Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization
| Autor: | Sibel Kalyoncu, James C. Gumbart, Jennifer A. Maynard, David P. Heaner, Jeongmin Hyun, Jennifer L. Johnson, Aly Sheppard, Ivan A. Morales, Kevin C. Entzminger, Raquel L. Lieberman |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Protein Conformation Crystallography X-Ray Epitopes Immunoglobulin Fab Fragments Mice Protein structure Structural Biology Animals Peptide sequence biology Chemistry Membrane Proteins General Medicine Protein engineering Directed evolution Research Papers Crystallography Beta barrel Membrane protein Chaperone (protein) Biophysics biology.protein Crystallization Single-Chain Antibodies |
| Popis: | Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach. |
| Databáze: | OpenAIRE |
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