The modular structure of many protein families, such as β-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms.Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical β-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.protein evolution | computational protein design | self-assembly | β-propeller | protein crystallography
The overall structure of CysB(88-324) is strikingly similar to those of the periplasmic substrate-binding proteins. A similar fold has also been observed in the cofactor-binding domain of Lac repressor, implying a structural relationship between the Lac repressor and LysR families of proteins. In contrast to Lac repressor, in CysB the twofold axis of symmetry that relates the monomers in the dimer is perpendicular rather than parallel to the long axis of the cofactor-binding domain. This seems likely to place the DNA-binding domains at opposite extremes of the molecule possibly accounting for CysB's extended DNA footprints.
Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial β-trefoil lectin that recognises Burkitt’s lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called “Mitsuba”, is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind α-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning “three-leaf” in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Galα(1,4)Galβ(1,4)Glc) on the surface, but the cytotoxicity is abolished.
Two artificial β-propeller proteins with eight identical blades were designed, purified and crystallized. X-ray crystallography confirmed the perfectly symmetrical structures of these highly stable proteins.
The cavity of the toroidal protein TRAP (trp RNA-binding attenuation protein) is modified to capture gold nanodots in solution. By engineering a titanium-binding peptide onto one surface of the ring it is also possible to bind it specifically and tightly to a TiO2 surface. TRAP bound in this way is then used to capture gold nanodots and attach them to prepared surfaces. Gold-protein complexes are observed using atomic force microscopy and transmission electron microscopy. The modified TRAP is used to build gold nanodots into the SiO2 layer of a metal oxide semiconductor. This is the first use of a ring protein, rather than the more commonly used spherical protein cages, to constrain nanodots to a surface. This method is an important addition to the current range of bionanotechnology tools and may be the basis for future, multicomponent electronic devices.
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