Engineering a light-controlled F1ATPase using structure-based protein design

The F 1 sub-complex of ATP synthase is a biological nanomotor that converts the free energy of ATP hydrolysis into mechanical work with an astonishing efficiency of up to 100% ( Kinosita et al., 2000 ). To probe the principal mechanics of the machine, I re-engineered the active site of E.coli F 1 ATPase with a structure-based protein design approach: by incorporation of a site-specific, photoswitchable crosslinker, whose end-to-end distance can be modulated by illumination with light of two different wavelengths, a dynamic constraint was imposed on the inter-atomic distances of the α and β subunits. Crosslinking reduced the ATP hydrolysis activity of four designs tested in vitro and in one case created a synthetic ATPase whose activity can be reversibly modulated by subsequent illumination with near UV and blue light. The work is a first step into the direction of the long-term goal to design nanoscaled machines based on biological parts that can be precisely controlled by light.