Enzyme-free Phosphorylation with Kinetic Gating in a De Novo Coiled-coil system

Phosphorylation is among the most ubiquitous and essential post-translational modifications in biological systems. It is regulated by highly complex enzymatic networks. Here, we explore enzyme-free phosphorylation in a designed peptide system. Specifically, we use phosphorylation to modulate coiled-coil (CC) assembly and dynamics. This exploits a non-biological reaction cycle to phosphorylate and dephosphorylate a histidine residue in an a-helix, enabling or disabling CC formation with a second helix. Dephosphorylation is kinetically gated-it is 25x faster in the CC compared to the non-assembled state. As a result, the cycle of phosphorylation, CC formation, dephosphorylation, and CC disassembly is ratcheted. The minimal synthetic phosphorylation cycle provides design principles and fulfills requirements to perform work mimicking the function of molecular motors, walkers, and pumps.

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