Single kinesin molecules studied with a molecular force clamp

Abstract

Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

Kinesin motors as molecular machines

“…(29) ATP hydrolyzed. (33,34) Thermal fluctuations are expected to play an important role in the motor mechanism, either by driving diffusional movements of the motor to its next binding position (35) or by promoting diffusive structural changes that drive force-producing conformational changes. (30) The structural elements that undergo strain have not been identified with certainty for any molecular motor, but are likely to have spring-like or elastic properties that allow them to extend or rotate, then recoil back into their original conformation.…”

On the use of in vivo cargo velocity as a biophysical marker

“…A more specialized version of the model further proposes that run lengths are determined by random detachment of engaged motors, promoted by this load. As mentioned above, in vitro molecular motor studies have shown that motor velocity depends on load [19][20][21] such that motors move faster when under less load. Further, there is evidence that when multiple motors work together under negligible opposing load, the catalytic rate of each individual motor is not altered [26].…”

“…biochemical regulation, are of secondary importance. This proposal stems from in vitro observations [19][20][21] showing that load on a motor decreases its processivity and velocity. Thus, if motors share the load opposing cargo motion, then removal of a motor from the engaged motor pool will increase load on a per-motor basis, and thus decrease cargo velocity.…”

Enhancement of cargo processivity by cooperating molecular motors

“…Single molecule imaging methods have been pivotal in the experimental study of molecular motor dynamics 8,9 . Such advanced techniques have allowed researchers to dissect various aspects of molecular motor mediated transport 10,11,12,13,14,15 .…”