Compared to other biopolymers, polypeptide nanostructures are able to form highly complex structures that are defined by the aminoacid sequence. Nature explored only a small portion of all protein folds, while huge regions of 'protein space' remain unexplored. There are several approaches to discovering or creating new protein folds – coiled-coil protein origami (CCPO) is one of such. CCPO is de novo rationally designed protein fold from linked coiled-coil (CC) forming modules. CCPO is modular, composed of CCs that form the edges around the internal hydrophilic cavity and can form diverse polyhaedral shapes. In the presented thesis we explored the strategies of presenting protein domains at the vertices of tetrahedral CCPOs by direct genetic fusion. As a proof of principle, four tetrahedral CCPO (TET12SN) constructs were decorated either with one green fluorescent protein (GFP), one green and one red fluorescent protein (RFP) or four GFPs or four RFPs. All constructs expressed well and were characterised by small-angle X-ray scattering (SAXS), which, coupled with in silico modelling, revealed that there was some undesired flexibility of the fourth presented protein domain. The mobility was reduced by modifying the insertion site – and to further explore the developed system, seven additional constructs, with different ratios of GFP : RFP were prepared and analysed with SAXS and analytical gel filtration. Moreover, TET12SN-RRRR, tetrahedral CCPO decorated with four RFPs, was used as a vaccine for antigen presentation. Mice immunised with an antigen, presented on a scaffold (TET12SN-RRRR), developed IgG antibodies against RFP faster and with higher titer compared to mice immunised with a monomeric antigen. As an alternative strategy of protein domain grafting onto the CCPO scaffold, TET12SN was decorated with SpyCatcher/SpyTag protein domains, that were used to create bigger protein complexes of a tetrahedron decorated with (i) one, (ii) two or (iii) three additional ligand tetrahedral CCPO.
Currently, the complexity of polyhaedral CCPOs are limited by the number of available orthogonal CCs. We investigated if the same CC pair could be used several times within the same structure. We successfully designed and characterised a tetrahedral CCPO with one (TET12SN(2CC)), two (TET12SN(22CC)) or three (TET12SN(222CC)) CC pair that occured two times in the sequence. In order to prove that protein domains could still be presented on CCPO with repeated CC pairs, seven protein constructs with varying ratios of GFP : RFP were created utilizing newly designed CCPO TET12SN(22CC). All successfully isolated proteins from the last series were also characterized by an analytical gel filtration and SAXS; moreover, protein construct with four RFPs was characterised in more detail with cryoelectron microscopy. The developed system for presenting protein domains at the vertices of tetrahedral CCPO offers a powerful tool in expanding toolkit of protein origami, that could one day be used in medical, industrial and/or biotechnological applications.
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