IN VIVO SELF-ASSEMBLING COILED-COIL-BASED PROTEIN ORIGAMILapenta, Fabio (Avtor) Jerala, Roman (Mentor) Protein de novo designcoiled coilprotein cagesProteins are complex biopolymers that fold in a large array of nanostructures. Although resolved natural protein structures span over a large conformational landscape, a much wider sampling space, formed by polypeptides that nature has not sampled, is yet to be explored. The ability of designing novel protein structures from scratch, de novo protein design, offers a way to sample the unknown part of protein sequence-structure landscape and test new protein architectures. This thesis encompasses some of the recent advances concerning a modular approach to protein de novo design, coiled coil protein origami (CCPO) design, a strategy based on specific arrangements of coiled coil units within a polypeptide chain, which fold into polyhedral cages. Solubility and overall expression level of these cages were increased, which allowed the implementation of a purification protocol that did not require refolding passages and in turn permitted large-scale isolations and biophysical characterization. Variants of a soluble 12 coiled coil segment tetrahedral cage served as starting scaffold for initial characterization and for refining the design principles. Consequently, larger coiled coil cages such as a 16-segment coiled coil square-pyramid and an 18-segment trigonal prism were isolated, characterized and their conformation was analysed via Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM) analysis. Ultimately, choosing to work on an 18-segment trigonal bipyramidal cage, we designed such fold both as a single chain polypeptide and as heterodimeric complex. Different strategies for oligomeric assembly were tested and a correctly folded heterodimeric bipyramidal complex was obtained upon self-assembly of different heterodimers. In addition, we devised a proteolysis-triggered conformational rearrangement of two subunits into a heterodimeric bipyramid. SAXS analysis confirmed the CCPO cages folding in accordance with our design. Overall, these implementations represented a leap forward in the increasingly expanding field of protein design, demonstrating the potential of a modular coiled coil based design.20192020-01-03 15:50:19Doktorsko delo/naloga113428sl