PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Theme

Chaperone-assisted crystallography

PSI-SGKB [doi:10.1038/th_psisgkb.2009.8]
Technical Highlight - March 2009
Short description: Addition of single-domain antibodies to target proteins increases crystallization success and provides phasing information, making the structure easier to solve.Prot. Sci. 17, 1175-1187 (2008)

It's not through lack of trying that the problem of reliably obtaining protein crystals suitable for X-ray diffraction is not yet solved. Reductive methylation, 'genome pool' strategies and analysis of strong determinants for crystallization have all helped improve crystallization rates. Addition of readily crystallizable proteins that bind to the target protein has also paid dividends.

The antigen-binding fragments of antibodies and synthetic binding proteins such as ankyrin repeats can 'chaperone' proteins into crystals. They improve crystal packing by providing additional contacts between molecules and by reducing conformational differences between individual protein molecules. And as a bonus, the chaperone adds model-based phasing information needed for resolving the X-ray pattern.

Many of the structures solved using crystallization chaperones have used antibody fragments such as Fab. But Fab fragments are quite large, at around 500 amino acids, and are often produced at only low levels in bacterial expression systems. Now, Tereshko et al. 1 from PSI ISFI propose that an alternative antibody fragment will make life easier.

They show that the antigen-binding domain (VHH) of camelid single-domain antibodies is a versatile crystallization chaperone. The VHH domain has a highly structured β-sheet core that provides a rigid scaffold for stabilizing potential intra-crystal contacts. At around 125 amino acids it is smaller than Fab, and might be the chaperone of choice for smaller protein targets. VHH is also easily modified to incorporate selenomethionine, useful for phasing experiments.

Another advantage of VHH, is that it can be readily adapted by selection in both phage and yeast display systems to generate a range of different high-affinity binding domains, thus increasing the likelihood of obtaining usable crystals. The VHH domain described by Tereshko et al. could well prove to be an excellent scaffold for producing effective crystallization chaperones.

Maria Hodges

References

  1. B. Tereshko et al. Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold.
    Prot. Sci. 17, 1175-1187 (2008).

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health