Protein−protein interactions control a wide variety of natural biological processes. α-Helical coiled coils frequently mediate such protein− protein interactions. Due to the relative simplicity of their sequences and structures and the ease with which properties such as strength and specificity of interaction can be controlled, coiled coils can be designed de novo to deliver a variety of non-natural protein−protein interaction domains. Herein, several de novo designed coiled coils are tested for their ability to mediate protein−protein interactions in Escherichia coli cells. The set includes a parallel homodimer, a parallel homotetramer, an antiparallel homotetramer, and a newly designed heterotetramer, all of which have been characterized in vitro by biophysical and structural methods. Using a transcription repression assay based on reconstituting the Lac repressor, we find that the modules behave as designed in the cellular environment. Each design imparts a different property to the resulting Lac repressor-coiled coil complexes, resulting in the benefit of being able to reconfigure the system in multiple ways. Modification of the system also allows the interactions to be controlled: assembly can be tuned by controlling the expression of the constituent components, and complexes can be disrupted through helix sequestration. The small and straightforward de novo designed components that we deliver are highly versatile and have considerable potential as protein−protein interaction domains in synthetic biology where proteins must be assembled in highly specific ways. The relative simplicity of the designs makes them amenable to future modifications to introduce finer control over their assembly and to adapt them for different contexts.

De novo peptides and proteins that switch state in response to chemical and physical cues would advance protein design and synthetic biology. Here we report two designed systems that disassemble and reassemble upon site-specific phosphorylation and dephosphorylation, respectively. As starting points, we use hyperthermostable de novo antiparallel and parallel coiled-coil heterotetramers, i.e., A 2 B 2 systems, to afford control in downstream applications. The switches are incorporated by adding protein kinase A phosphorylation sites, R-R-X-S, with the phosphoacceptor serine residues placed to maximize disruption of the coiled-coil interfaces. The unphosphorylated peptides assemble as designed and unfold reversibly when heated. Addition of kinase to the assembled states unfolds them with half-lives of ≤5 min. Phosphorylation is reversed by Lambda Protein Phosphatase resulting in tetramer reassembly. We envisage that the new de novo designed coiled-coil components, the switches, and a mechanistic model for them will be useful in synthetic biology, biomaterials, and biotechnology applications.

De novo designed protein domains are increasingly being applied in biotechnology, cell biology and synthetic biology. Therefore, it is imperative that these proteins are robust to superficial changes, i.e., small changes to their amino-acid sequences should not cause gross structural changes. In turn, this allows properties such as stability and solubility to be tuned without affecting structural attributes like tertiary fold and quaternary interactions. Reliably designed proteins with predictable behaviors may then be used as scaffolds to incorporate function, e.g., through the introduction of features for small-molecule, metal or macromolecular binding, and enzyme-like active sites. Generally, achieving this requires the starting protein fold to be well understood. Herein, we focus on designing α-helical coiled coils, which are well studied, widespread and often direct protein-protein interactions in natural systems. Our initial investigations reveal that a previously designed parallel, homotetrameric coiled coil, CC-Tet, is not robust to sequence changes that were anticipated to maintain its structure. Instead, the alterations switch the oligomeric state from tetramer to trimer. To improve the robustness of designed homotetramers, additional sequences based on CC-Tet were produced and characterized in solution and by X-ray crystallography. Of these updated sequences, one is robust to truncation and to changes in surface electrostatics; we call this CC-Tet*. Variants of the general CC-Tet* design provide a set of homotetrameric coiled coils with unfolding temperatures in the range 40 ˚C to >95 ˚C. We anticipate that these will be of use in applications requiring robust and well defined tetramerization domains.

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Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide−protein or protein−protein interactions, de novo designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs. Typically, however, such designs are tested in vitro and function in cells is not guaranteed. Here, we describe the design, characterization, and application of de novo helical hairpins that heterodimerize to form 4-helix bundles in cells. Starting from a rationally designed homodimer, we construct a library of helical hairpins and identify complementary pairs using bimolecular fluorescence complementation in E. coli. We characterize some of the pairs using biophysics and X-ray crystallography to confirm heterodimeric 4-helix bundles. Finally, we demonstrate the function of an exemplar pair in regulating transcription in both E. coli and mammalian cells.