Proteins are the most abundant type of macromolecules that participate in almost every process in the cell. The sequences of simple amino acid monomers enable the formation of millions of complex protein structures with diverse functions. Field of de novo protein design is based on physical laws and protein sequences unrelated to those in nature. One of the design approaches is based on a modular noncovalent pairing of α-helices in coiled-coil secondary structures. Helices within the coiled-coil motif can be coupled in parallel or antiparallel fashion and the specificity of joining is determined by hydrophobic and electrostatic interactions. Analogous to the complementary pairing of DNA molecules, complex modular protein structures can be assembled with the intramolecular pairing of α-helices into coiled-coils. Orthogonal coiled-coil peptides are connected into a single polypeptide chain that upon pairing folds into polyhedron shaped protein cage. Known types of protein cages include a tetrahedron formed by 12 coiled-coil segments, square pyramid formed by 16 and a triangular prism and triangular bipyramid formed by 18 coiled-coil segments. In this thesis, we describe the preparation and characterization of bipyramidal protein cage for binding of nanobodies. Two edges of designed protein cage are formed by APH peptide segments, that were recognized to bind different nanobodies. The bipyramidal protein cage was successfully isolated and biophysically characterized. Protein is folded in solution and has expected size and shape. Binding analysis showed that protein cage could be targeted by individual nanobodies as well as multiple ones simultaneously, presenting an attractive tool for the development of delivery systems. According to the binding results, complexes of nanobodies with bipyramidal protein cage were prepared for crystallization trials, but unfortunately, we were unable to obtain quality protein crystals.
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