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Toward chaperone-assisted crystallography: Protein engineering enhancement of crystal packing and x-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold

¹ University of Chicago;
² The University of Chicago

(RECEIVED February 12, 2008; ACCEPTED April 15, 2008)

A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing and provides high quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (VHH) as a crystallization chaperone. Using a yeast surface display system for VHH we successfully introduced additional Met residues in the core of the VHH scaffold. We identified a set of SeMet labeled VHH variants that collectively produced six new crystal forms as the complex with the model antigen, RNaseA. The crystals exhibited monoclinic, orthorhombic, triclinic and tetragonal symmetry, have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched VHH chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and VHH model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength MAD data. Together with the presence of high-throughput selection systems (e.g. phage display libraries) for VHH, the enhanced VHH domain described here will be an excellent scaffold for producing effective crystallization chaperones.

Keywords: Protein crystallization; SeMet phasing; VHH; camelid single-domain antibody; chaperone-assisted crystallography; yeast surface display

³ E-mail: koss{at}cummings.uchicago.edu

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