Design and selection of heterodimerizing helical hairpins for synthetic biology

DOI: 10.1021/acssynbio.3c00231

Authors: Abigail J. Smith (University of Bristol) , Elise A. Naudin (University of Bristol) , Caitlin L. Edgell (University of Bristol) , Emily G. Baker (University of Bristol) , Bram Mylemans (University of Bristol) , Laura Fitzpatrick (AstraZeneca) , Andrew Herman (University of Bristol) , Helen M. Rice (University of Bristol) , David M. Andrews (AstraZeneca) , Natalie Tigue (AstraZeneca) , Derek N. Woolfson (University of Bristol) , Nigel J. Savery (University of Bristol)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Acs Synthetic Biology

State: Published (Approved)
Published: May 2023
Diamond Proposal Number(s): 23269

Abstract: 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.

Journal Keywords: coiled coil; in-cell library screening; protein−protein interactions; rational peptide design; synthetic biology

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-Macromolecular Crystallography

Added On: 28/05/2023 09:32

Documents:
acssynbio.3c00231.pdf

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)