Abstract
F1-ATPase is a rotary motor protein in which the central γ-subunit rotates inside the cylinder made of α3β3 subunits. To investigate interactions between the γ shaft and the cylinder at the molecular scale, load was imposed on γ through a polystyrene bead by three-dimensional optical trapping in the direction along which the shaft penetrates the cylinder. Pull-out event was observed under high-load, and thus load-dependency of lifetime of the interaction was estimated. Notably, accumulated counts of lifetime were comprised of fast and slow components. Both components exponentially dropped with imposed loads, suggesting that the binding energy is compensated by the work done by optical trapping. Because the mutant, in which the half of the shaft was deleted, showed only one fast component in the bond lifetime, the slow component is likely due to the native interaction mode held by multiple interfaces.
Highlights
To dissect how a protein operates as an independent molecular device, the quaternary structure is crucial to realize its unique function
Α3β3γ subcomplex, which we refer hereafter as F1-ATPase, is the world’s smallest rotary motor ever found: the γ shaft rotates against the α3β3 cylinder in a counterclockwise manner when viewed from the protruded side of γ driven by the chemical energy of hydrolysis of ATP
The experimental setup allows to impose a constant load to a single protein that is immobilized on the glass surface
Summary
To dissect how a protein operates as an independent molecular device, the quaternary structure is crucial to realize its unique function. One good example is a molecular motor, myosin: the single ‘subfragment-one’ domain is enough to drive the motility[1] and the unitary step with ~5 nanometer (nm)[2], but the function is further enhanced to move without dissociation with larger steps of 36 nm by taking a dimer form as a more complex quaternary structure[3,4]. Another extreme is a rotary molecular motor: engines that are symmetrically arranged induce one-directional rotation of the other component[5]. It was experimentally proven that the hydrolysis reactions in the three catalytic sites occur not www.nature.com/scientificreports/
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