Crystal structure of dimeric
(Adapted from Kozielski et al., Cell 1997)
Kinesin is one of the world's tiniest motors.
Kinesin molecules use the energy of ATP hydrolysis to
move in discrete steps along protein filaments called microtubules in
cells. Each molecule of kinesin consists of two catalytically active
motor domains, called "heads" that bind both a microtubule and ATP. The
two heads are joined by short polypeptide chain, called the "neck
linker" to a long coiled-coil stalk that terminates in a distal cargo
A single (dimeric) protein molecule -- consisting of
only ~800 amino acids, or roughly ten thousand atoms -- is sufficient
to move long distances along a microtubule, even against considerable
load. By studying the motion of single molecules of kinesin, our aim is
to understand how this tiny machine works. Historically, most
measurements of kinesins have focused on conventional kinesin
(Kinesin-1 or, more commonly, "kinesin"), the founding member of a
diverse family of related motor proteins. Kinesin proteins are
essential for vesicle transport in neurons, and chromosome movement in
dividing cells. By learning how different kinesin proteins work, we
take an important step toward comprehending the dynamics of living
We use optical traps to probe the stepping of single
molecules of kinesin.
In order to track kinesin motion, we attach the
molecules to microscopic beads. Kinesin itself is much too small to see
in the optical microscope, so the beads serve as markers that can be
tracked with very high precision (to 1 nm or better). The beads also
act as "handles", through which we can apply force using an optical
trap. Applying tension reduces Brownian motion of the bead, and allows
us to observe that kinesin moves in a stepwise fashion, in many
sequential increments (thus, kinesin is a processive enzyme).
The step size is 8-nm, and corresponds to the spacing of the tubulin
dimers that make up the microtubule. For each 8-nm step, kinesin uses a
single fuel molecule, hydrolyzing one ATP molecule into ADP and
inorganic phosphate. By observing an individual step of kinesin, we
observe a single enzymatic turnover of ATP hydrolysis.
Cartoon of a kinesin experiment.
The kinesin walks along the microtubule towards its plus-end, while
being subjected to a retarding force by the optical trap.
Kinesin steps under constant
forward load. The position of a kinesin motor in nanometers versus
time at low ATP concentration. In this graph, the 8-nm steps that
kinesin takes along the microtubule can readily be seen.
Lang et al., Biophysical Journal, 2002 ).
|We can apply controlled forces to
kinesin with the optical trap.
Our optical traps have become very
sophisticated, with automatic feedback control that allows us to apply
forces of constant magnitude and direction (any direction in the plane
of the coverslip) to the kinesin motors while they move. Studying the
effect of load on the speed and regularity of kinesin's stepping motion
provides clues about how motion is coupled to the biochemical events of
For example, our recent data suggests that at least 3 nm
of each 8-nm step occurs all at once, during a single biochemical
transition (Block et al.,
PNAS, 2003). This "working stroke" must be well-aligned with the
microtubule. The remaining 5 nm may occur simultaneously, or may occur
after a slight (< 1 ms) delay. We also find evidence for smaller
(< 1 nm) side-to-side motions occurring at other times in the
Read more about this work in:
trap provides new insights into motor molecules -- nature's
ultimate nanomachines" in the Stanford Report.
|MOVIE: Kinesin-driven bead movement along a microtubule |
(Credit: J. Shaevitz)
[FreeMover.avi 741 KB]
In this clip, a micron-scale kinesin-coated bead moves along a microtubule.
MOVIE: Kinesin movement against an optical trap
(Credit: J. Shaevitz)
[TrappedBead.avi 1311 KB]
In this clip, a freely diffusing kinesin-coated bead is grabbed by the optical trap and positioned above a
microtubule. As the bead is moved away from the center of the trap (indicated by the crosshairs),
the force on the kinesin increases.
Eventually the motor releases microtubule and the bead returns to the trap center. Multiple such "runs" can be observed.
Overview of Current Research Areas:
- How exactly do kinesin's two heads move during stepping?
Although the center of mass position of a bead-bound kinesin motor can be measured with exquisite precision
using an optical trap, the relative position of each motor head is more difficult to determine.
Two broad classes of models have been invoked to explain the movement of kinesin's heads during a step.
In hand-over-hand models, the heads exchange leading and trailing roles with every step. By contrast, in inchworm
models, one head always leads. By measuring the stepwise motion of individual enzymes,
we find that some kinesin molecules exhibit a striking alternation in the
dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping
implies that kinesin molecules strictly alternate between two different conformations as they step,
indicative of a hand-over-hand mechanism. We are currently investigating the structural origins of limping, and
further exploring the details of kinesin's walk along a microtubule.
- How do kinesin's heads remain
synchronized with each other during processive stepping?
Kinesin is more than the sum
of its parts
When one head of kinesin is removed by using biochemical techniques,
the remaining head can still bind and release microtubules and
hydrolyze ATP. Each head can
therefore function as an independent enzyme. However, arbitrarily
gluing the two individual heads back together would not form a
processive motor. Doing that would create a
molecule where each head randomly cycled through states with different
conformations and affinities for the microtubule track independently of
what the other head was doing. It wouldn't take long
before both heads happened to let go of the microtubule at the same
time. Whenever both heads let go, kinesin diffuses away into the
surrounding solution, ending its processive run. Since kinesin is
known to take ~100 steps on average before detaching from the microtubule,
there must be some mechanism to allow the heads to communicate to each
other the status of their enzymatic cycling and thereby make sure at
least one is holding on to the microtubule at all times.
Studies with nucleotide
Evidence of head-head communication is therefore an important goal of
our research on kinesin. We've approached the problem by watching
how kinesin steps in the presence of nucleotide analogs, such as AMP-PNP
and BeFx. Nucleotide analogs are small molecules like
ATP that the kinesin motor normally uses as its fuel. Unlike ATP,
analogs bind to kinesin's rear head and pause
its motion since they cannot be used as fuel. Such pauses are
evident in records of kinesin stepping in the figure below.
Furthermore, the pauses are interspersed by short-lived backsteps, shown in red and
orange. To our surprise, the pauses only ended immediately after
the motor took one of these backsteps. Since the pauses were
induced by binding of nucleotide analogs, we concluded that the analogs
can only unbind
from kinesin when it takes a backstep and the analog is bound to the
front head. This result means that there must be some way the heads
communicate with each other to determine which is in front and which is in back.
A likely mechanism is the internal strain in the protein thought to build up
whenever both heads are attached to the microtubule.
records in the presence of 2 mM ATP and either 1 mM BeFx
(traces 1, 2, and 4) or 1 mM AMP-PNP (trace 3). Each record shows
stepping (black) interrupted by pauses (blue). Each pause is
punctuated by backsteps (red and orange). The pauses end only
after the motor takes a backstep and releases the bound nucleotide
To learn more about this project, see Guydosh et al.
- Besides conventional kinesin, what are
the biophysical properties of other kinesin-family proteins?
(Top) A kinesin dimer binds vesicular cargo and
moves along a single microtubule;
by contrast tetramers of Eg5 likely crosslink microtubules during cell
division and generate sliding forces that help move the two
duplicated daughter cells apart. (Bottom) Force vs. velocity curves for
kinesin and Eg5. Eg5 walks more slowly, and is less
sensitive to force than kinesin.
|The mitotic motor protein, Eg5
Although much is now known of the stepping mechanism of conventional
kinesin, very little is known of the biophysical and biochemical
properties of the other members of the diverse family of
kinesin-related enzymes. One important subset of this large family of
kinesin-related molecular motors generates the forces that drive cell
division (or mitosis). Eg5 is one such mitotic kinesin. In its native
state, Eg5 consists of four catalytically-active motor heads, each one
of which can bind microtubules and use the energy of ATP hydrolysis to
To better understand how Eg5 functions in cells, we
performed the first single-molecule experiments of Eg5 and demonstrated
that individual dimers are mechanically processive, even under
significant load. Like kinesin, Eg5 moves in 8-nm steps toward the
plus-end of the microtubules, but moves ~10-fold slower, and takes
~10-fold fewer steps, on average, before detaching from the
Additionally, by measuring Eg5's velocity as a function of ATP
concentration and applied force, we found that Eg5 is significantly
less sensitive to force. We suspect these differences have evolved to
satisfy the distinct physiological demands of cell division and cargo
transport. Additional measurements of Eg5 detachment kinetics and
force-dependencies are underway.
To learn more about this work, see
Valentine et al. Nature Cell Biology, 2006, and Valentine
et al. Cell Division, 2006.
- Recent Technical Advances:
Polymer-based surface treatments that reduce
nonspecific surface interactions in single-molecule assays
Optical trapping assays have stringent requirements for the controlled
attachment of microtubules and motors to glass coverslips and polymeric
colloidal spheres. Many single-molecule assays have traditionally
relied upon fortuitous nonspecific interactions between motors,
filaments, and surfaces, but extension to new classes of kinesin family
motors has proven difficult, limiting the application of optical
trapping techniques to new motor proteins. We have developed protocols
using a variety of novel polymer-based surface chemistries that allow
for robust, stereospecific attachment of motors and filaments while
inhibiting undesirable nonspecific interactions. For more detail, see
Fordyce et al., (2007) "Single-Molecule Techniques: A Laboratory
Manual" in Cold Spring Harbor Laboratory Press .
Combined optical tweezers and single-molecule
New tools will be required to determine exactly which of kinesin's
biochemical steps correspond to physical motions. Toward this end, we
are developing a combined instrument for optical trapping and single
molecule fluorescence. By attaching fluorescent reporter molecules to
kinesin or ATP, we hope to establish the timing of biochemical events
relative to the mechanical steps, and to determine the conformational changes of
kinesin during processive stepping. For instrument design and preliminary
Lang, et al., Nature Methods 2004, and
Lang et al., Journal of Biology, 2003.
Automated two-dimensional force-clamp
To better study the effects of load on single kinesin proteins moving
along a microtubule, we constructed a new instrument that can be
operated as a two-dimensional force clamp, allowing the application of
loads of fixed magnitude and direction to motor-coated microscopic
beads moving in vitro. Flexibility and automation in experimental
design are achieved by computer control of both the trap position,
using acousto-optic deflectors, and the sample position, using a
three-dimensional piezo stage. Sophisticated calibration routines
reduce potential sources of error, and improve force and position
resolution. For more detail, see
Lang et al., Biophysical Journal (2002).
Meet "Team Kinesin":
||Assistant Professor, UC Santa Barbara
||Postdoctoral Fellow, UC San Francisco
||Assistant Professor of Physics, Princeton
||Assistant Professor of Physiology and Biophysics,
University of Washington
||Assistant Professor of Mechanical and Biological
||Assistant Professor of Biological Sciences and
Applied Physics, Stanford University
||Associate Professor of Physics, University of
||Associate Professor, Developmental and Cell
Biology and Biomedical Engineering, U.C. Irvine
||Professor of Physics, University of
||Group Leader, HHMI/Janelia Farm
Selected Block Lab Publications on
- Block, S.M. Kinesin motor mechanics:
Binding, stepping, tracking, gating, and limping. Biophysical
Journal 92: 2986-2995 (2007)
- Guydosh, N.R., Block, S.M. Not so lame after all:
Kinesin still walks with a hobbled head. The Journal of General
Physiology 130(5): 441-444 (2007) ( Full
- Valentine, M.T., Fordyce, P.F., Block, S.M. Eg5 steps
it up! Cell Division 1 31 (2006) (Full
- Valentine, M.T., Fordyce, P.M., Krzysiak, T.C.,
Gilbert, S.P., Block, S.M. Individual dimers of the mitotic kinesin
motor Eg5 step processively and support substantial loads in vitro. Nature
Cell Biology 8(5):470-477 (2006) (Full
- Guydosh, N.R., Block, S.M. Backsteps induced by
nucleotide analogs suggest the front head of kinesin is gated by
strain. PNAS 103(21):8054-8059 (2006) (Full
Click here for Supplemental Materials).
- Asbury, C.L., Fehr, A.N., Block, S.M. Kinesin Moves
by an Asymmetric Hand-Over-Hand Mechanism. Science 302:2130.
- Rosenfeld S.S., Fordyce P.M., Jefferson G.M., King
P.H., Block S.M. Stepping and stretching: How kinesin uses internal
strain to walk processively. J. Biol. Chem. 278:18550-18556
- Block, S.M., Asbury, C.L., Shaevitz, J.W., Lang, M.J.
Probing the kinesin reaction cycle with a 2D optical force clamp. PNAS
100:2351-2356 (2003) (Full
- Schnitzer, M.J., Visscher, K. and Block, S.M.
Mechanism of force production by single kinesin motors. Nature
Cell Biology 2 (2000) (Full
- Visscher, K., Schnitzer, M.J. and Block, S.M. Single
kinesin molecules studied with a molecular force clamp. Nature
400: 184-189 (1999) (Full
- Block, S.M. Kinesin: What gives? Cell 93:
5-8 (1998) (Full
- Block, S.M. Leading the procession: new insights into
kinesin motors. J. Cell Biol. 140: 1281-1284 (1998)
- Schnitzer, M.J. and Block, S.M. Kinesin hydrolyses
one ATP per 8-nm step. Nature 388: 386-390 (1997) (Full
- Block, S.M. Nanometers and picoNewtons: the
macromolecular mechanics of kinesin. Trends Cell Biol. 5:
169-175 (1995) (Full
- Svoboda, K., Mitra, P. and Block, S.M. Fluctuation
analysis of kinesin movement. Biophysical Journal 68:
- Svoboda, K. and Block, S.M. Force and velocity
measured for single kinesin molecules. Cell 77: 773-784
- Svoboda, K., Schmidt, C.F., Schnapp, B.J., and Block,
S.M. Direct observation of kinesin stepping by optical trapping
interferometry. Nature 365: 721-727 (1993).
- Block, S.M., Goldstein, L.S.B., and Schnapp, B.J.
Bead movement by single kinesin molecules studied with optical
tweezers. Nature 348: 348-352 (1990).