In nature, we encounter a multitude of intricate biological structures and materials capable of self-assembly, serving as an inspiration for the development of novel man-made nanomaterials with diverse applications. Synthetic biology offers a promising avenue to engineer molecular interactions at the nanometer scale, and one such approach involves the de novo design of protein building blocks based on the sequence of α-helical peptide segments. These segments, forming orthogonal pairs that create coiled-coil helices (CC), facilitate the self-assembly of the polypeptide chain. Our research group has successfully showcased the design of stable cages with various geometries (tetrahedron, pyramid, bipyramid) from a single polypeptide chain, where orthogonal pairs of peptide segments represent the edges. We aim to design larger and more intricate structures, relying on CC interactions for various applications like molecular encapsulation. One strategy to achieve this goal involves connecting multiple chains which presents many challenges and addressing them is the primary focus of this Ph.D.
During the doctoral work, we expanded the utilization of peptide segments forming CC to design and prepare nanostructures composed of multiple polypeptide chains. The envisioned multi-chain nanostructures included discrete cages of regular (tetrahedron, trigonal bipyramid) and irregular shapes (concave octahedron), as well as larger structures with periodic repetition features (protein nanofibers and protein lattices). Our attempts to guide the assembly of nanostructures towards the intended types involved the preorganization of protein components using various strategies, such as chain cyclization with intein splicing, introduction of flexible or rigid linkers, and incorporation of trimerization domains and intra-chain CC connections.
The first of the three concepts developed during the doctoral research focused on designing lattices assembled from multiple subunits connected via CC-forming segments. The planar orientation of these connecting segments was intended to be dictated by trimerization domains on the subunits. However, the utilized foldon and collagen trimerization domains did not provide sufficient rigidity and planar orientation, leading to the assembly of discrete 6-mer structures instead of the intended periodically repeating networks. Nevertheless, we have structurally analyzed these novel multimeric structures using small-angle X-ray scattering (SAXS).
The second concept involved two subunits with complementary CC-forming segments designed to self-assemble into nanofibers. We aimed to achieve periodic stacking of the subunits to form two connected parallel chains. The strategy also involved additional stabilization of these threads through the formation of covalent bonds between terminal ends of the individual units, which we sought to achieve using split inteins capable of protein splicing. While this concept did not succeed in assembling longer threads, we explored various orthogonal pairs of split inteins and optimized conditions for successful cis splicing, which we applied in designs involving cyclized subunits.
The third concept envisioned designing multi-chain protein structures based on cyclized peptide chains. Polypeptides with three or six CC-forming segments were cyclized in bacterial cells using split inteins, initiating cyclization through splicing of the N- and C-terminal ends of the subunit. This approach resulted in subunits preorganized into stable forms of triangles or bi-trigons, featuring defined internal angles between segments, providing greater predictability for designing multi-chain structures. Attempts to assemble various structures from cyclized triangular subunits proved unsuccessful. Still, cyclized rhombs, containing one intra-chain CC and four free segments for CC formation, emerged as successful subunits for constructing multi-chain nanostructures. We demonstrated that cyclized rhombs are crucial for the successful assembly of structures such as a two-chain tetrahedron and a three-chain concave octahedron.
In summary, our doctoral work successfully extended the use of peptide segments forming CC to design nanostructures composed of multiple polypeptide chains. By utilizing trimerization domains, we prepared discrete multi-chain structures, while attempts to design periodically repeating lattices using foldon and collagen trimerization domains proved unsuccessful. Our exploration of nanofiber self-assembly with short linear peptides and intein stabilization primarily served to optimize intein reactions, which we applied to the design of multi-chain structures from cyclized subunits. Using split inteins we cyclized peptide chains to preorganize them into triangles or bitrigonal shapes. The latter proved to be suitable subunits for successfully assembling multi-chain structures such as a two-chain tetrahedron and a three-chain concave octahedron. This research contributes valuable insights into the intricate world of de novo-designed protein structures and their potential applications in nanotechnology.
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