high-speed atomic force microscopy

High-speed atomic force microscopy
of protein dynamics: myosin on actin
and rotary enzyme F1-ATPaseToshio Ando, Department of Physics, Kanazawa University, JapanINTRODUCTION
One frequent way to study protein function isto examine protein structure, as there is tight
relationship between structure and function.
The detailed structures of over 60,000 proteins
have been revealed to date, however, thesestructures are merely static snapshots of theproteins, whereas proteins are highly dynamic.
The dynamic behaviour of proteins in action hasbeen studied by single-molecule fluorescencemicroscopy, but the protein molecules themselves
are invisible in the observations. Hence, thesimultaneous assessment of structure anddynamics has not been feasible, meaning that wehave to infer how proteins function from gleaned
data with significant resolution gaps. Directly
visualizing functioning protein molecules at highspatiotemporal resolution has long been desired.
To materialize this long-quested dream,
two decades ago Hansma’s and my groups had
independently embarked on the development ofhigh-speed atomic force microscopy (HS-AFM).
AFM enables the high resolution visualizationof biological samples in solutions. However, it
usually takes minutes to capture an image, andtherefore moving objects cannot be imaged or canonly be blurrily imaged. Through the creationof new techniques and the improvement of thefirst version of our instrument [1], HS-AFM
has now been established [2] and an instrument
is now commercially available from RIBM.
The current HS-AFM can generally capture animage of protein molecules within 100 ms or less.
Importantly, the function of fragile molecules is
not disturbed by the interaction with a cantilevertip. As has been demonstrated in recent imaging
studies [3-6], the visualized dynamic images ofproteins can provide information inaccessible
with other approaches, giving a great insight into
how the proteins function.
Here we describe our experimental set-up
for HS-AFM and report on its application to
the study of two biological mechanotransducer
systems: the movement of myosin V on actin
filaments and the conformational changes inrotorless F1-ATPase.

displaced by up to 1 μm, 4 μm, and 800 nm in
the X-, Y- and Z-directions, respectively. The
resonant frequency of the Z-scanner is 450 kHz.
The Z-piezoactuator (2x2x2 mm3) is glued to theinner surface of a solid hollow cylinder at its fourrims parallel to the displacement direction (insetin Figure 2) so that the Z-piezoactuator’s centre ofmass is maintained stationary [7].
To reduce the quality factor and hence increase
the response speed of the Z-scanner, we developed
a new Q-control method [8]. In a conventional
Q-control method, a damping signal proportional
to –dz/dt is added to the driving signal. However,to measure the displacement or speed of theZ-scanner in real time is difficult. Instead, weadded the output signal from a ‘mock Z-scanner’
constructed of an LRC circuit, as shown in Figure
2. The mock Z-scanner is characterized witha transfer function similar to that of the realZ-scanner. The amplitude detector can output
amplitude signals every cycle of cantilever
oscillation [2]. HS-AFM cantilevers made ofsilicon nitride are 10–20-times smaller thanconventional ones. They are 6 μm long, 2 μm
wide and 90 nm thick, which give the following
mechanical properties: resonant frequency in air,
3.5 MHz; resonant frequency in water, 1.2 MHz;spring constant, 0.2 N/m; quality factor in water,
2. Small cantilevers are available from Olympus
and NanoWorld.
The feedback bandwidth fB representing thespeed performance of the HS-AFM system reaches
110 kHz. The maximum possible imaging rate
Rmax is a function of fB and imaging conditions(the scan size in the X-direction W; the numberof scan lines N; the spatial frequency of sample
height corrugation to be imaged 1/l) as well as ofthe maximum possible phase delay qmax in thefeedback operation that depends largely on thesample fragility:
Rmax = 2qmax l fB/(pWN) [9].
For example, under a realistic condition for
imaging protein molecules by our HS-AFM( fB = 110 kHz, qmax = 20°, l = 10 nm, W = 150 nm,
and N = 100), Rmax becomes 16.3 frames per second(fps).

MATERIALS AND METHODS
High-speed atomic force
microscope SYSTEM
A schematic of our tapping mode HS-AFM
system is shown in Figure 1. A small samplestage (1.5 mm in diameter) is placed upside down
and glued to the Z-scanner. The scanner can be

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Figure 1
Schematic of the high-speed atomic force
microsope system [9].

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high-speed atomic force microscopy

SAMPLE PREPARATION
Substrate surfaces onto which samples are placed
are a key to successful HS-AFM imaging. For theobservation of a motor protein myosin V (M5)walking on an actin filament, we used planar
phospholipid bilayers containing biotin-lipid,
formed on a mica surface [4]. We added a small
amount of positively charged lipid in the bilayer
to facilitate observing the characteristic sideways
figure of M5. Partially biotinylated actin
filaments were immobilized onto the lipid bilayer
surface via streptavidin molecules with a low
surface density.
For the observation of rotorless F1-ATPase,
we used a mica surface which was first treatedwith aminosilane and then with glutaraldehyde[5]. (Lys)7 tags were introduced to the N-terminiof the b subunits of F1-ATPase. The tags werecovalently reacted to the modified mica surface,resulting in the immobilization of F1-ATPasewith the C-terminal side at the top.

Figure 2
Active Q-control for damping Z-scanner vibrations by the use of a mock Z-scanner.

RESULTS AND DISCUSSIONWALKING MYOSIN V
Double-headed M5 functions as a cargotransporter in cells and moves processively
along an actin filament toward the plus end of
the filament in a hand-over-hand manner witha 36-nm advance for every ATP hydrolysis cycle(Figure 3a shows tail-truncated M5 (M5HMM)).
‘Hand-over-hand’ means that the two heads
exchanges leading and trailing roles at each step,
like ‘bipedal walking’. We observed M5HMM
interacting with actin using HS-AFM [4]. Figure
3b shows HS-AFM images of unidirectionallymoving M5HMM (see Movie 1 [10]). The
translocation velocity was 70% of that measured
by fluorescence microscopy. When no positively
charged lipid was included in the substrate, thevelocity increased to 100 %, indicating no effect oftip-sample interaction on the motor activity. The
velocity did not decrease with time throughout
the imaging for 23 seconds, at 7 fps, despite thefact that each motor domain was tapped with theoscillating tip more than 60,000 times during thisimaging.
In this experiment, the stepping behaviour
could not be resolved because a step completed
within a frame time. To slow down the stepping,
we added more streptavidin on the substratesurface as a moderate obstacle to the advance. As
shown in Figure 3c (see Movie 2 [10]), after trailinghead detachment, the leading head spontaneously
rotated from the reverse arrowhead orientation
towards the arrowhead orientation but briefly
halted by colliding with a streptavidin molecule
placed in the way of its natural path, while thedetached trailing head was distant from the actin
filament and slightly rotated around the neckneck junction. Then, the leading head overcame
the streptavidin blockade to completely rotate to
the arrowhead orientation. Accompanied by thisfurther rotation, the trailing head was bound toa forward site of the filament to become a newleading head. The rotation of the leading headis the swinging lever-arm motion proposed by
Hugh Huxley for muscle myosin.
When two-headed bound M5HMM wasobserved in the nucleotide-free condition, theleading head was often sharply bent, as shown inFigure 4a, whereas it was straight in the presence
MicroscopyandAnalysis | April 2013 Scanning Probe Microscopy Issue

Figure 3(a) Schematicof actin-bound
M5HMM. (b, c)
HS-AFM images
showing (b)unidirectional
movement and(c) hand-overhand movementof M5HMM on an
actin filament [4].
Frame rate, 7 fps.

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high-speed atomic force microscopyof nucleotide, as shown in Figure 4b. Therefore,by observing the leading-head conformation,
we can judge whether or not the leading headcontains nucleotides. To estimate the rate
constant of ADP dissociation from the leading
head, we observed two-headed bound M5HMM
in various ADP concentrations. It came out tobe 0.1/s, meaning that ADP dissociates fromthe leading head every 10 seconds, on average.
However, in the presence of ATP, M5HMM
walks many steps in 10 seconds. Therefore, when
M5HMM is walking in the presence of ATP, ADP
never dissociates from the leading head. Thus,
we conclude that ADP dissociation, subsequent
ATP binding, and the resulting dissociation fromactin only occur at the trailing head. This is thebasis underlying the processive hand-over-handmovement of M5.
When two-headed bound M5HMM wasobserved in ADP, the coiled-coil tail was
sometimes unfolded, as shown in Figure 5. Upon
unfolding, the leading head rotated from thereverse arrowhead orientation to the arrowheadorientation, similar to the swinging lever-arm
motion, indicating that intramolecular tension
for the lever-arm swing exists in the twoheaded bound M5HMM in ADP. This meansthat external energy is not required for tension
generation. We observed a M5 behaviour (named‘foot stomp’) in the presence of ATP, which
reinforced this inference; the leading head briefly
detached from actin and then rebound to actin,
whereas the molecule remained at approximately
the same position on the filament. After footstomp, M5HMM stepped forward. Because
phosphate (Pi) release occurs very quickly whenthe ADP–Pi-bound head is bound to actin, theleading head does not carry Pi before and after thefoot stomp. These observations suggest that thechemical energy liberated by ATP hydrolysis is
not used for force generation as well as forward
movement of M5. External energy for forward
movement is only required for the trailing headdetachment from actin. The energy of ATP
binding is used to detach the trailing head. TheATP hydrolysis reaction (not energy) is used tomake it sure that the mechanical process proceeds
unidirectionally. This idea, which should beverified henceforth, will overturn the common
view that the energy of ATP hydrolysis is used formotor proteins to move.
ROTARY CATALYSIS OF ROTORLESS
F1-ATPASE
The minimum unit of F1-ATPase, the a3b3g
subcomplex, is a rotary motor. The rotary shaft
g is inserted in the central cavity formed by thestator ring a3b3, where the three a and three b
subunits are alternately arranged. g rotates in
the counterclockwise direction as viewed fromthe C-terminal side of a3b3. Three ATP bindingsites are located at the a-b interfaces, mainly inthe b subunits. In the ATPase cycle, three bs takedifferent chemical states: one ATP-bound, one
ADP-bound, and one nucleotide-free (empty).
Thus, there is strong co-operativity between
the three bs even though they are not in directcontact. It has been proposed that interactions
with g determine the conformational andcatalytic states of the individual bs because g has
no symmetry and hence the three b-g interactions
are different.

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Figure 4
HS-AFM images showing (a) sharply bent leading head in nucleotide-free condition and (b) straight leadinghead in ADP [4].

Figure 5
HS-AFM images showing (a) before and (b) after unfolding of coiled-coil tail of two-headed bound M5HMM
in the presence of ADP [4].

We investigated this issue by observing a3b3(Figure 6a) using HS-AFM [5]. When nucleotides
were absent, alternately arranged three subunits
had higher protrusions than the other three(Figure 6b). From the crystal structure of a3b3(Figure 6c), the three subunits with higher
protrusions were identified as bs. In the presenceof AMP-PNP, a3b3 looked triangular, because
two of the alternately arranged three subunits
were inwardly retracted. Moreover, only thesubunit that was outwardly extended had ahigher protrusion (Figure 6d). This subunit wasidentified to be nucleotide-free b from the crystal
structure of nucleotide-bound a3b3 (Figure 6e).
When imaged in ATP at 12.5 fps, each b
exhibited a conformational transition between
the outwardly extended high state (O state) andthe retracted low state (C state), as shown in Movie
3 [10]. Only one b assumed the O state. Whenan O-to-C transition occurred at one subunit, aC-to-O transition occurred simultaneously at thesubunit’s counterclockwise neighbour. Thus, theO-conformation propagated counterclockwise,

as shown in Figure 6f. These dynamic transitionsindicate that the O-to-C transition occurs
when ATP is bound to an empty b subunit,
while the C-to-O transition occurs when anADP-bound b subunit releases ADP. Therefore,
the three subunits take different states in acounterclockwise arrangement, explaining therotary catalysis by the a3b3 subcomplex. Thus,
we can conclude that the intrinsic co-operativity
responsible for torque generation to rotate g iselicited through b-b interplay alone, and that gpassively undergoes torque to rotate.
CONCLUSIONs
High-speed atomic force microscopy allows usto visualize directly protein molecules in action,
at subsecond to sub-100-millisecond temporaland submolecular spatial resolution, without
disturbing their function. Visualized dynamic
images of molecules can provide information
inaccessible with other approaches, giving agreat insight into how the proteins function.
The dynamics of molecules that appear in AFM

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high-speed atomic force microscopy

biography

Toshio Ando is a
Professor of Physics andBiophysics, and Directorof the Bio-AFM Frontier
Research Center atKanazawa University inJapan. Toshio received aBEng in applied physics
and a DSc in physics
from Waseda University. After working fornearly seven years as a postdoctoral fellow
and then an assistant research biophysicist
at University of California San Francisco, hejoined the faculty at Kanazawa University.
He has been working on high-speed AFM
for two decades.

abstractFigure 6
HS-AFM images of rotorless F1-ATPase a3b3 subcomplex [5]. (a) Schematic of a3b3 subcomplex. (b) AFM
image of a3b3 in nucleotide-free condition. (c) Crystal structure of nucleotide-free a3b3. (d) AFM imageof a3b3 in AMP-PNP. (e) Crystal structure of nucleotide-bound a3b3. (f) HS-AFM images showing theconformational change of b subunits in ATP. The highest pixel in each image is indicated by the red circle.
Frame rate, 12.5 fps.

movies can be interpreted straightforwardly
without intricate analyses and interpretations,making it possible to attain firm conclusions.
references
1. Ando, T., N. Kodera, E. Takai, D. Maruyama,
K. Saito, and A. Toda. A high-speed atomicforce microscope for studying biologicalmacromolecules. Proc. Natl. Acad. Sci. USA
98:12468–12472, 2001.
2. Ando, T., T. Uchihashi and T. Fukuma.
High-speed atomic force microscopy for nanovisualization of dynamic biomolecular processes.
Progress Surface Science 83:337–437, 2008.
3. Shibata, M. et al. High-speed atomic force
microscopy shows dynamic molecular processesin photo-activated bacteriorhodopsin. Nature
Nanotechnology 5:208–212, 2010.
4. Kodera, N., D. Yamamoto, R. Ishikawa, andT. Ando. Video imaging of walking myosin V
by high-speed atomic force microscopy. Nature
468:72–76, 2010.
5. Uchihashi, T., R. Iino, T. Ando, and H. Noji.
High-speed atomic force microscopy reveals
rotary catalysis of rotorless F1-ATPase. Science
333:755–758, 2011.
6. Ando, T. High-speed atomic force microscopy
coming of age. Nanotechnology 23: 062001 (27pp),
2012.

7. Fukuma, T., Y. Okazaki, N. Kodera, T.
Uchihashi, and T. Ando. High resonancefrequency force microscope scanner usinginertia balance support. Applied Physics Letters
92:243119 (3 pp), 2008.
8. Kodera, N., H. Yamashita and T. Ando. Active
damping of the scanner for high-speed atomic
force microscopy. Review Scientific Instruments
76: 053708 (5 pp), 2005.
9. Uchihashi, T. et al. Guide to video recordingof structure dynamics and dynamic processes ofproteins by high-speed atomic force microscopy.
Nature Protocols 7:1193–1206, 2012.
10. Movies 1, 2 and 3 may be viewed on theauthor’s website: http://www.s.kanazawa-u.ac.jp/phys/biophys/index.htm

AFM allows the visualization of biologicalsamples under physiological solution
conditions, at high spatial resolution.
However, captured images are limited
to snapshots because it takes at least 30
seconds to capture an image, whereas
biological phenomena are highly dynamic.
To overcome this limitation, my group has
developed high-speed AFM. The dynamic
processes and structural dynamics ofprotein molecules in action can now bevisualized using HS-AFM. Here we describe
our experimental set-up for HS-AFM andreport on its application to the study of two
biological mechanotransducer systems:
the movement of myosin V on actin
filaments and the conformational changes
in rotorless F1-ATPase.
acknowledgements
This work was supported by JST/CREST andMEXT Japan.
Corresponding author details
Professor Toshio Ando
Department of Physics and Bio-AFM Frontier
Research Center,Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan
Tel: +81 (0)76 264 5663
E-mail: tando@staff.kanazawa-u.ac.jp
www.s.kanazawa-u.ac.jp/phys/biophys/index.htm
Microscopy and Analysis 27(3):10-13 (AP),2013©2013 John Wiley & Sons, Ltd

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