45 research outputs found
Recovery of state-specific potential of molecular motor from single-molecule trajectory
We have developed a novel method to evaluate the potential profile of a
molecular motor at each chemical state from only the probe's trajectory and
applied it to a rotary molecular motor F-ATPase. By using this method, we
could also obtain the information regarding the mechanochemical coupling and
energetics. We demonstrate that the position-dependent transition of the
chemical states is the key feature for the highly efficient free-energy
transduction by F-ATPase.Comment: 5 pages, 5 figure
Direct Observation of the Myosin Va Recovery Stroke That Contributes to Unidirectional Stepping along Actin
Myosins are ATP-driven linear molecular motors that work as cellular force
generators, transporters, and force sensors. These functions are driven by
large-scale nucleotide-dependent conformational changes, termed
“strokes”; the “power stroke” is the force-generating
swinging of the myosin light chain–binding “neck” domain
relative to the motor domain “head” while bound to actin; the
“recovery stroke” is the necessary initial motion that primes, or
“cocks,” myosin while detached from actin. Myosin Va is a processive
dimer that steps unidirectionally along actin following a “hand over
hand” mechanism in which the trailing head detaches and steps forward
∼72 nm. Despite large rotational Brownian motion of the detached head about
a free joint adjoining the two necks, unidirectional stepping is achieved, in
part by the power stroke of the attached head that moves the joint forward.
However, the power stroke alone cannot fully account for preferential forward
site binding since the orientation and angle stability of the detached head,
which is determined by the properties of the recovery stroke, dictate actin
binding site accessibility. Here, we directly observe the recovery stroke
dynamics and fluctuations of myosin Va using a novel, transient caged
ATP-controlling system that maintains constant ATP levels through stepwise
UV-pulse sequences of varying intensity. We immobilized the neck of monomeric
myosin Va on a surface and observed real time motions of bead(s) attached
site-specifically to the head. ATP induces a transient swing of the neck to the
post-recovery stroke conformation, where it remains for ∼40 s, until ATP
hydrolysis products are released. Angle distributions indicate that the
post-recovery stroke conformation is stabilized by ≥5
kBT of energy. The high kinetic
and energetic stability of the post-recovery stroke conformation favors
preferential binding of the detached head to a forward site 72 nm away. Thus,
the recovery stroke contributes to unidirectional stepping of myosin Va