PT  - JOURNAL ARTICLE
AU  - Elise A. Naudin
AU  - Katherine I. Albanese
AU  - Abigail J. Smith
AU  - Bram Mylemans
AU  - Emily G. Baker
AU  - Orion D. Weiner
AU  - David M. Andrews
AU  - Natalie Tigue
AU  - Nigel J. Savery
AU  - Derek N. Woolfson
TI  - From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles
AID  - 10.1101/2022.08.04.502660
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.08.04.502660
4099  - http://biorxiv.org/content/early/2022/08/05/2022.08.04.502660.short
4100  - http://biorxiv.org/content/early/2022/08/05/2022.08.04.502660.full
AB  - The design of completely synthetic proteins from first principles—de novo protein design—is challenging. This is because, despite recent advances in computational protein-structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles, which, in turn, could be used in chemical and synthetic biology to direct protein-protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg—i.e., the sequence signature of many helical bundles—the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive a single-chain 4-helix-bundle protein for recombinant production in E. coli. All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design.Competing Interest StatementThe authors have declared no competing interest.