(The FASEB Journal. 1999;13:S201-S208.)
© 1999 FASEB
Real time imaging of rotating molecular machines
KAZUHIKO KINOSITA, JR1
Department of Physics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan; and CREST "Genetic Programming" Team 13, Teikyo University Biotechnology Research Center 3F, Kawasaki 216-0001, Japan
1Correspondence: Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokahama 223-8522, Japan. E-mail: kazuhiko{at}phys.keio.ac.jp
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ABSTRACT
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Observation of true rotation has been relatively rare in living systems,
but there may be many molecular machines that rotate. Molecular
rotations accompanying function can be imaged in real time under an
optical microscope by attaching to the protein machine either a small
tag such as a single fluorophore or a tag that is huge compared with
the size of the protein. As an example of the former approach, axial
rotation of an actin filament sliding over myosin has been measured
quantitatively by attaching a fluorophore rigidly to the filament and
imaging the orientation of the fluorophore continuously by polarization
microscopy. As a huge tag in the latter approach, an actin filament
turned out to be quite useful. Using this tag, the enzyme
F1-ATPase has been shown to be a rotary stepper motor made
of a single molecule. Further, the efficiency of this ATP-fueled motor
has been shown to reach almost 100%. The two examples above
demonstrate that one can now image conformational changes, which
necessarily involve reorientation, in a single protein molecule during
function. Single-molecule physiology is no longer a dream.—Kinosita,
Jr., K. Real time imaging of rotating molecular machines.
Key Words:
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INTRODUCTION
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ROTATION OVER INFINITE angles of one part
against another requires that the two parts slide against each other
and hence that the two be disconnected from each other. Thus, rotation
is inconceivable at the level of an individual, except for apparent
rotations such as the swing of an arm, which is actually a combination
of two torsional motions. Connectivity, however, is ill defined at the
level of molecules, because noncovalent bonds between molecules are
continually broken and formed at body temperature. Lipid bilayer is a
typical example in which molecules freely diffuse against one another
yet keeping the overall integrity of the membrane. As a result, one
part of a cell can rotate indefinitely against the other without
breaking the cell membrane, as has been well demonstrated for a
protozoan (1)
and sperm (2)
. Such rotations
at the cellular level, however, appear to be rare exceptions, because
in most cases the cell membrane is backed up by cytoskeletal network
that does not readily disintegrate into pieces.
Protein-protein interactions are stereospecific, involving many
noncovalent bonds that act cooperatively. Relatively strong
associations between proteins, however, are often modified by specific
ligands such as a nucleotide, leading to rapid alternation of binding
and unbinding. If one of the partners is an array of protein molecules,
sliding may ensue, as exemplified by molecular motors. Sliding is an
essential ingredient of rotation. On the other hand, protein arrays are
often helical, including disks and rings, because Nature has created
only limited kinds of proteins and because repeated use of identical
molecules in identical manners results, in general, in a helical
symmetry. Sliding along a helical array produces rotation. Thus,
rotation may be quite common at the level of protein molecules.
However, there have been few protein machines that have been proved to
rotate, possibly because of technical difficulty in observing rotation.
Here we demonstrate that molecular rotations can be visualized under an
optical microscope by the use of either a small or huge tag.
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HELICAL ROTATION OF A SLIDING ACTIN FILAMENT
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An actin filament is made of a two-start, right-handed helix with
a pitch of ~72 nm (ref. 3
; Fig. 1a
). When the helical filament is moved by myosin, a linear motor, one
may expect that the filament rotates around its axis. Indeed, Nishizaka
et al. (4)
have shown that a sliding actin filament does
rotate as a right-handed screw as anticipated from the sense of the
actin helix. They, however, were unable to quantitate the axial
rotation because of the lack in spatial resolution in their microscopic
images. We therefore attempted to visualize the axial rotation by the
use of a small tag, a single fluorophore (5)
.
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Figure 1. Visualization of the axial rotation of a sliding actin filament.
a) Schematic diagram of the experiment. Violet bars
represent the orientation of the fluorophore attached to the actin
filament. b) Polarized dual-view observation of the
orientations of fluorophores. C) Sequential images, at
33-ms intervals, of a single tetramethylrhodamine fluorophore bound to
an actin filament sliding from bottom right toward top left. Adapted
from ref. 5
(copyright, 1997, National Academy of
Sciences, USA).
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As shown in Fig. 1b
, the orientation of a fluorophore can be
assessed by decomposing the fluorescence from the fluorophore into
vertically (V) and horizontally
(H) polarized components and imaging the two
components side by side on a video camera (6)
. When the
fluorophore is vertically oriented, it shows up in the vertical
(V) image, and when it is in the
horizontal orientation, it appears in the horizontal
(H) image. In this way we can determine the
fluorophore orientation continuously in real time until the fluorophore
is eventually photobleached.
The fluorophore tetramethylrhodamine covalently bound to Cys374 of
actin turned out to lie at ~45° from the filament axis without
wobbling much on actin (5)
(Fig. 1a
). If we let
an actin filament slide at 45° in the image plane, and if the
filament rotates as it slides over myosin molecules distributed on a
glass surface, then the orientation of the fluorophore in the image
plane should alternate between V and H (Fig. 1a
). Indeed, we observed alternate appearance in the
V and H images of the fluorescence from a single
fluorophore on an actin filament sliding from bottom right toward top
left (Fig. 1c
). The regular alternation indicates regular
rotation at a rate of about two turns per second.
Sliding of a helical array of actin against myosin thus produced
rotation as expected. The pitch of rotation, however, did not match the
pitch of the actin helix. Analysis of 17 actin filaments showed an
average speed of linear sliding of ~2 µm/s, whereas the average
rotational rate was ~2 turns/s (5)
. Thus, the actin
filaments make a turn every sliding distance of 1 µm. This rotational
pitch of 1 µm is ~15 times as large as the helical pitch of 72 nm.
If myosin molecules had tenaciously followed the helical track on an
actin filament, the filament should have rotated much faster, ~15
turns/µm or ~30 turns/s. The observed inefficiency in rotation
implies that a myosin molecule interacts with an actin filament
intermittently, skipping at least ~36 nm (half pitch of the actin
helix) between successive bindings. On the surface of an actin
filament, a myosin molecule with its two ‘feet’ (the two
actin-binding domains of myosin usually referred to as ‘heads’)
‘runs’ rather than ‘walks.’ If myosin were to walk without
detaching its two feet simultaneously, as in the case of kinesin, it
would stay on one of the two helical strands of an actin filament,
because simultaneous landing of the two feet on the opposing two
strands is sterically improbable.
Note that a running myosin molecule does not jump to the next landing
site if the myosin is alone. A myosin molecule can move to a distant
site on actin because other myosin molecules pull the actin filament
while the former is detached. If a single myosin molecule not attached
to glass interacts with actin, the myosin will only hop into the
solution and will then undergo random diffusion.
Our result that the axial rotation of an actin filament was rather slow
also implies that the force generated at the myosin-actin interface is
primarily along the filament axis, because an oblique force would
rotate the filament efficiently. One might argue that the rotational
component of the oblique force may be obstructed by the other myosin
molecules that are simultaneously bound to the same filament, but the
obstruction also operates on the linear sliding. The driving force,
either linear or rotational, is the net sum of the forces by all myosin
molecules bound, of which some exert positive and the others negative
forces.
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CIRCULARLY ROTATING MOLECULAR MACHINES
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Helical structures are abundant in cells. Actin and DNA are
typical examples, and microtubules are also helical although their
normal pitch is long. Thus, helical rotation may well be found in many
places in a cell. Protein machines working along DNA are most likely to
rotate, although direct evidence has not been obtained.
Rings and disks are relatively scarce. Until recently, the almost sole
example of circularly rotating molecular machines was the bacterial
flagellar motor (7)
. This motor is assembled at the root
of a flagellum and consists of several rings containing a total of
~102 protein molecules (8
, 9)
.
Flow of protons through the motor causes rotation of the flagellum,
which propels the bacterium. The rotation was first revealed in a
tethered cell (10)
where the cell body served as a huge
indicator of the motor rotation; when the flagella were attached to a
glass surface, the cell body was seen to rotate under an optical
microscope. Using a similar technique, we have proved the existence of
another circularly rotating machine, the F1-ATPase (11)
.
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F1-ATPase: A ROTARY MOTOR MADE OF A SINGLE MOLECULE
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The fit among subunits of a protein molecule is generally
considered to be quite snug. Normally they do not separate from one
another. Thus, when P. Boyer proposed almost 20 years ago that a single
molecule of the enzyme ATP synthase may be a rotary motor in which one
or more subunits rotate against the others (12)
, few
people would believe it. Now we know he was right.
The ATP synthase synthesizes ATP from ADP and phosphate using the flow
of protons as the energy source. This enzyme is ubiquitous from animals
to plants and bacteria. In animals, ATP synthase resides in the inner
mitochondrial membrane. Protons are ejected from mitochondria by the
action of a chain of protein machines, and the protons flow back into
mitochondria through the ATP synthase, where ATP is produced
(13)
. As shown in Fig. 2
, the ATP synthase consists of a membrane-embedded portion called
F0 and a protruding portion F1. Protons flow through the
F0 portion, and the flow somehow leads to the
synthesis of ATP in the F1 portion. The ATP
synthase is a completely reversible molecular machine in that the
hydrolysis of ATP in F1 drives protons in the
reverse direction through F0.
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Figure 2. Schematic diagram of the ATP synthase. The gray part is the putative
stator for the F0 rotor. Adapted from ref. 21
(copyright, 1998, Cell Press).
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The mystery is how the proton translocation in F0
is coupled to the chemical synthesis/hydrolysis of ATP in
F1. Boyer’s proposal (12
, 14
, 15)
was that the coupling is mechanical. F0 and
F1 are each rotary motors, one driven by the
proton flow and the other driven by ATP hydrolysis, and the rotating
shaft is common to the two. When the energy driving the proton flow
(the difference in the electrochemical potential of protons between the
two sides of the membrane) is larger than the free energy obtained from
ATP hydrolysis, F0 drives the shaft in its
genuine direction; the ATP-driven motor, F1, is
forced to rotate in its reverse direction, resulting in ATP synthesis.
If the energy for ATP hydrolysis is larger, the shaft rotates in the
opposite direction, and the reverse rotation of the
F0 motor drives protons back against the
electrochemical gradient. (In cells, this back reaction tends to be
inhibited by a built-in mechanism.)
One can isolate the F1 portion as a soluble
protein, and then it only catalyzes ATP hydrolysis, not synthesis.
Hence, the isolated F1 is called
F1-ATPase. The subunit composition of F1-ATPase is
3ß3
, and the
ATPase activity resides in the three ßs (one catalytic site per ß,
close to an -ß interface). It was this subunit composition,
together with the fact that the three ßs participate equally in
steady-state ATP synthesis/hydrolysis, that constituted a basis for the
Boyer’s proposal. The single-copy subunits—, , and —cannot
each have a three-fold symmetry, yet the three ßs are equivalent in
long-term catalysis. In particular, is essential for catalysis and
thus is not a peripheral accessory. For the asymmetric to interact
with the three ßs impartially, has to rotate, visiting the three
one by one.
When a crystal structure of F1-ATPase was solved
by J. Walker et al. (16)
, Boyer’s proposal became
realistic. As shown schematically in Fig. 2
,
3ß3 hexamer forms a
cylinder, and occupies the central hole (part of , and and
were not resolved in the atomic structure). The interface between
the central and the hexamer wall is mostly hydrophobic, suggesting
that the wall constitutes an oily sleeve for the putative rotor
(16)
. F1 alone would thus work as a
motor when fueled with ATP.
The structure immediately suggested ingenious experiments that would
support the rotational catalysis model. In one series (17
, 18)
, a particular residue on was crosslinked to one of the
three ßs. Then the link was cut and the enzyme was allowed to
catalyze ATP hydrolysis (F1 preparation)
(17)
or synthesis (whole ATP synthase) (18)
.
After that, the residue was again crosslinked to a ß. The second
crosslinking was found to be made to any of the three ßs, indicating
that the residue on faces all three ßs equally during catalysis.
In experiments where catalysis was not allowed between the two
crosslinking treatments, the second target was the same as the first
one. In another set of experiments (19
, 20)
, a fluorescent
dye was attached to . Time-resolved polarization measurement showed
that the fluorophore changes its orientation over many degrees when the
enzyme undergoes ATP hydrolysis reaction. In the most recent experiment
(20)
, the fluorophore appeared to adopt three distinct
orientations.
These experiments indicate that can rotate within the
3ß3 cylinder to the
extent that the three ßs are impartially visited. This, however, does
not necessarily imply that continues to rotate in one direction
without fluctuating back and forth. Also, the symmetry consideration
above that led to the Boyer’s proposal does not necessarily point to
unidirectional rotation; to-and-fro motions are equally acceptable.
Observation of a single F1-ATPase molecule
established that the rotation is mostly unidirectional with only
occasional (but significant) backward movement (11
, 21
, 22)
.
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DIRECT OBSERVATION OF THE ROTATION IN F1-ATPase
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To observe rotation, we constructed a subcomplex of F1,
3ß3, from a
thermophilic bacterium (11)
. This subcomplex, lacking
and , is sufficient for the ATPase activity and, as shown below, is
also sufficient for rotation. Hereafter we refer to this complex simply
as F1.
The putative stator part of F1, the 3ß3 cylinder, was
fixed to a glass surface (11)
or, more recently, to the
surface of a plastic bead (22)
such that the binding site
for F0 was away from the surface (Fig. 3a
). To the putative rotor , a fluorescent actin filament was attached
as a huge marker through streptavidin-biotin links. A desired
concentration of ATP was added, and the sample was examined under a
fluorescence microscope. Then we saw fluorescent filaments rotating in
one direction (Fig. 3b
) invariably counterclockwise when
viewed from above in Fig. 3a
. There were many more filaments
that did not rotate, presumably because they were bound by more than
one F1 molecules or were obstructed by the
surface. But those filaments that rotated continued to do so over many
revolutions, often well over hundreds of turns (11)
. The
counterclockwise rotation was powered by ATP; in its absence,
continuous rotation was not observed.
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Figure 3. a) The experimental system for the observation of the
rotation of F1-ATPase. The space-filling model of
F1 is from ref. 16
(blue, ; green, ß;
red, ), actin filament (orange) from ref. 3
, and
streptavidin (brown) from ref. 23
. Streptavidin has four
strong binding sites for biotin, and the actin filament is heavily
decorated with biotin. There is, however, only one biotin on .
Presumably, free rotation between streptavidin and is prevented by
steric hindrance. The diameter of the gray disk, representing the
surface to which the three ß subunits are bound through histidine
tags, is ~22 nm. B) Sequential images, at 33-ms
intervals, of a rotating actin filament. The average rate of rotation
was 1.3 turns/s. Bar = 5 µm. Adapted from ref. 11
(copyright, 1997, Macmillan Magazines).
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The filament in Fig. 3b
rotated around its center, where a
tiny F1 presumably held and drove the filament.
This motion cannot be a pseudo rotation without sliding (as in the case
of the swinging of an arm). When one holds the middle of a rod much
longer than one’s height, one cannot rotate it without releasing the
grip occasionally. Thus, a single molecule of F1
is indeed a molecular motor in which the central subunit
rotationally slides against the stator hexamer
3ß3 over infinite
angles. With the size of the stator cylinder of ~10 nm and the
diameter of the rotor of ~2 nm, F1 is the
world’s smallest rotary motor ever known.
The counterclockwise rotation is consistent with the crystal structure
of F1 (16)
. In the crystal, the
three ß subunits carried an analog of ATP, ADP, and none in the
clockwise order (Fig. 2)
. If this structure represented a kinetic
intermediate in the hydrolysis reaction, the ATP in the first ß
would, in the next step, be converted to ADP, the ADP in the second ß
would be released, and the third ß would bind ATP from solution. Then
the surface of that favors the empty ß (convex in Fig. 2
,
bottom), for example, would rotate toward the second ß (i.e., in the
counterclockwise direction). It is likely that the crystal structure,
obtained in the presence of MgADP in the medium, represents the
so-called MgADP-inhibited form of the enzyme (16)
. This is
a kind of product inhibition that cells probably exploit to prevent
futile consumption of ATP. The clockwise order of bound nucleotides
consistent with the counterclockwise rotation, then, suggests that the
inhibited form arises from a normal intermediate. ADP to be released is
somehow trapped in the binding site possibly by premature release of
phosphate, which leads to tighter binding of ADP.
The observation of individual molecules under a microscope has
demonstrated that F1 is a rotary motor that
rotates counterclockwise when it hydrolyzes ATP. We note again,
however, that many of the filaments we observed did not rotate, or only
fluctuated, and that there were many more F1
molecules to which an actin filament failed to bind. That is, the
conclusions we draw from these experiments apply to those molecules
that we somewhat arbitrarily selected to analyze. What we show here is
the ‘best’ performance of F1 on our artificial
surface. Such selection is a weak point of most single-molecule studies
but is also a strong point in that one can focus on the best part,
which is often buried in experiments that report only the average
behavior. It is quite possible, for example, that most of the molecules
being measured in a conventional experiment, either in vitro
or in vivo, are in fact dormant without being noticed to be
so. Single-molecule observation will detect such heterogeneity and will
allow the selective characterization of active ones.
The selection process might cast some doubt on the objectivity of the
conclusions. For example, an actin filament bound to a single myosin
molecule on a glass surface rotates plus or minus several turns just by
diffusion (24)
, presumably because myosin is a very
flexible molecule. If one selects from the video of this fluctuating
actin a particular short sequence, one could show that the filament
makes a complete, apparently regular, turn, which would be
indistinguishable from the rotation driven by F1-ATPase. Conversely, an actin filament
presumably bound to F1 often exhibits random
rotation, and there are occasions where it makes a turn or two, over
time, of clockwise rotation. We are tempted to ascribe these irregular
motions to thermal diffusion. In contrast, when we saw by eye the
long-lasting counterclockwise rotation of an actin filament, we almost
immediately believed that it must represent the authentic motor motion,
because the motion was too consistent to be of thermal origin. This may
not be a perfectly scientific judgement, but those who watch the video
may find seeing to be believing.
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F1-ATPase IS A STEPPER MOTOR WITH A NEARLY 100% EFFICIENCY
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Now we report more on the properties of ‘well-behaving’
F1. First, it is a 120°-stepper motor, as may
be expected from its structure. Stepping is not resolved in Fig. 3b
obtained at a high ATP concentration (2 mM). At nanomolar
concentrations of ATP, however, the actin rotation became clearly
stepwise, as shown in Fig. 4
(22)
. Between steps, the F1 waited
for the next ATP molecule to come. The structure of
F1 in the waiting phase appears to be stable for
many seconds, at least as long as the orientation of is concerned.
Metastable orientations of other than those separated by 120°
were not noticeable at the time resolution of 5 ms.
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Figure 4. Stepping rotation of F1 at 20 nM ATP. Inset shows the trace
of the centroid of the actin filament. Adapted from ref.
22
(copyright, 1998, Cell Press).
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The intervals between steps were irregular, as expected for stochastic
binding of ATP. The histogram of the intervals was exponential with
short intervals being more abundant than long ones. This indicates that
each step is driven by one ATP molecule (22)
. If two or
more ATP molecules were required per step, short intervals would be
scarce because simultaneous binding of two ATP molecules should be a
rare event.
Each step is made in a time of the order of 0.1 s. If
F1 is scaled to the size of a person, the person
standing at the bottom of a swimming pool would be rotating a rod some
300 m long at the speed of 120° per 0.1 s! The hydrodynamic
friction against rotation is enormous. F1 is
really a powerful motor. Naturally, though, the stepping velocity is
slower for a longer filament that is subject to a greater friction
(Fig. 5
). The stepping velocities measured in individual steps are highly
variable, but, on the whole, they are distributed around the solid line
in Fig. 5
. The line shows the velocity expected when
F1 produces a constant torque of 44 pN·nm
irrespective of the frictional load (actin length). It appears that
F1 is designed to be a constant-torque machine.
If the torque of 44 pN·nm is produced at the interface between ß
and , at the radius of 1 nm, the sliding force between ß and
amounts to 44 pN, far greater than the force produced by various linear
motors (21)
.
The torque of 44 pN·nm times 2
/3 radians (120°), ~90 pN·nm,
is the work done in a step against the viscous load. Because the torque
is constant, the work per step is also constant. In situ,
probably, the F1 motor linked to F0 can do this much of work against
F0 to pump protons. In cells, the free energy for
ATP hydrolysis is ~80 pN·nm per one molecule of ATP. Thus,
F1 is expected to work at ~100% efficiency.
We also measured the torque and work at high ATP concentrations where
steps were no longer resolved (22)
. Figure 6
summarizes the average velocities of actin filaments of different
lengths (abscissa) at various ATP concentrations (distinguished by
color). The smooth lines in the figure are calculated on the
assumptions that 1) F1 produces a
constant torque of 40 pN·nm irrespective of the load or ATP
concentration, 2) three ATP molecules are consumed per turn,
and 3) the time per one third of revolution is simply the
sum of the ATP cycle time at no load (measured without actin) and the
time needed to rotate the actin filament by 120° under the torque of
40 pN·nm. The fairly good agreement between the data and calculated
lines indicate again that the F1 motor produces a
constant torque of ~40 pN·nm or a constant work per step of ~80
pN·nm. In most of these experiments and those in Fig. 5
, the free
energy of ATP hydrolysis was not well defined because the
concentrations of ADP and phosphate were not controlled. Figure 6
,
however, contains two data sets where the free energy per one molecule
of ATP was set at 110 pN·nm (+ in dark red) and 90 pN·nm (+ in
red). These data also agree with the red line, indicating that
F1 under these conditions indeed worked at a near
100% efficiency.
In summary, the F1 motor is a 120° stepper
motor that produces a constant torque of ~40 pN·nm, or a constant
work per step of ~80 pN·nm, over a broad range of load and speed.
It consumes one ATP molecule per step, and it can operate even when the
free energy of ATP hydrolysis is almost equal to the work to be done.
Another interesting feature is that the motor occasionally makes back
steps, as seen in Fig. 4
. The back steps are as fast as forward ones
(red symbols in Fig. 5
), suggesting that these back steps also consume
an ATP molecule.
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MECHANISM OF F1 MOTOR
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The rotation of F1 is clearly stepwise at
nanomolar ATP concentrations. In this concentration range,
F1 in the waiting phase between steps is
considered to carry only one nucleotide among the three ßs, in an
equilibrium between ATP and ADP plus phosphate. When a second ATP binds
to an empty ß, it induces rapid release of ADP and phosphate from the
first site, resulting in the net hydrolysis of one ATP molecule. The
central is supposed to rotate during this cooperative catalysis or
during the seesaw action between the two binding sites (14
, 15)
.
To ensure the counterclockwise rotation, the second ATP has to bind to
the correct ß, the one in the counterclockwise direction from the
first ß. Presumably, it is the central, asymmetric that controls
the affinity of peripheral ßs for nucleotides. The affinity of the
correct ß for ATP is made higher, but, probably, the affinity of the
other empty ß is also appreciable and binding of ATP to this wrong
ß causes the backward step.
Conversely, the orientation of is dictated by the peripheral ßs.
The ßs change their conformations depending on the bound nucleotide,
and the conformations of the three ßs together determine the most
stable orientation of . In the F1 in the
waiting phase, only one out of the three equivalent ßs binds a
nucleotide, and this ß stabilizes in a particular orientation.
Binding of the second ATP eventually shifts the stable orientation to
one 120° ahead, but precisely when and how this happens is not yet
clear. Logically, it is most likely that the binding of the second ATP
immediately shifts the stable orientation (the minimum of the
rotational potential for ) forward, because some movement of
would be required to promote the release of the hydrolysis products
from the first site. Whether the binding of the second ATP alone,
before the release of the hydrolysis products from the first site,
moves the potential minimum 120° ahead remains to be seen. Complete
120° shift may require the product release, which, in addition to the
ATP binding, is also expected to confer some energy to the
F1 motor. In this case, addition of sufficient
ADP in the medium would stop the rotation at an intermediate angle. So
far, we have been unable to observe pauses at intermediate angles,
because the formation of the MgADP-inhibited form interferes with this
kind of experiment. It is also possible that the ATP binding alone
shifts the potential minimum by 120°, and the product release makes
the minimum deeper.
Analysis of our stepping data suggests that, at least by the time the
hydrolysis products are released (completion of a hydrolysis cycle),
the rotational potential for is set up such that the minimum is
120° ahead of the previous minimum and the minimum is ~80 pN·nm
deep (22)
. This depth determines the constant work output
of this motor. The potential should be mostly downhill toward the
minimum, because otherwise the rapid stepping cannot be explained.
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TOWARD SINGLE-MOLECULE PHYSIOLOGY
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We have shown that molecular rotations can be imaged continuously
in real time, under an optical microscope, by the use of either a small
tag such as a single fluorophore or a huge tag such as the actin
filament. This implies that one can now image conformational changes,
which necessarily involve reorientation (partial rotation), in a single
protein machine at work. In fact, the rotation of
F1 is a conformational change over infinite
angles. The stage has been set for ‘single-molecule physiology,’
where one observes and analyzes the behavior of single protein machines
individually to elucidate the mechanism underlying their function.
A widely-used tag that is huge compared to protein machines is a
plastic bead of a micrometer size. Individual steps of the linear
motors kinesin (25)
and myosin (26)
have been
measured with this tag. Consider an example where a plastic bead is
attached at the tail end of an actin filament interacting with myosin
on a glass surface (27)
. The bead is held with optical
tweezers, and stepwise movement of actin caused by a myosin molecule is
revealed as the displacement of the bead. A popular view is that the
step arises entirely from a conformational change of myosin while it is
bound to actin. Regardless of whether this is true or not, the
experiment suggests that one can detect conformational changes in a
protein molecule by attaching to it a plastic bead through a string of
actin.
The measurement using a huge tag is precise, because it gives an
intense optical signal. But a huge tag does not reveal which part of
the molecule undergoes the conformational change. To answer this
question, one could attach a small tag, a single fluorophore, at a
desired site and measure its orientation as we have done in the
detection of the axial rotation of an actin filament.
The huge tag we used to observe the rotation of
F1, the actin filament, is a smart probe in that
it magnifies the nanometer motion without the aid of sophisticated
tools. An angular resolution of 30° is easily obtained even in direct
observation by eye. The angle of 30° corresponds to the sliding of
against the 3ß3
cylinder of only 0.5 nm. This tag gave us an additional bonus in that
it allowed the measurement of force produced by the
F1 motor. The translational speed of the tip of
the actin filament was tens of micrometers per second, more than an
order of magnitude faster than the motion of a bead driven by linear
motors. The fast motion accompanies a large enough frictional drag that
counteracts the motor force. However, we were unable to measure the
performance of the F1 motor at no load. This can
be achieved by the use of a small tag, as done in the
single-fluorophore polarization measurement by Häsler et al.
(20)
.
A huge probe also serves as a handle for manipulation. A plastic bead
can easily be manipulated with optical tweezers (28
, 29)
.
By attaching a plastic bead to the actin filament bound to
F1, one could hopefully rotate
F1 in the reverse direction and synthesize ATP.
Or, one could measure the rotational potential from the movement of the
bead in the optical trap. Also, the use of single fluorophores is not
limited to the detection of molecular orientations. For example, one
can measure chemical reaction (ATP hydrolysis) in a single protein
machine (30)
simultaneously with the movement and force it
produces (31)
. Applied to F1, one
could determine how the orientation of regulates the hydrolysis
kinetics and how the bound nucleotides determine the rotational torque
on . An optical microscope has hitherto served as one of the most
important tools of cell biology. Now it opens up a new field of
science, the single-molecule physiology.
|
ACKNOWLEDGMENTS
|
|---|
I thank my colleagues for the work cited here and Y. Harada and H.
Itoh for critically reading the manuscript. This work was supported in
part by grants-in-aid from Ministry of Education, Science, Sports and
Culture of Japan and a Keio University special grant-in-aid.
|
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TOP
ABSTRACT
INTRODUCTION
, where 
L3/[ln(L/2r)-0.447]
is the rotational frictional drag coefficient, L and
r are the length and radius of the actin filament, and
120°), where