Description
Protein G is a well-characterized immunoglobulin binding protein expressed in Streptococcus bacteria. Protein G binds to the constant region of antibody. The β1 domain of Protein G (Gβ1) is a thermally stable protein module that has been used as a tag for recombinant protein expression, as a sensor for protein stability, in folding studies, and as a template for novel protein design. As part of an effort to computationally design novel Protein Gβ1 variants with unique properties of self-association, Colin, et al. generated a mutant version of Protein G, called "monomer A", that displays exceptionally high thermal stability and shows a propensity to spontaneously assemble into homodimers in solution. The subsequent introduction of pairs of histidine amino acid residues against this monomer A background resulted in proteins that assemble into higher order oligomers in a zinc-dependent manner in solution. In this study we have crystallized two different di-histidine Protein Gβ1 monomer A mutant proteins in the presence of zinc and determined their high resolution structures by x-ray crystallography. The structure of the first of di-histidine mutant, mutant 3, was solved by molecular replacement and refined against data to 1.48 Å resolutions. Initial experimental electron density maps reveal clear peaks revealing the positions of zinc ions bound throughout the crystal unit cell. The second mutant, referred to as mutant 1, was solved by single wavelength anomalous dispersion (SAD) phasing using the absorption edge of zinc and refined to 1.49Å resolution. This allowed for the direct calculation of zinc ion positions within the protein unit cell and calculation of experimental electron density maps from these initial zinc positions. Both refined structures reveal that zinc ions are, in fact, bound by the engineered histidine pairs. However, it is not clear from the structures alone how this metal binding might contribute to higher order oligomerization of monomer A dimers in solution. We propose that redesigning the dimer interface for higher affinity metal binding might help stabilize the dimer interface.
