Team:Slovenia/Coiled-coil polyhedra Idea Approach.html

Summary

We set to prepare complex polyhedra as well as two-dimensional lattice; both can be constructed from interacting rigid polypeptide rods, assuming the polypeptide being equivalent of DNA origami. This could produce structures that do not exist in nature. Coiled-coil interactions represent the best candidate for this type of assembly as their sequence dictates the selectivity and orientation for interactions with other coiled-coil segments. In the section on modeling we analyzed the potentials of this approach and designed amino acid sequences of their building blocks.
We tested experimentally the feasibility of manufacturing this type of structures as nothing similar had been shown before. We designed, prepared and characterized a polypeptide composed of designed pair of complementary parallel coiled-coil-forming segments and a parallel homodimeric coiled-coil-forming segment in between. This polypeptide could, depending on its concentration, assemble a box or cover the surface with hexagonal packing. We designed a procedure for slow chemical annealing of the assembly and confirmed that the designed polypeptide forms the expected secondary structure. At low concentrations of the polypeptide small assemblies with nanometer dimensions were observed by AFM. Self-assembly based on slow annealing at higher polypeptide concentration moreover produced a two dimensional polypeptide lattice with edges below 10 nm, which was detected by a TEM (Figure 1). Results confirm our design and that it is indeed possible to manufacture this type of polypeptide assemblies. This opens exciting prospects for even more complex assemblies and a whole new range of potential applications.

Figure 1: TEM image of the self-assembled nanostructure made of K2

Idea & Approach

Based on modeling of self-assembly of interacting coiled-coil-forming segments, we selected one of the simplest combinations that could theoretically form a box as well as cover the plane in a polyhedral lattice. This combination comprised two parallel coiled-coil heterodimers (designed P1 and P2) and one parallel homodimer (Gcn4-p1(I-L)). Between each coiled-coil-forming domain we introduced a dipeptide linker Gly-Ser, to allow the limited flexibility of coiled-coil segments. Additionally the polypeptide construct (Figure 1) included a hexahistidine peptide tag, to facilitate purification, detection and attachment site of additional functions, such as metal or fluorophore binding, to the assembled product. We designed artificial heterodimeric parallel coiled-coil-forming segments (P1 and P2), as described in the modeling section. For parallel homodimeric segment we used Gcn4-p1(I-L) domain described in the literature (Harbury et al., 1993). P1 and P2 were designed to form a stable parallel coiled-coil heterodimer, which strongly prefers this combination and at the same time destabilizes formation of parallel coiled-coil homodimers, antiparallel heterodimers or pairs with central GCN domain. The GCN parallel homodimer coiled-coil was selected for the similar properties. The proposed combination of three coiled-coil segments should enable the self-assembly of a box or a hexagonal network as shown on Figure 2.

Figure 1: Scheme of the K2 polypeptide construct. Gene coding for the described polypeptide was cloned into the vector according to the standard BBC RFC37. Polypeptide consists of a histidine peptide tag, P1, a designed parallel heterodimeric coiled-coil-forming segment, dipeptide linker SG, a parallel homodimeric coiled-coil-forming segment GCN, another dipeptide linker SG and P2, a designed parallel heterodimeric coiled-coil-forming segment that can pair with P1 in a parallel orientation.

Figure 2: Scheme of the self-assembly of a single type of a polypeptide chain comprising three coiled-coil-forming segments (A) where two of them form parallel heterodimers (a-a’) and one forms a parallel homodimer (b). Two chains can form dimers (B, C), which can further assemble into a box (D) or into a hexagonal lattice (E). Arrows denote the orientation of the interacting coiled-coil-forming segments in the assembly. a and a’ denote complementary segments of parallel heterodimer and b denotes the parallel homodimeric segment.