Engineered, Peptide BINDING Chaperones for Membrane PROTEIN Crystallization

Hydrophobic membrane proteins perform a variety of important functions in the cell, but their structures are notoriously difficult to solve.  Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. We aim to develop a toolbox of orthogonal single-chain antibody fragments engineered for hyper-crystallizability and specific recognition of short peptides. These peptide sequences can be introduced into flexible internal loops or at the termini of membrane proteins without interfering with protein function. The resulting scFv-membrane protein complex is expected to form a crystal lattice mediated by chaperone interactions, resulting in high-resolution crystal structures.

We have developed candidate scFv chaperone proteins binding hexa-histidine, EYMPME (EE) and FLAG tags.  The scFv’s were engineered via site-directed and random mutagenesis in conjunction with phage display to improve biophysical features influencing crystallization propensity, including peptide affinity and specificity, stability, solubility and expression level.    Selected variants exhibit high solubility (up to 16.6 mg/ml) and nanomolar peptide binding affinities; complexes of one chaperone with the signal peptide peptidase integral membrane proteins harboring an internal EE tag has been isolated by gel filtration. Three of these scFv chaperones have been crystallized. The 3.1 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ~52 Å channel in the crystal lattice. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.