The symmetric designer protein Pizza as a scaffold for metal coordination.

Symmetric proteins are currently of interest as they allow creation of larger assemblies and facilitate the incorporation of metal ions in the larger complexes. Recently this was demonstrated by the biomineralization of the cadmium-chloride nanocrystal via the Pizza designer protein. However, the mechanism behind this formation remained unclear. Here, we set out to investigate the mechanism driving the formation of this nanocrystal via truncation, mutation, and circular permutations. In addition, the interaction of other biologically relevant metal ions with these symmetric proteins to form larger symmetric complexes was also studied. The formation of the initial nanocrystal is shown to originate from steric strain, where His 58 induces a different rotameric conformation on His 73, thereby distorting an otherwise perfect planar ring of alternating cadmium and chlorine ions, resulting in the smallest nanocrystal. Similar highly symmetric complexes were also observed for the other biological relevant metal ions. However, the flexibility of the coordinating histidine residues allows each metal ion to adopt its preferred geometry leading to either monomeric or dimeric ß-propeller units, where the metal ions are located at the interface between both propeller units. These results demonstrate that symmetric proteins are not only interesting to generate larger assemblies, but are also the perfect scaffold to create more complex metal based assemblies. Such metal protein assemblies may then find applications in bionanotechnology or biocatalysis.

Development and applications of artificial symmetrical proteins.

Since the determination of the first molecular models of proteins there has been interest in creating proteins artificially, but such methods have only become widely successful in the last decade. Gradual improvements over a long period of time have now yielded numerous examples of non-natural proteins, many of which are built from repeated elements. In this review we discuss the design of such symmetrical proteins and their various applications in chemistry and medicine.

Molecular assemblies built with the artificial protein Pizza.

Recently an artificial protein named Pizza6 was reported, which possesses six identical tandem repeats and adopts a monomeric ß -propeller fold with sixfold structural symmetry. Pizza2, a truncated form that consists of a double tandem repeat, self-assembles into a trimer reconstructing the same propeller architecture as Pizza6. The ability of pizza proteins to self-assemble to form complete propellers makes them interesting building blocks to engineer larger symmetrical protein complexes such as symmetric nanoparticles. Here we have explored the self-assembly of Pizza2 fused to homo-oligomerizing peptides. In total, we engineered five different fusion proteins, of which three appeared to assemble successfully into larger complexes. Further characterization of these proteins showed one monodisperse designer protein with a structure close to the intended design. This protein was further fused to eGFP to investigate functionalization of the nanoparticle. The fusion protein was stable and could be expressed in high yield, showing that Pizza-based nanoparticles may be further decorated with functional domains.