De Novo Designed Protein-Interaction Modules for In-Cell Applications

Caitlin L Edgell, Abigail J Smith, Joseph L Beesley, Nigel J Savery, Derek N Woolfson

Research output: Contribution to journalArticle (Academic Journal)peer-review

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Abstract

Protein–protein interactions control a wide variety of natural biological processes. α-Helical coiled coils frequently mediate such protein–protein interactions. Due to the relative simplicity of their sequences and structures and the ease with which properties such as strength and specificity of interaction can be controlled, coiled coils can be designed de novo to deliver a variety of non-natural protein–protein interaction domains. Herein, several de novo designed coiled coils are tested for their ability to mediate protein–protein interactions in Escherichia coli cells. The set includes a parallel homodimer, a parallel homotetramer, an antiparallel homotetramer, and a newly designed heterotetramer, all of which have been characterized in vitro by biophysical and structural methods. Using a transcription repression assay based on reconstituting the Lac repressor, we find that the modules behave as designed in the cellular environment. Each design imparts a different property to the resulting Lac repressor-coiled coil complexes, resulting in the benefit of being able to reconfigure the system in multiple ways. Modification of the system also allows the interactions to be controlled: assembly can be tuned by controlling the expression of the constituent components, and complexes can be disrupted through helix sequestration. The small and straightforward de novo designed components that we deliver are highly versatile and have considerable potential as protein–protein interaction domains in synthetic biology where proteins must be assembled in highly specific ways. The relative simplicity of the designs makes them amenable to future modifications to introduce finer control over their assembly and to adapt them for different contexts.
Original languageEnglish
Article number2
Pages (from-to)427-436
Number of pages10
JournalACS Synthetic Biology
Volume9
DOIs
Publication statusPublished - 6 Feb 2020

Structured keywords

  • BrisSynBio
  • Bristol BioDesign Institute

Keywords

  • de novo protein design
  • coiled coil
  • transcriptional control
  • Lac repressor
  • artificial transcription factor

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