Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

Autor: Johnson JL; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA., Entzminger KC; McKetta Department of Chemical Engineering, University of Texas at Austin, MC0400, 1 University Station, Austin, TX 78712, USA., Hyun J; McKetta Department of Chemical Engineering, University of Texas at Austin, MC0400, 1 University Station, Austin, TX 78712, USA., Kalyoncu S; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA., Heaner DP Jr; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA., Morales IA; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA., Sheppard A; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA., Gumbart JC; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA., Maynard JA; McKetta Department of Chemical Engineering, University of Texas at Austin, MC0400, 1 University Station, Austin, TX 78712, USA., Lieberman RL; School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA.
Jazyk: angličtina
Zdroj: Acta crystallographica. Section D, Biological crystallography [Acta Crystallogr D Biol Crystallogr] 2015 Apr; Vol. 71 (Pt 4), pp. 896-906. Date of Electronic Publication: 2015 Mar 27.
DOI: 10.1107/S1399004715001856
Abstrakt: 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: MEDLINE
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