2358

Biophysical Journal

Volume 97

October 2009

2358–2367

Streptavidin 2D Crystal Substrates for Visualizing Biomolecular
Processes by Atomic Force Microscopy
Daisuke Yamamoto,†‡ Naoki Nagura,† Saeko Omote,† Masaaki Taniguchi,† and Toshio Ando†‡*†Department of Physics, Kanazawa University, Kanazawa, Japan; and ‡Core Research for Evolutional Science and Technology of the Japan
Science and Technology Agency, Tokyo, Japan

ABSTRACT Flat substrate surfaces are a key to successful imaging of biological macromolecules by atomic force microscopy(AFM). Although usable substrate surfaces have been prepared for still imaging of immobilized molecules, surfaces that are moresuitable have recently been required for dynamic imaging to accompany the progress of the scan speed of AFM. In fact, the stateof-the-art high-speed AFM has achieved temporal resolution of 30 ms, a capacity allowing us to trace molecular processes
played by biological macromolecules. Here, we characterize three types of streptavidin two-dimensional crystals as substrates,
concerning their qualities of surface roughness, uniformity, stability, and resistance to nonspeciﬁc protein adsorption. These
crystal surfaces are commonly resistant to nonspeciﬁc protein adsorption, but exhibit differences in other properties to someextent. These differences must be taken into consideration, but these crystal surfaces are still useful for dynamic AFM imaging,as demonstrated by observation of calcium-induced changes in calmodulin, GroES binding to GroEL, and actin polymerizationon the surfaces.

INTRODUCTION
Atomic force microscopy (AFM) (1) has become a powerfultool for high-resolution visualization of biological samples inaqueous solutions (2–4). Immobilization of biological molecules onto a planar support is prerequisite for the imaging,and therefore, selecting or preparing appropriate substrates
is a key to successful imaging. Thus far, a number of immobilization methods have been developed to this end (5–7).
Bare mica surface has been frequently used for reproducible
imaging of biological samples because it is atomically flatover a wide area and many proteins attach to the surface
under buffer solutions. However, because of the electrostaticadsorption, its attachment is often weak and selective attachment in controlled orientation cannot be achieved, except forrare cases. To achieve a firm attachment, the technique ofchemisorption of biomolecules onto self-assembled monolayers of molecules, terminated with chemically reactive
groups, has been used (8,9). This immobilization can avoid
displacement of biomolecules by a scanning tip. Thesemethods for sample attachment have contributed to the success of still imaging and slow time-lapse imaging (10–13) of
biological samples.
Recent advent of high-speed AFM expands the usefulnessof AFM for biological research (14,15). Its maximum
imaging rate of 33 frames/s enables direct imaging of biomolecular processes in real time. However, for dynamic AFM
imaging of molecular processes, previously used substratesurfaces are often useless. Biomolecular processes often
contain dynamic interactions between molecules. When allmolecules are attached to a surface, they have almost no
chance to interact with each other. In addition to such generSubmitted April 30, 2009, and accepted for publication July 20, 2009.
*Correspondence: tando@kenroku.kanazawa-u.ac.jp
Editor: Peter Hinterdorfer.
Ó 2009 by the Biophysical Society
0006-3495/09/10/2358/10 $2.00

ally required conditions for the surfaces as a small amount ofroughness for identifying the molecules of interest, and ahigh affinity for the sample, dynamic AFM imaging also
requires selective attachment of a specific component ina multicomponent sample, controlled orientation of immobilized molecule, and a lack of perturbation to the biological
function of the sample (i.e., absence of nonspecific interactions).
In a pioneering study (16), streptavidin two-dimensional(2D) crystals formed on biotin-containing planar lipid layers(17–20) was imaged by AFM, and their use for biomolecule
attachment in AFM studies was proposed. Here, we examined whether the surfaces of streptavidin 2D crystals canfulfill the above-mentioned requirements. Streptavidin iscomprised of four identical subunits, each of which specifically binds to one biotin molecule with a strong affinity(Ka z 1013 M1) (21). In the 2D crystals, the two biotin
binding sites face the free solution, and therefore, can bindto biotinylated samples to be imaged (18,20). Since biotinylated Ni-NTA is commercially available, the surface also canimmobilize His-tag conjugated recombinant proteins without
lateral diffusion (note that His-tag conjugated proteins can bedirectly attached to a Ni-NTA containing lipid bilayer surface, but lateral diffusion occurs because of fluidity of thelipid bilayer). Importantly, streptavidin is resistant to nonspecific binding to many proteins (21), which possibly guarantees the surface-bound proteins against dysfunction (22).
On biotinylated planar lipid layers, streptavidin selfassembles into three distinct crystalline arrangements (i.e.,
types 1–3 with P2 symmetry), which depend on crystallization conditions such as pH (23) and ionic strength (24). Here,we characterize these different 2D crystals taking into
consideration the stability, degree of order, surface roughness, and nonspecific binding. Although the surfaces of thesedoi: 10.1016/j.bpj.2009.07.046

Streptavidin Substrata for Dynamic AFM

crystals satisfy the required conditions for dynamic imaging
of molecular processes, our characterization results indicatethat it would be better to consider their different properties
depending on the sample to be imaged.
Using high-speed AFM, we demonstrate the applicabilityof these crystal surfaces to dynamic AFM imaging by observing three different samples. For immobilized calmodulin(CaM), we detected its structural changes upon calcium
binding. GroEL was immobilized in a controlled orientation(i.e., side-on orientation) by introducing biotin at the outersurface of the equatorial domain of GroEL. Using thisorientation (in which both ends of GroEL double rings areaccessible to GroES), binding of GroES to GroEL wassuccessfully visualized. The process of actin polymerization
was also visualized. Thus, the streptavidin 2D crystals areshown to be a useful tool for high-speed AFM imaging of
biomolecular processes.
MATERIALS AND METHODS
Materials
We obtained following materials and used without further purification;
streptavidin, Wako Pure Chemical (Osaka, Japan); bovine brain CaM, Calbiochem (Darmstadt, Germany); lipids, Avanti Polar Lipids (Alabaster, AL);
biotin-(AC5)2-sulfo-osu and biotin-PEAC5-maleimide, Dojindo Laboratories (Kumamoto, Japan); Cy3-osu, GE Healthcare (Buckinghamshire, UK);and phalloidin, Invitrogen (Carlsbad, CA). Coverslips and natural muscovite
mica were purchased from Matsunami (Osaka, Japan) and Nilaco (Tokyo,
Japan), respectively.

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the chemical fixation of the streptavidin crystals, a 10 mM glutaraldehydecontaining crystallization solution was applied and incubated for 5 min.
The reaction was quenched using 20 mM Tris mixed in the crystallizationbuffer. To distinguish the crystal types in this article, we used the notations
type-1, type-2, and type-3. (Note that, for the type-1 P2 crystal, C222 hasbeen used before in a 2D image in the literature (16,20,25). Also please
note that, although the notation P1 has been used for the type-3 P2 crystal(23,24), it certainly has P2 symmetry because the top view of the surfaceattached streptavidin tetramer itself has a twofold rotation symmetry.)

Biological samples
CaM
To prepare biotinylated CaM, CaM was reacted with biotin-(AC5)2-sulfoosu as described previously (26). For the fluorescent labeling, nonbiotinylated or biotinylated CaM was reacted with Cy3-osu as described (26).
The molar ratio of biotin to CaM in the biotinylated CaM was determined
to be 0.6 based on the avidin-2-(40 -Hydroxybenzeneazo)benzoic acid assay(27). The molar ratio of conjugated Cy3 to CaM was 0.7–1.9 throughout the
study. Before the immobilization of biotinylated CaM, the streptavidin 2D
crystals were washed with buffer A (20 mM imidazole, 100 mM KCl,
2 mM MgCl2, 1 mM EGTA, pH 7.6). To distinguish specific and nonspecificbinding, 0.1 nM of biotinylated or nonbiotinylated Cy3-CaM in buffer A
was infused to the flow cell. After incubating for 3 min, the sample waswashed with buffer A to remove excess Cy3-CaM. To further examine thenonspecific adsorption, a 100-times higher concentration (i.e., 10 nM) of
nonbiotinylated Cy3-CaM was infused to the flow cell and then observed
by TIRFM. For AFM observations, 200 nM biotinylated or nonlabeled
CaM in buffer A was deposited onto the streptavidin type-3 P2 crystal
formed on mica-supported lipid bilayer. After 3 min, the sample was washedwith buffer A to remove unattached CaM. The AFM measurements were
performed in buffer A in the presence or absence of 2 mM Ca2þ.

GroEL and GroEL-GroES complexes

Coverslips
Coverslips were washed for 30 min with detergent in an ultrasonic bath, andthen boiled in H2SO4/H2O2 (80:20, v/v) to remove hydrocarbon contaminants. After rinsing with MilliQ water (Millipore, Billerica, MA), thecoverslips were immersed in 6 M KOH for 12 h. Subsequently, the cleaned
coverslips were sonicated in MilliQ water.

Preparation of streptavidin 2D crystals
For the measurement by total internal reflection fluorescence microscopy(TIRFM), coverslips are used as the solid support for biotinylated lipid bilayers; a flow cell was built using two coverslips (24  36 mm2 and 18 
18 mm2), between which a space was created by two strips of double-sided
sticky tape, forming a center channel for injecting samples and buffer solutions. For AFM imaging, either mica (1-mm diameter) or a coverslip (2 
2 mm2) were used as the solid support. Streptavidin 2D crystals were formedon supported biotin-containing lipid bilayers as described (25). Briefly,
a supported biotin-containing lipid bilayer was first obtained by applying
small unilamellar vesicles (0.125 mM) of lipids to a solid support. The lipid
compositions were dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)(biotinylated lipid) at 7:2:1 (w/w). After washing out the excess lipids
with a crystallization buffer solution, a 0.1–0.2 mg/ml streptavidin solution
was placed on the biotinylated lipid bilayer and incubated at room temperature in a sealed container. The crystallization buffers used were 10 mM
HEPES, 150 mM NaCl, and 2 mM CaCl2 (pH 7.4) for the type-1 P2 crystal;
10 mM MES, 450 mM NaCl, and 2 mM CaCl2 (pH 5.8) for the type-2 P2
crystal; and 10 mM acetate, 450 mM NaCl, and 2 mM CaCl2 (pH 4.0) for
the type-3 P2 crystal. After the incubation for at least 2 h, the excess streptavidin was washed out with the buffer solution used for crystallization. For

The GroEL D490C mutant was produced by site-directed mutagenesis.
D490C GroEL and wild-type GroES were expressed in Escherichia coli
XL1-Blue and purified as described (28). D490C GroEL was dialyzed
against buffer B (50 mM HEPES-KOH, 50 mM KCl, 10 mM MgCl2,
pH 7.4) containing 2 mM Tris (2-carboxyethyl) phosphine hydrochloride
and then biotinylated by the reaction with biotin-PEAC5-maleimide (molar
ratio of reagent to subunit equal to 1) for 30 min at 25 C. The reaction was
quenched with 5 mM dithreitol (DTT). To remove unreacted biotin, thesample was dialyzed against buffer B containing 2 mM DTT. The molar
ratio of biotin to GroEL subunit was 0.8. The streptavidin type-1 P2 crystal
formed on mica-supported lipid bilayer was used as a substrate after fixing
with glutaraldehyde. The streptavidin crystal was rinsed with buffer B. For
high-speed AFM observation, the biotinylated GroEL was diluted to 25 nM
in buffer B and deposited to the streptavidin crystal. After 3 min, the sample
was washed with buffer B to remove unattached GroEL. High-speed AFM
measurement of GroEL-GroES complex was performed in buffer B in thepresence of 1 mM GroES, 1 mM ADP, and 2 mM DTT.

Actin
Actin was purified from rabbit skeletal muscles as described (27). Partially biotinylated F-actin was prepared as follows. G-actin was reactedwith biotin-(AC5)2-sulfo-osu as described (29). The molar ratio of conjugated biotin to G-actin was 2.0. The biotinylated G-actin was copolymerizedwith nonbiotinylated G-actin (1:9 molar ratio) in buffer A containing
0.2 mM ATP, and then stabilized with phalloidin. As a substrate, the streptavidin type-2 P2 crystal formed on mica-supported lipid bilayer was used
after fixing with glutaraldehyde. The streptavidin crystal was rinsed withbuffer A. One micromolar of partially biotinylated F-actin in buffer A wasdeposited to the 2D crystal. After 3 min, the sample was washed with buffer
A to remove excess actin. High-speed AFM visualization of polymerization
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processes of single actin filaments were performed in buffer A in the presence of 1 mM nonbiotinylated G-actin, 0.2 mM ATP, and 2 mM DTT.

TIRFM
Fluorescently labeled proteins were illuminated with a diode pumped green
laser (532 nm; CrystaLaser, Reno, NV). The fluorescence images were
captured at 10 frames/s using an inverted fluorescence microscope (IX71;
Olympus, Tokyo, Japan) equipped with a high NA objective lens (100Apo TIRF NA ¼ 1.49; Nikon, Tokyo, Japan) and a charge-coupled device
camera (Andor Technology, Belfast, Northern Ireland). The recorded
images were averaged over 10 frames.

AFM imaging
For scan sizes R500 nm, images were recorded with a commercial AFM(Nanoscope IVa; Veeco, Santa Barbara, CA) in the tapping mode, using
cantilevers (BioLever mini; Olympus) with a spring constant of 0.1–
0.3 N/m. The cantilever was oscillated at its resonant frequency (~30 kHz
in water). For scan sizes <500 nm, high-speed AFM imaging was carriedout in the tapping mode, using our laboratory-built instrument (15) and smallcantilevers (Olympus) with a spring constant of 0.1–0.2 N/m and a resonant
frequency of 0.8–1.2 MHz in water, as described previously (25).

RESULTS AND DISCUSSION
Surface homogeneity and topographiesof streptavidin 2D crystals
The surface homogeneity and small roughness of streptavidin 2D crystals are important factors for easy identification
by AFM of proteins immobilized on these surfaces. We
examined these properties by imaging streptavidin 2D
crystals by AFM. Fig. 1 shows low magnification AFM
images of three types of streptavidin 2D crystals formedon mica-supported lipid bilayers. The crystals showed P2
space group symmetry with different vector axes (for themolecular arrangements, see Fig. 3), i.e., type-1 (Fig. 1 A),
type-2 (Fig. 1 B), and type-3 (Fig. 1 C). Wide areas werecovered by ordered crystalline lattices, suitable as substrates
for AFM imaging of biological molecules. These crystals
were stable over a period >12 h.
Although not significant obstacles to practical use, thesurface of the type-3 P2 crystal contained a number of line
defects and cracks (Fig. 1 C, arrowheads), due to a relatively
smaller size of crystalline domains. In the type-1 and type-2
P2 crystals, the surfaces were continuous and smooth overa wide area (>1 mm2), except for a small number of defects
such as 1), large particles (Fig. 1, A and C, arrows); and 2),
noncrystallized region (Fig. 1 B, asterisk). These imperfections of the crystals are likely to be induced by the instabilityof lipid bilayers underneath the streptavidin crystals or by not
fully optimized crystallization conditions.
AFM images at medium magnification revealed moredetailed surface structures (Fig. 2). The type-1 P2 crystal(Fig. 2 A) and the type-2 P2 crystal (Fig. 2 B) showed asignificantly low density of vacancy defects. In contrast, asignificant defects such as point (Fig. 2 C, arrowheads)
and line defects (Fig. 2 C, outlined with dashed lines) were
Biophysical Journal 97(8) 2358–2367

FIGURE 1 Low magnification AFM images of streptavidin 2D crystals
formed on mica-supported lipid bilayer. The space group symmetries of
the crystals are (A) type-1 P2, (B) type-2 P2, and (C) type-3 P2. The arrows
indicate large particles found on the streptavidin 2D crystals. The asterisk inpanel B indicates a noncrystallized region. The arrowheads in panel C indicate cracks found on the type-3 P2 crystal. These AFM images wereobtained at a scan rate of 1 s/line (512 pixels). Z scale: 5 nm.

observed in the type-3 P2 crystal. However, these latticedefects cannot be severe obstacles to AFM imaging of proteins immobilized on such surfaces. Proteins immobilized
onto crystalline areas always protrude from the crystal
surface, whereas the vacancies and line defects can be seen
as depressions. Therefore, the proteins attached to the crystal
surface are easily identified. Note that, as mentioned later,
proteins do not attach to the cracks, because the exposed
surfaces are those of lipid bilayers.
Another type of defect is small particles (Fig. 2, arrows).
The height of these particles was 3.3 5 1.3 nm, which is comparable to diameters of many proteins. The similar dimensions might be an obstacle to identifying immobilized
proteins by AFM. However, the density of small particles issufficiently low (1–2 particles per 200  200 nm2) to allow
AFM imaging of immobilized proteins on the surfaces. The
origin of the small particles is uncertain. One possibility could
be misalignments of streptavidin in the crystalline arrays.

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imaging. The cross-sectional views of the 2D crystals(Fig. 3, bottom) indicate that the smallest roughness isobtained in the type-3 P2 crystal. The surface corrugation
amplitude of the type-3 P2 crystal was <1 nm, whereas itwas 2–3 nm in the other two crystals. The surface roughness
measurements are summarized in Fig. 4. For each crystal
surface, the root mean-square (RMS) roughness was calculated using corresponding AFM images from which maximum diffraction spots better than 0.67 nm1 were obtained.
For a comparison, the values of RMS roughness of glass andmica surfaces are also given in Fig. 4. The values of RMS
roughness of the type-1 and type-2 P2 crystals were comparable to that of bare glass surfaces, while the surface roughness of the type-3 P2 crystal was only slightly larger thanthat measured for atomically flat mica.
High-speed AFM imaging of the crystals also showeda different aspect of the crystals. As found by our previous
study with the type-1 P2 crystal (25), the diffusion ofvacancy defects were also observed in the type-2 P2 crystal.
In contrast, the vacancies in the type-3 P2 crystal did not
diffuse, indicating that the intermolecular interactions ofstreptavidin are considerably stronger in the type-3 P2
crystal than those in the other two crystals. In fact, thetype-3 P2 crystal was highly resistant to an increased tapping
force exerted by an oscillating tip. Although the type-1 andtype-2 P2 crystals are somewhat fragile, we can treat themby glutaraldehyde when higher mechanical strength isrequired. After the treatment, the crystal surfaces still retain
sufficiently strong affinity for biotinylated proteins.
FIGURE 2 Medium magnification AFM images of streptavidin 2D crystals formed on mica-supported lipid bilayer. The space group symmetries of
the crystals are (A) type-1 P2, (B) type-2 P2, and (C) type-3 P2. The arrowheads indicate vacancies. The dashed line in panel C outlines a line defect.
The arrows indicate small particles. These AFM images were obtained ata scan rate of 1 s/line (512 pixels). Z scale: 3.5 nm.

The molecular arrangements of streptavidin in the different 2D crystals are clearly identified in the high magnification AFM images (Fig. 3, top). The lattice constants
obtained from the images are a ¼ 5.8 5 0.1 nm, b ¼ 5.8 5
0.2 nm, and g ¼ 89 5 1 for the type-1 P2 crystal (Fig. 3 A);
a ¼ 5.8 5 0.1 nm, b ¼ 11.3 5 0.1 nm, and g ¼ 102 5 3for the type-2 P2 crystal (Fig. 3 B); and a ¼ 5.5 5 0.1 nm,
b ¼ 4.6 5 0.1 nm, and g ¼ 110 5 2 for the type-3 P2
crystal (Fig. 3 C). These values well agree with those obtained in previous studies (18,30,31), when the unit cells
are consistently selected. The Fourier transformation of the
AFM images showed characteristic diffraction patterns forrespective streptavidin 2D crystals (Fig. 3, middle). The
maximum diffraction spots ranging 0.83–0.9 nm1 were obtained for all three types of streptavidin 2D crystals, although
they do not necessarily represent the spatial resolution of theimages (32).
The small roughness of the crystal surfaces is an importantfactor when they are served as substrate surfaces for AFM

Resistance to nonspeciﬁc protein adsorptionof streptavidin 2D crystals
Resistance of substrate surfaces to nonspecific binding ofproteins is important for the immobilized proteins to retain
their native activities and also for selectively attachinga specific protein contained in a multicomponent sample.
Using TIRFM, we examined this issue by measuring nonspecific adsorption of fluorescently labeled CaM to the streptavidin 2D crystals. For the TIRFM measurements, we usedcoverslips as a substitute for mica.
First, we confirmed that streptavidin could be crystallizedon glass-supported lipid bilayers using the same crystallization procedures used for mica. Fig. 5 A shows an AFM imageof the type-1 P2 crystal formed on a glass-supported lipid
bilayer. The type-2 and type-3 P2 crystals were also formed.
The lattice constants of these crystals were consistent withthat formed on mica-supported lipid bilayer. However, thesurfaces of the crystals formed on glass-supported lipid bilayers showed a slight undulation, reflecting the rugged surface of the coverslip underneath the supported lipid bilayer.
Next, TIRFM measurements were performed to examine
nonspecific binding of proteins to the crystal surfaces.
A 0.1 nM Cy3-CaM solution was applied to a flow cell, on
the surface of which the type-1 P2 crystals were formed.
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FIGURE 3 High magnification surface topographies ofthe streptavidin 2D crystals formed on mica-supported
lipid bilayer. The space group symmetries of the crystals
are (A) type-1 P2, (B) type-2 P2, and (C) type-3 P2.
(Top) AFM images of the streptavidin 2D crystals. These
AFM images were obtained at an imaging rate of 1 s/framewith 256  256 pixels. (Middle) Calculated diffraction
patterns from the AFM images shown in the top panels.
The large circles indicate the position of 1 nm1. The small
circles surround diffraction spots with the apparent highest
resolution detected. In the reciprocal lattice, the diffraction
spots can be discerned up to (5, 1) for the type-1 P2, (4, 4)
for the type-2 P2, and (3, 4) for the type-3 P2, corresponding to the apparent resolutions of 1.14, 1.19, and1.17 nm, respectively. Note that these values do not necessarily represent the spatial resolution of the images becauseof an effect of tip-contouring (32). (Bottom) Cross sectionsof the surface profiles along the lines indicated in the AFM
images in the top panels.

After washing the flow cell with buffer-A solution, no fluorescent spots were observed (Fig. 5 B). When 100-times
higher concentration of Cy3-CaM was added without being
followed by washing, a low density of fluorescent spots wasobserved (Fig. 5 C). On the other hand, when 0.1 nM biotinylated Cy3-CaM was used, a significant density of fluorescent spots was observed (Fig. 5 D). Even when CaM was
replaced with bovine serum albumin, we obtained similar
results. Thus, the type-1 P2 crystal surface is resistant tononspecific binding to proteins. In addition, the chemical
fixation of the type-1 P2 crystal with glutaraldehyde had
no effects on this resistance. Comparable results were also
obtained for the type-2 and type-3 P2 crystals althoughdensely packed fluorescent regions were frequently observed
for these crystals. This nonspecific binding is likely due to

FIGURE 4 RMS surfaces roughness of glass, mica, and streptavidin 2D
crystals (type-1, type-2, and type-3) formed on mica-supported lipid bilayer.
The data were obtained from several high magnification AFM images (scanning area of 75  75 nm2) of respective surfaces. The error bars are SD.
Biophysical Journal 97(8) 2358–2367

the partial disruption of lipid bilayer on coverslips underthe buffer conditions used for these types of crystals, since
examinations by AFM imaging for the type-2 and type-3
P2 crystals formed on mica-supported lipid bilayers did

FIGURE 5 Specific and nonspecific adsorption of CaM to the type-1 P2
crystal of streptavidin formed on glass surface. (A) AFM image of thetype-1 P2 crystal formed on a glass surface. The AFM image was obtainedat an imaging rate of 1 s/frame with 256  256 pixels. Z scale: 3.3 nm. (B–D)
TIRFM images of Cy3-CaM attached to the type-1 P2 crystal of streptavidin. (B) After incubating in the presence of 0.1 nM Cy3-CaM for 3 min,the flow cell was washed with buffer A. (C) The flow cell was incubatedin the presence of 10 nM Cy3-CaM without being followed by washingout free Cy3-CaM. (D) Specific binding of biotinylated Cy3-CaM after
the incubation in the presence of 0.1 nM biotinylated Cy3-CaM for 3 min.

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A

B

C

FIGURE 6 Biotinylated CaM immobilized on the type-3
P2 crystal of streptavidin. The successive AFM images
were obtained in the absence (A and C) or presence (B)of Ca2þ, at an imaging rate of 1 s/frame with 256  256
pixels. Z scale: 6 nm. The three sets of imaging for thesame sample were performed in the alphabetical order.
Before taking images of panels B and C, the buffer solution
was replaced with 2 mM Ca2þ-containing buffer-A andbuffer-A alone, respectively. Numbers in the AFM images
indicate frame numbers. (D) Histogram of the height of theimmobilized CaM in the presence or absence of Ca2þ.

D

not show such densely assembled regions of applied proteins(CaM, GroEL, GroES, and actin).
Ca2þ-induced conformational change in CaM
As the type-3 P2 crystal shows the smallest roughness
among the three types of crystals (Fig. 4), it is a suitable
substrate when structural changes of relatively small proteins
are imaged by AFM. Here, we used CaM (16.7 kDa) as asmall protein to be visualized on the type-3 P2 crystal.
CaM is a Ca2þ binding protein with the N-terminal andC-terminal globular domains connected by a central linker
helix (33). Each globular domain contains two Ca2þ bindingsites. CaM undergoes a conformational change upon Ca2þbinding (34,35).

Fig. 6 A shows successive AFM images captured at
1 s/frame of biotinylated CaM specifically bound to thetype-3 P2 crystal in the absence of Ca2þ. Rather than a pairof two globular domains as expected from earlier structural
studies (33,34), the immobilized CaM was observed as single
globes. This probably arises from lack of immobilization ofeither of the globular domains due to the low content of biotin(0.6 per molecule) and the intrinsic flexibility of the central
linker helix. In fact, noise spikes frequently appeared aroundthe globes, indicating rapidly moving moieties around theglobes. The addition of Ca2þ to the sample changed theappearance of CaM, resulting in larger lateral dimensions
and greater height (Fig. 6 B). The average height wasincreased from 1.9 5 0.3 nm to 2.6 5 0.4 nm (Fig. 6 D), while
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the average half-width was increased from 4.3 5 1.0 nm to
5.9 5 1.1 nm. This change in height and width was fully
reversible in the same sample (Fig. 6 C), representing theconformational change of CaM.
Previous structural studies have indicated that the conformational change of CaM upon binding to Ca2þ at each
globular domain is drastic (33,34). The binding of Ca2þ
induces an opening of the two EF-hand motifs in each
globular domain. As a result, the distances between two
helices in each EF-hand substantially increase (2.5–6.2 Å)
upon binding to Ca2þ. The increase in the dimension of
CaM observed in Fig. 6 B is close to the opening of theEF-hands in the immobilized globular domain. This result
indicates that AFM measurements on the streptavidin
crystal enable the detection of conformational change ofthe immobilized proteins in the z dimension at subnanometer
precision.
Immobilization of GroEL with controlledorientation
Streptavidin 2D crystals will benefit AFM imaging studies, if
the orientation of the immobilized proteins can be controlled.
This control can be achieved by the introduction of morethan two biotin molecules at specific sites of a protein. Here
we immobilized GroEL on the type-1 P2 crystal surface ina side-on orientation. GroEL and GroES are chaperonin
proteins that assist folding of other proteins in vivo andin vitro (36,37). GroEL is a cylindrical protein composedof two heptameric rings stacked back to back, each ofwhich is comprised of seven identical ATPase subunits(38). During the ATPase cycle, GroES transiently binds tothe ends of the double rings (39,40).
Previously, the association and dissociation between
GroEL and GroES were directly observed by AFM (11,12).
In these studies, GroEL was adsorbed onto a mica surface, inwhich the GroEL has a strong tendency to take an end-up
orientation. In this orientation, only one GroEL ring is
exposed to the aqueous buffer and accessible to GroES
floating in the solution. To solve this problem, previous
TIRFM studies introduced biotin to the Cys-490 residue
located at the outer surface of the equatorial domain of the
D490C GroEL mutant (41,42). The resulting biotinylated
GroEL is expected to be anchored on a streptavidin layer
in a side-on orientation, but no evidence for this side-onorientation of the anchored GroEL was ever provided fromother studies.
Fig. 7 shows successive AFM images of the biotinylated
GroEL mutant immobilized on the type-1 P2 crystal in thepresence of GroES and ADP. Two species with different
appearances were observed. One exhibited a rectangle shape(Fig. 7, arrowhead) (rather than a circular shape, as has been
observed with GroEL on a mica surface), indicating GroEL
alone in a side-on orientation, and the other exhibited a bullet
shape (Fig. 7, arrow), indicating a 1:1 complex of GroEL
Biophysical Journal 97(8) 2358–2367

FIGURE 7 AFM images of GroEL alone and GroEL-GroES complex.
The GroEL is D490C mutant, the Cys-490 residues of which are labeledwith biotin, and is immobilized in a side-on orientation on the streptavidin
type-1 P2 crystal that is chemically fixed with glutaraldehyde. The arrowhead indicates GroEL alone and the arrow indicates the GroEL-GroES
complex. The dashed lines indicate the end positions of the GroEL. The
asterisks indicate GroES bound to one of the GroEL rings. The AFM images
were obtained at an imaging rate of 2 s/frame with 200  200 pixels. Z scale:
19 nm. Numbers in the AFM images indicate frame numbers.

and GroES (39). Interestingly, the protruded side of thebullet-shaped GroEL is switched (asterisks; compare theimages in Fig. 7), indicating GroES release from one endof the two rings, which is followed by GroES binding tothe other end. This behavior occurred reversibly in the
GroEL molecule, although most GroEL molecules wererather stable and did not show such dynamic association/
disassociation events during the imaging period. Note that
GroES never bound to the streptavidin crystal surface evenwhen its concentration was relatively high. As exemplified
here, it is possible to immobilize proteins in a desired orientation by introducing multiple biotins to a protein at designed
sites.
Visualization of actin polymerization
Here, we further verified the applicability of the streptavidin
2D crystal surfaces to dynamic AFM imaging by directly
observing actin polymerization on the type-2 P2 crystal
surface. The polymerization of actin is a key process ina variety of cellular functions (43). To date, many studies
have demonstrated that the polymerization of actin consistsof nucleation and growth steps followed by dynamic equilibrium in the steady state. It is also well known that actin filaments have a structural polarity, so that the polymerization
kinetics is different at the two ends of the filaments; theStreptavidin Substrata for Dynamic AFM

2365

A

B

polymerization rate is higher at the barbed end than at the
pointed end. Previous TIRFM imaging studies (44,45) on
the polymerization dynamics with individual actin filaments
have suggested a polymerization unit not to be composed ofmonomers alone but, instead, an average of monomers, oligomers, and fragments. However, analysis of precise molecular events occurring at the filament edges has been difficult
due to the limited spatial resolution of optical microscopy. In
contrast, high-speed AFM allows direct observation of thepolymerization process at the molecular level, thanks to thehigh spatial resolution and subsecond temporal resolution.
Fig. 8 A shows AFM images captured at 0.25 s/frame fora polymerizing actin filament in the presence of nonbiotinylated G-actin (also see Movie S1 in the Supporting Material).
Instead of finding actin filament ends, the ends were
produced by breaking an actin filament (Fig. 8 A, 0 s)
upon applying an increased tapping force (Fig. 8 A, 2.0–
2.5 s). This procedure allowed simultaneous visualizationof both barbed and pointed ends in the relatively small

FIGURE 8 Polymerization process at actin filament ends
observed by high-speed AFM. (A) High-speed AFM
images of partially biotinylated actin filaments on the streptavidin type-2 P2 crystal that is chemically fixed withglutaraldehyde. The dashed lines and dot-dash lines indicate the initial positions of the barbed and pointed ends,
respectively. The ends were produced by partially dissecting the filament by an oscillating cantilever tip. The arrows
indicate the positions of the growing barbed end of thedissected actin filament. The successive images were obtained at an imaging rate of 0.25 s/frame with 256  128
pixels. Z scale: 11 nm. (B) Change with elapsed time in
the barbed-end position along the longitudinal axis of thefilament. The position was measured from the successive
AFM images. The arrows indicate decrease in the lengthof the filament.

scan area. The actin filament showed stepwise elongation
at one end (Fig. 8 A, arrows at 6.75 s–13.5 s, and Fig. 8 B),
whereas at the other end, elongation was not observed in theimaging period. From this difference, the end on the lefthand is assigned to be the barbed end. The observed elongation rate at the barbed end was 0.11 mm/min, which iscomparable to the values obtained previously (44,45). Interestingly, a stepwise decrease (3.5 5 1.4 nm) in the length
was occasionally observed (Fig. 8 B, arrows). An actin filament is composed of two filamentous actin strands organized
into an apparent double helix. The actin subunits are spaced
5.5 nm apart along each helix. The two strands are axiallystaggered relative to each other by half the subunit repeat(i.e., 2.75 nm). Based on this structure, we can make acorrelation between the observed elongation step lengths(Fig. 8 B: 3.1 5 0.9 nm and 5.2 5 1.1 nm) and the numbersof the molecules incorporated. The smaller step length isclose to the half-length of an actin subunit, whereas the larger
one agrees well with the size of an actin subunit. The former
Biophysical Journal 97(8) 2358–2367

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result indicates monomer binding to the end of one strand thatis recessed relative to the end of the other strand. On the other
hand, the latter result indicates dimer binding; the barbed endof each strand is bound by an actin subunit, so that the filament
elongates by the size of an actin subunit. Therefore, bothmonomers and dimers are incorporated into the barbed endof actin filaments during the polymerization process.
In a previous TIRFM study on actin polymerization,
the polymerization unit was estimated to be six monomers
on average (44). This value is significantly larger than thatobtained in this study. One possible reason for this discrepancy may be attributed to the annealing of actin fragments, inwhich fragments associate to each other at their ends to forma longer filament. The annealing rate is comparable to thepolymerization rate as reported previously (46). Therefore,
annealing events will make the apparent polymerization
unit larger. We did not observe the annealing process inthis study. A further examination of actin polymerization
by high-speed AFM will provide deeper insights into theactin polymerization process.
CONCLUSION
The characterization of the three types of streptavidin 2D
crystals showed their appropriate properties as substrates towhich proteins are immobilized for dynamic AFM imaging,i.e., uniformity over relatively wide areas, small roughness,and resistance to nonspecific binding to proteins. Amongthe three types of crystals, the type-3 P2 crystal seems themost appropriate choice for a wide range of biologicalsamples because of the smallest roughness and the highest
stability (glutaraldehyde treatment is unnecessary). Although
type-3 P2 crystals often contain line cracks, they are also
resistant to nonspecific binding to proteins because theirsurfaces are those of lipid bilayers. When the presence ofline cracks is undesired in dynamic AFM imaging of a biological sample, we can use the type-1 or type-2 P2 crystalsof which surfaces have high uniformity over a wide area.
The stable immobilization of oligomerized proteins such as
GroEL and actin filaments to the surfaces can be achieved
easily by introducing multiple biotin molecules. The abilityof the crystal surfaces to specifically bind to biotinylated
proteins (or His-tag conjugated proteins) without nonspecificbinding to proteins allows studying dynamic interaction
processes between protein molecules, as demonstrated here
in the observation of GroES-GroEL binding and actin polymerization.
SUPPORTING MATERIAL
A movie is available at http://www.biophysj.org/biophysj/supplemental/
S0006-3495(09)01309-5.
This work was supported by the Core Research for Evolutional Science andTechnology project and the project of Development of System and Technology for Advanced Measurement and Analysis (Japan Science and TechBiophysical Journal 97(8) 2358–2367

Yamamoto et al.
nology Agency, Japan), and a Grant-in-Aid for Basic Research (S) (No.
20221006; Japan Society for the Promotion of Science, Japan).

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Biophysical Journal 97(8) 2358–2367