5 research outputs found
Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies
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