Clocked stepping of an artificial protein walker along a DNA track.

Molecular motors are fundamental to life because they transduce free energy into mechanical work, a capability rooted in the chemical and structural complexity of their constituent proteins. Although motors based on small molecules and DNA have been developed, the creation of an artificial protein motor has remained an elusive goal in synthetic biology. Here we report the realization of an artificial, externally controlled protein motor, termed Tumbleweed (TW). TW was engineered using a modular design strategy that combines proteins with well-characterized properties to produce emergent motor function and directionality. TW comprises three 'legs', each containing a ligand-gated DNA-binding domain that enables selective interaction with specific sites along a DNA track. Using single-molecule fluorescence assays in conjunction with a programmable microfluidic device, we show that TW takes directional 16 nm steps along a designed DNA substrate in response to a defined sequence of ligand inputs. Moreover, both the timing and direction of stepping can be precisely controlled on a timescale of seconds. This approach provides a versatile platform for engineering dynamic and sophisticated protein-based nanomachines, as well as for probing the physical principles governing protein walkers with precisely defined architectures.