title: Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.
abstract: 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.
authors: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
authors: Jennifer L
authors: JL
authors: Johnson
authors: McKetta Department of Chemical Engineering, University of Texas at Austin, MC0400, 1 University Station, Austin, TX 78712, USA.
authors: Kevin C
authors: KC
authors: Entzminger
authors: McKetta Department of Chemical Engineering, University of Texas at Austin, MC0400, 1 University Station, Austin, TX 78712, USA.
authors: Jeongmin
authors: J
authors: Hyun
authors: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
authors: Sibel
authors: S
authors: Kalyoncu
authors: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
authors: David P
authors: DP
authors: Heaner
authors: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
authors: Ivan A
authors: IA
authors: Morales
authors: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
authors: Aly
authors: A
authors: Sheppard
authors: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
authors: James C
authors: JC
authors: Gumbart
authors: McKetta Department of Chemical Engineering, University of Texas at Austin, MC0400, 1 University Station, Austin, TX 78712, USA.
authors: Jennifer A
authors: JA
authors: Maynard
authors: School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
authors: Raquel L
authors: RL
authors: Lieberman
mesh_terms: D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D057127:Single-Chain Antibodies
keywords: Fab fragment; GPCRs; crystallization chaperones; membrane proteins; β-barrel