In this thesis, we aimed to investigate the applicability of synthetic protein nanostructures for cryo-electron microscopy. Our research focused on protein bio nanostructures that form stable, precisely designed conformations.
The primary objective was to design and synthesize protein markers that enhance the recognition of target structures. To achieve this, we utilized protein origami, based on coiled-coil helices, which self-assemble into predefined modules. In addition to coiled-coil dimers, we expanded the set of building blocks to include bundles of three and four helices. This allowed us to broaden the range of possible topologies, conformations, and permutations, increase the molecular mass, and improve certain properties of the designed proteins.
In the first part of the study, we focused on the preparation of simple shapes such as triangles and stars with different arrangements of building blocks within the polypeptide chain. Although these proteins were expressed and folded into the intended structures, they were too small and too flexible or heterogeneous for structural determination and application in cryo-electron microscopy.
In the second part, we designed tetrahedral structures, whose stability and distingushed shape could enable better molecular visualization. We employed various characterization methods, and all proteins exhibited a high α-helical content, high thermal stability, and strong refolding capability. Furthermore, we demonstrated that all isolated proteins folded into the designed shapes. This enabled us to determine the structure of the first designed tetrahedral protein cage using cryo-electron microscopy. The study was further extended to include two four helical bundles within a single polypeptide chain and the self-assembly of dimeric structures composed of two identical polypeptide chains. This demonstrated a high degree of flexibility and the potential for the controlled self-assembly of this type of structures.
Our findings show that it is possible to design modular proteins with precisely defined conformations that fold stably into predefined polyhedral structures. The introduction of four helical bundles contributes to both flexibility of the design and stability of the proteins. Based on these results, such proteins could be utilized as delivery systems, for compartmentalization, or for the development of new markers for cryo-electron microscopy.
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