From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles.

The design of completely synthetic proteins from first principles- 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 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 4-helix bundles. In turn, this 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, -, the sequence signature of many helical bundles-the key features that we identify are: = Leu, = Ile, = Ala, = 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 in one step a single-chain 4-helix-bundle protein for recombinant production in . 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.