Dataset Information

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies.

ABSTRACT: 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., A₂B₂ 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.

SUBMITTER: Thompson HF

PROVIDER: S-EPMC10127268 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies.

Thompson Harry F HF Beesley Joseph L JL Langlands Hannah D HD Edgell Caitlin L CL Savery Nigel J NJ Woolfson Derek N DN

ACS synthetic biology 20230329 4

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., A2B2 systems, to afford control in downstream applications. T ...[more]

PMID: 36988263

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Project description:Owing to their synthetic accessibility and protein-mimetic features, peptides represent an attractive biomolecular building block for the fabrication of artificial biomimetic materials with emergent properties and functions. Here, we expand the peptide building block design space through unveiling the design, synthesis, and characterization of novel, multivalent peptide macrocycles (96mers), termed coiled coil peptide tiles (CCPTs). CCPTs comprise multiple orthogonal coiled coil peptide domains that are separated by flexible linkers. The constraints, imposed by cyclization, confer CCPTs with the ability to direct programmable, multidirectional interactions between coiled coil-forming "edge" domains of CCPTs and their free peptide binding partners. These fully synthetic constructs are assembled using a convergent synthetic strategy via a combination of native chemical ligation and Sortase A-mediated cyclization. Circular dichroism (CD) studies reveal the increased helical stability associated with cyclization and subsequent coiled coil formation along the CCPT edges. Size-exclusion chromatography (SEC), analytical high-performance liquid chromatography (HPLC), and fluorescence quenching assays provide a comprehensive biophysical characterization of various assembled CCPT complexes and confirm the orthogonal colocalization between coiled coil domains within CCPTs and their designed on-target free peptide partners. Lastly, we employ molecular dynamics (MD) simulations, which provide molecular-level insights into experimental results, as a supporting method for understanding the structural dynamics of CCPTs and their complexes. MD analysis of the simulated CCPT architectures reveals the rigidification and expansion of CCPTs upon complexation, i.e., coiled coil formation with their designed binding partners, and provides insights for guiding the designs of future generations of CCPTs. The addition of CCPTs into the repertoire of coiled coil-based building blocks has the potential for expanding the coiled coil assembly landscape by unlocking new topologies having designable intermolecular interfaces.

Dataset Information

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies.

Publications

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies.

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