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 novodesigned
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 testedin vitroand function
in cells is not guaranteed. Here, we describe the design, characterization,
and application ofde novohelical 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
inE. 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 bothE. coliand mammalian cells.