Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies

Harry F Thompson; Joe L Beesley; Hannah D Langlands; Caitlin L Edgell; Nigel J Savery; Dek N Woolfson

doi:10.1021/acssynbio.3c00064

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies

Harry F Thompson, Joe L Beesley, Hannah D Langlands, Caitlin L Edgell, Nigel J Savery^*, Dek N Woolfson^*

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

2 Citations (Scopus)

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.

Original language	English
Pages (from-to)	1308–1319
Number of pages	12
Journal	ACS Synthetic Biology
Volume	12
Issue number	4
DOIs	https://doi.org/10.1021/acssynbio.3c00064
Publication status	Published - 29 Mar 2023

Bibliographical note

Funding Information:
HFT, HDL and CLE were supported by the EPSRC/BBSRC-funded Centre for Doctoral Training in Synthetic Biology (EP/L016494/1). JLB was supported by a European Research Council Advanced Grant awarded to DNW (340764). CLE was supported by BBSRC grant awarded to NJS and DNW (BB/S002820/1). DNW held a Royal Society Wolfson Research Merit Award (WM140008). We thank BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (BB/L01386X/1), for access to facilities, and the University of Bristol School of Chemistry Mass Spectrometry Facility for use of the EPSRC-funded Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1). We thank Drs. Will Dawson, Elise Naudin, and Fabio Pirro for discussions and critical reading of the manuscript.

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

Research Groups and Themes

Bristol BioDesign Institute
BrisSynBio

Keywords

synthetic biology

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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.",

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note = "Funding Information: HFT, HDL and CLE were supported by the EPSRC/BBSRC-funded Centre for Doctoral Training in Synthetic Biology (EP/L016494/1). JLB was supported by a European Research Council Advanced Grant awarded to DNW (340764). CLE was supported by BBSRC grant awarded to NJS and DNW (BB/S002820/1). DNW held a Royal Society Wolfson Research Merit Award (WM140008). We thank BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (BB/L01386X/1), for access to facilities, and the University of Bristol School of Chemistry Mass Spectrometry Facility for use of the EPSRC-funded Bruker Ultraflex MALDI-TOF/TOF instrument (EP/K03927X/1). We thank Drs. Will Dawson, Elise Naudin, and Fabio Pirro for discussions and critical reading of the manuscript. Publisher Copyright: {\textcopyright} 2023 The Authors. Published by American Chemical Society.",

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AU - Savery, Nigel J

AU - Woolfson, Dek N

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N2 - 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.

AB - 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.

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JO - ACS Synthetic Biology

JF - ACS Synthetic Biology

IS - 4

ER -

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies

Abstract

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