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

Naudin, E.A.; Albanese, Katherine I.; Smith, Abigail J.; Mylemans, B.; Baker, Emily G.; Weiner, Orion D.; Andrews, David M.; Tigue, Natalie J.; Savery, Nigel J.; Woolfson, Derek N.

doi:10.1039/d2sc04479j

Cited by 8 publications

(21 citation statements)

References 97 publications

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“…On this basis, and for simplicity, we renamed these apCC-Tet-A and apCC-Tet-B, respectively, and the resulting complex apCC-Tet-A 2 B 2 . This is a new de novo designed CC component, although we have recently reported another example of this design target, which we have named apCC-Tet*-A 2 B 2 . These recently published designs differ from the original apCC-Tet systems in two ways: the heterotetramer, apCC-Tet*-A 2 B 2 , has 3 heptads, which limits design variations; and the residues used for core packing at the a and d sites are different, with the apCC-Tet designs having Leu-Leu cores and the apCC-Tet* designs having Leu-Ile cores.…”

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

“…This is a new de novo designed CC component, although we have recently reported another example of this design target, which we have named apCC-Tet*-A 2 B 2 . 48 These recently published designs differ from the original apCC-Tet systems in two ways: the heterotetramer, apCC-Tet*-A 2 B 2 , has 3 heptads, which limits design variations; and the residues used for core packing at the a and d sites are different, with the apCC-Tet designs having Leu-Leu cores and the apCC-Tet* designs having Leu-Ile cores. Among other things, we anticipate that these will be useful for mimicking or substituting for antiparallel PPIs, which are common in natural systems.…”

Section: Resultsmentioning

confidence: 99%

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Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies

et al. 2023

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De novo peptides and proteins that switch state in response to chemical and physical cues would advance protein design and synthetic biology. Here we report two designed systems that disassemble and reassemble upon site-specific phosphorylation and dephosphorylation, respectively. As starting points, we use hyperthermostable de novo antiparallel and parallel coiled-coil heterotetramers, i.e., A 2 B 2 systems, to afford control in downstream applications. The switches are incorporated by adding protein kinase A phosphorylation sites, R-R-X-S, with the phosphoacceptor serine residues placed to maximize disruption of the coiled-coil interfaces. The unphosphorylated peptides assemble as designed and unfold reversibly when heated. Addition of kinase to the assembled states unfolds them with half-lives of ≤5 min. Phosphorylation is reversed by Lambda Protein Phosphatase resulting in tetramer reassembly. We envisage that the new de novo designed coiled-coil components, the switches, and a mechanistic model for them will be useful in synthetic biology, biomaterials, and biotechnology applications.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies

et al. 2023

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“…Previously, we and others have successfully used de novo designed homo and heterodimeric and tetrameric CCs as PPI modules in E. coli, for instance to control transcription when fused to DNA binding domains. ,,,,, For the new study presented here, we were interested in (1) breaking the CC symmetry of homomeric assemblies to generate specific heteromers, and (2) screening the hydrophobic-core composition to explore a wider range of CC sequences and, potentially, topologies. As a starting scaffold, we aimed to design a helical hairpin that homodimerized to form a 4HB.…”

Section: Resultsmentioning

confidence: 99%

“…Variation of the hydrophobic residues within the core direct natural and designed CCs to assemble into a variety of oligomerization states and helix orientations . Dimers, trimers, and tetramers tend to dominate natural CC structures, but through de novo design higher-order oligomers can be accessed, as well as uniform homodimers or heterodimers in both parallel or antiparallel orientations. , This depth and quality of data and understanding has led to a large number of highly successful CC design programs, , and the development of rationally designed “toolkits” of CC peptides. ,,− The application of these toolkits is beginning to realize the vision of building increasingly complex synthetic biological systems from simpler components. ,,,, …”

Section: Introductionmentioning

confidence: 99%

Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology

et al. 2023

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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.

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

et al. 2023

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In photosynthesis, pigment–protein complexes achieve outstanding photoinduced charge separation efficiencies through a set of strategies in which excited states delocalization over multiple pigments (“excitons”) and charge‐transfer states play key roles. These concepts, and their implementation in bioinspired artificial systems, are attracting increasing attention due to the vast potential that could be tapped by realizing efficient photochemical reactions. In particular, de novo designed proteins provide a diverse structural toolbox that can be used to manipulate the geometric and electronic properties of bound chromophore molecules. However, achieving excitonic and charge‐transfer states requires closely spaced chromophores, a non‐trivial aspect since a strong binding with the protein matrix needs to be maintained. Here, we show how a general‐purpose artificial protein can be optimized via molecular dynamics simulations to improve its binding capacity of a chlorophyll derivative, achieving complexes in which chromophores form two closely spaced and strongly interacting dimers. Based on spectroscopy results and computational modeling, we demonstrate each dimer is excitonically coupled, and propose they display signatures of charge‐transfer state mixing. This work could open new avenues for the rational design of chromophore–protein complexes with advanced functionalities.

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

Abstract: Rules for designing 4-helix bundles are defined, tested, and used to generate de novo peptide assemblies and a single-chain protein.

Cited by 8 publications

References 97 publications

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies

Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology

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

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