The Use of Engineered Antibody Fragments to Promote Crystallization of Membrane Proteins

Abstract

Membrane proteins have a variety of functions in the cell, which are important from both biological and therapeutic standpoints. However, structure determination of membrane proteins is still challenging due to their hydrophobic nature. Techniques such as chaperone-based crystallization, tailoring the lipid and/or detergent environment and mutagenesis studies have emerged to make crystallization of a target membrane protein tractable. However, most of those techniques are specific to target protein; a more general approach would be beneficial. Our potentially generalizable approach uses engineered single chain antibody fragments (scFvs) specific to a short peptide epitope that can be inserted into any extramembranous loop of target membrane protein. Tight binding of engineered scFv variants to the membrane protein will provide a more hydrophilic crystal contacts leading to more stable crystal lattices, and better diffracting crystals.A toolbox of such scFv ‘crystallization chaperones' is being developed based on the scaffold of anti-His6 3D5, which employs non-CDR residues in crystal contacts and has a ∼75A channel in its crystal lattice. We used protein engineering techniques to improve biophysical characteristics of the parent scFv and to alter the binding specificity to the EE epitope (EYMPME). Here we present our first- and second-generation crystallization chaperones, as well as progress toward complexation and co-crystallization with both α-helical and β-barrel membrane proteins.