together may account for approximately 70% of the free energy of binding. Hence these data support the theory that a small number of residues form the 'functional epitope' and are most important for binding of NC10 to neuraminidase. The salt link between antibody residue (Asp)H56 and (Lys)N432 from neuraminidase was demonstrated to be important for affinity, since substitution of (Asp)H56 with Asn caused a large reduction in the free energy of binding (deltadeltaG = +2.8 kcal/mol). Hydrogen bonds provided by (Tyr)L32 and (Asp)H56 were also important for binding: mutation of (Tyr)L32 to Phe resulted in a significant reduction in binding affinity (deltadeltaG = +1.7 kcal/mol). Disruption of hydrophobic interactions (van der Waals contacts) led to significant reductions in affinity also ((Tyr)H99 to Ala, deltadeltaG = +1.5 kcal/mol; (Leu)L94 to Ala, deltadeltaG > +3.0 kcal/mol). An attempt to increase binding affinity by filling a cavity in the interface with a larger antibody side chain was unsuccessful, as the free energy gained by new antibody-antigen interactions did not compensate for the removal of cavity-bound water molecules. |
| المشرفين على المادة: | 0 (DNA Primers) 0 (Immunoglobulin Fab Fragments) 0 (Recombinant Proteins) EC 3.2.1.18 (Neuraminidase) |
| تواريخ الأحداث: | Date Created: 19980514 Date Completed: 19980610 Latest Revision: 20190909 |
| رمز التحديث: | 20231215 |
| DOI: | 10.1093/protein/11.1.65 |
| PMID: | 9579662 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 0269-2139 |
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| DOI: | 10.1093/protein/11.1.65 |