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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. 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, 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 in one step 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.
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Interhelical E@ g ‐N@ a interactions modulate coiled coil stability within a de novo set of orthogonal peptide heterodimers
The designability of orthogonal coiled coil (CC) dimers, which draw on well‐established design rules, plays a pivotal role in fueling the development of CCs as synthetically versatile assembly‐directing motifs for the fabrication of bionanomaterials. Here, we aim to expand the synthetic CC toolkit through establishing a “minimalistic” set of orthogonal, de novo CC peptides that comprise 3.5 heptads in length and a single buried Asn to prescribe dimer formation. The designed sequences display excellent partner fidelity, confirmed via circular dichroism (CD) spectroscopy and Ni‐NTA binding assays, and are corroborated in silico using molecular dynamics (MD) simulation. Detailed analysis of the MD conformational data highlights the importance of interhelical E@g‐N@ainteractions in coordinating an extensive 6‐residue hydrogen bonding network that “locks” the interchain Asn‐Asn′ contact in place. The enhanced stability imparted to the Asn‐Asn′ bond elicits an increase in thermal stability of CCs up to ~15°C and accounts for significant differences in stability within the collection of similarly designed orthogonal CC pairs. The presented work underlines the utility of MD simulation as a tool for constructing de novo, orthogonal CCs, and presents an alternative handle for modulating the stability of orthogonal CCs via tuning the number of interhelical E@g‐N@acontacts. Expansion of CC design rules is a key ingredient for guiding the design and assembly of more complex, intricate CC‐based architectures for tackling a variety of challenges within the fields of nanomedicine and bionanotechnology.
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- Award ID(s):
- 2112675
- PAR ID:
- 10527309
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Journal of Peptide Science
- Volume:
- 30
- Issue:
- 2
- ISSN:
- 1075-2617
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/psc.3540
