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

Abstract

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.

Engineering excitonically coupled dimers in an artificial protein for light harvesting via computational modeling

“…They offer, however, a particular challenge, since all attempts to crystallize BT6 have so far failed, and thus no structural model is available from experiments (Ennist, 2017). Nevertheless, the simplicity and strong foldability of maquette proteins render them good candidates for structure prediction software (Naudin et al, 2022). Indeed, all tested programs (Baek et al, 2021; Du et al, 2021; Jumper et al, 2021) predict four alpha helix bundles.…”

“…As an alternative, the de novo design of artificial cofactor–protein complexes has seen substantial progress in recent years, resulting in bioinspired systems able to act, for instance, as ion channels (Scott et al, 2021) or multi‐electron oxidases/reductases (Kaplan & DeGrado, 2004; Koebke & Pecoraro, 2018; Lombardi et al, 2019). This strategy generally follows a bottom‐up approach, where the complexes are generated from scratch following simple design rules, yet drawing inspiration from natural systems (Chalkley et al, 2022; Naudin et al, 2022). Excitingly, the growing knowledge on protein design could provide a vast array of tools to adjust the energy landscapes as necessary—for instance, to implement the design principles and reach high charge separation efficiencies.…”

Engineering Excitonically-Coupled Dimers in an Artificial Protein for Light Harvesting via Molecular Dynamics Simulations

“…34 Nevertheless, the simplicity and strong foldability of maquette proteins render them good candidates for structure prediction software. 13 Indeed, all tested programs 32,35,36 predict four alpha helix bundles. These, however, only produce structures for apo-proteins; to generate holo forms, we developed a computational protocol (described in detail in the SI Section 2.3) in which we mimic the binding process with steered MD simulations, followed by relaxation to allow the complexes to find a stable conformation.…”

“…[9][10][11] This strategy generally follows a bottom-up approach, where the complexes are generated from scratch following simple design rules, yet drawing inspiration from natural systems. 12,13 Excitingly, the growing knowledge on protein design could provide a vast array of tools to adjust the energy landscapes as necessaryfor instance, to implement the design principles and reach high charge separation efficiencies. However, this will only be possible if the protein binds cofactors with a high packing density, in order for them to display excitonic interactions and low-lying charge-transfer states.…”