The coiled-coil dimer is a widespread protein structural motif and, due to its designability, represents an attractive building block for assembling modular nanostructures. CC are useful as interactive domains that enable to bring other fused protein domains into a proximity. The specificity of coiled-coil (CC) dimer pairing is mainly based on hydrophobic and electrostatic interactions between residues at positions a, d, e, and g of the heptad repeat. Binding affinity, on the other hand, can also be affected by surface residues that face away from the dimerization interface. Here we show how design of the local helical propensity of interacting peptides can be used to tune the stabilities of CC dimers over a wide range. By designing intramolecular charge pairs, regions of high local helical propensity can be engineered to form trigger sequences, and dimer stability is adjusted without changing the peptide length or any of the directly interacting residues. This general principle is demonstrated by a change in thermal stability by more than 30 °C as a result of only two mutations outside the binding interface. The same approach was successfully used to modulate the stabilities in an orthogonal set of coiled-coils without affecting their binding preferences.
COBISS.SI-ID: 6191642
Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures encoded by the linear sequence of their building units. DNA nanotechnology can repurpose DNA for the rational de novo design of complex assemblies unseen in nature. A similar modular strategy can be applied to construct protein origami cages using coiled-coil (CC) dimers as building modules, where the fold is defined by the long-range interactions between pairs of modules arranged in a polypeptide chain in a defined order rather than by the hydrophobic core, as in globular natural proteins. In this study we present a de novo design of second-generation CC protein origami cages, based on a toolbox of supercharged CC dimer building modules and on a computational design platform, which enabled the construction of protein origami cages composed of more than 700 amino-acid residues that folded efficiently in vivo. Solution small-angle X-ray scattering (SAXS), electron microscopy, and biophysical analysis confirmed the folding of tetrahedra, four-sided pyramid, and triangular prism, in agreement with the design; in addition, the stability and folding kinetics of the CC protein origamis were comparable to natural proteins. Second-generation designs were also produced that self-assembled without the need for refolding in bacteria, in mammalian cells, and in mice without causing inflammation or other adverse pathological effects, thus opening a path toward new applications of designed protein cages.
COBISS.SI-ID: 6266906
A technology uses orthogonal pairwise interacting modules has recently been applied to protein design, using orthogonal dimerizing coiled-coil segments as interacting modules. When concatenated into a single polypeptide chain, they self-assemble into the 3D structure defined by the topology of interacting modules within the chain. This approach allows the construction of geometric polypeptide scaffolds, bypassing the folding problem of compact proteins by relying on decoupled pairwise interactions. This approach opens the way towards incorporation of designed foldamers in biological systems and their functionalization.
COBISS.SI-ID: 5889050