Good understandingof rules governing folding of natural proteins is the basis for de novodesign of protein nanostructures. These new structures can be applied in medicine, chemistry and biotechnology, i. e. for targeted drug delivery, vaccine design, tissue engineering,sensor design,biocatalysis and development of bionanomaterials.Only a few smaller domains are known well enough for de novo design. These can serve as modules for the design of polyhedric nanostructures. Protein origami is an example of modular topological nanostructure comprisedfrom coiled coils that represent modules.The polypeptide folds into the desired structure due to each module interacting exclusively with its partner.Coiledcoils arepolypeptide folds comprised of two chains that are characterized by a pattern of sevenamino acid residues called heptads. We designed, expressed and characterized three pairs of coiledcoil forming polypeptides: Tropo-Cn6, Tropo-Dn6, Tropo-Cn12, Tropo-Dn12, Tropo-Cn14 and Tropo-Dn14. These were designed at National Institute of Chemistry in Ljubljana as a part of fifth generation of 15 heptads long tropomyosin-basedcoiledcoilheterodimers. In the fifth generation we chose the most promising pair from the fourth generation of coiledcoils and introduced changes intonine previously unmodified heptads.The size of designed polypeptides was determined by dynamic light scattering (DLS). The measurements matched the predicted size of 15 nm. We also determined the presence of secondary structures by circular dichroism (CD). Against our expectations, the pair Tropo-Cn6/Tropo-Dn6 did not show any secondary structures. Pairs Tropo-Cn12/Tropo-Dn12 and Tropo-Cn14/Tropo-Dn14 produced better results. Measurements of the thermal stability of the secondary structures displayed bigger difference in Tmbetween individual polypeptide and the designed pairwith pair Tropo-Cn12/Tropo-Dn12 than Tropo-Cn14/Tropo-Dn14. We decided that further optimization of tropomyosin-based coiledcoil heterodimerswill thus continue on the basis of the Tropo-Cn12/Tropo-Dn12.