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Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix-turn-helix-turn-helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy., (© 2024. The Author(s).)

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.