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Design of Tunable Protein Interfaces Controlled by Post-Translational Modifications
Daniel L Winter1,2, Hasti Iranmanesh1, Douglas S Clark3,4
1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
ACS Synthetic Biology
|July 24, 2020
Summary
Synthetic biology designs protein interactions using post-translational modifications (PTMs). Researchers used serine phosphorylation to control peptide interactions, demonstrating a new method for engineerable protein complex assembly.
Area of Science:
- Synthetic biology
- Protein engineering
- Biochemistry
Background:
- Protein interaction interfaces are key in synthetic biology for assembling molecular complexes and nanostructures.
- Post-translational modifications (PTMs) naturally regulate protein interactions but are underutilized in synthetic protein design.
- Reversible PTMs offer a potential mechanism for dynamic control over engineered protein associations.
Purpose of the Study:
- To investigate the use of serine phosphorylation, a reversible PTM, to modulate peptide interactions.
- To design and characterize peptide pairs with engineered PKA recognition motifs for phosphorylation-dependent modulation.
- To expand the capabilities of synthetic biology by incorporating enzyme-mediated control over protein self-assembly.
Main Methods:
- Designed peptide pairs, including heterodimeric coiled coils, with protein kinase A (PKA) recognition motifs.
- Utilized mass spectrometry to confirm peptides as PKA phosphorylation substrates and lambda protein phosphatase for dephosphorylation.
- Measured binding kinetics (association and dissociation rates) before and after phosphorylation to assess interaction modulation.
Main Results:
- All designed peptides were efficiently phosphorylated by PKA and dephosphorylated by lambda protein phosphatase.
- Phosphorylation significantly altered the binding kinetics of peptide pairs, affecting both association and dissociation rates.
- Observed modulation ranged from interaction strengthening (up to 11-fold decrease in Kd) to weakening (up to 180-fold increase in Kd).
Conclusions:
- Serine phosphorylation can be effectively employed to dynamically control engineered peptide interactions.
- This PTM-modulated interface design provides a novel strategy for controlling protein association in biological systems.
- Enzyme-mediated control over protein complex assembly expands the paradigm of self-assembly in synthetic biology.
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