DOI: 10.1002/cphc.200800210

Visualization of Intrinsically Disordered Regions ofProteins by High-Speed Atomic Force Microscopy
Atsushi Miyagi,[a] Yasuo Tsunaka ,[b, c] Takayuki Uchihashi ,[a, d] Kouta Mayanagi ,[b, e, f]
Susumu Hirose,[g] Kosuke Morikawa ,[b, d] and Toshio Ando *[a, d]Intrinsically disordered (ID) regions of proteins are recognized to
be involved in biological processes such as transcription, translation, and cellular signal transduction. Despite the important rolesof ID regions, effective methods to observe these thin and flexible
structures directly were not available. Herein, we use high-speed
atomic force microscopy (AFM) to observe the heterodimeric
FACT (facilitates chromatin transcription) protein, which is predicted to have large ID regions in each subunit. Successive AFM
images of FACT on a mica surface, captured at rates of 5–17
frames per second, clearly reveal two distinct tail-like segmentsthat protrude from the main body of FACT and fluctuate in position. Using deletion mutants of FACT, we identify these tail segments as the two major ID regions predicted from the aminoacid sequences. Their mechanical properties estimated from the
AFM images suggest that they have more relaxed structures thanrandom coils. These observations demonstrate that this state-ofthe-art microscopy method can be used to characterize unstructured protein segments that are difficult to visualize with otherexperimental techniques.

1. Introduction
Proteins encoded in eukaryotic genomes often contain unstructured regions (> 50 residues).[1, 2] These disordered regions
lack intrinsically well-defined three-dimensional (3D) structures,but frequently fold into ordered conformations upon binding
to their extrinsic targets; this folding is termed “coupled folding”.[3] Such intrinsically disordered (ID) regions are highly conserved among species and are often involved in intra- and intermolecular recognition. Most unstructured segments comprise flexible linkers that play important roles in the assemblyof macromolecular complexes and in the recognition of smaller
biomolecules. The functional importance of ID regions was recently recognized, particularly in transcription, translation, andcellular signal transduction. However, the dynamics and functions of ID regions remain unclear at the molecular level. Thisis mainly due to a lack of useful techniques for analyzing unstructured segments of this sort at the single-molecule level.
Indeed, X-ray crystallography and electron microscopy do notallow us to observe ID regions directly. Atomic force microscopy (AFM) is a widely used technique for imaging biologicalmolecules at nanometer resolution.[4–6] The major advantage of
AFM is that it can operate under physiological conditions, andit provides unique information by direct visualization of individual molecules in solutions. However, due to the poor temporal resolution of conventional AFM (about one frame perminute), only time-averaged features of proteins lacking large
structural variation over time can be imaged. In solution, only
samples that are firmly attached to a substrate directly or
through adhesive chemicals can be observed; the alternative isto observe dried samples. Unfortunately, on drying, thin andflexible unstructured polypeptides tend to form lumps, or
become flattened due to their strong attachment to a surface.
In addition, they are not easily adsorbed onto substrate surfaChemPhysChem 2008, 9, 1859 – 1866

ces in solution, even with the assistance of adhesive chemicals.
On the other hand, high-speed AFM, which can capture
images at a rate surpassing ten frames per second, can directly
visualize structural changes in biological molecules evenagainst a background of Brownian motion.[7–10] Thus, highspeed AFM could potentially visualize unstructured ID regionsof proteins.
Examples of typical ID regions have been identified in proteins constituting the complex machinery involved in transcrip-[a] A. Miyagi, Dr. T. Uchihashi , Prof. Dr. T. Ando
Department of Physics
Kanazawa University
Kakuma-machi, Kanazawa 920-1192 (Japan)
Fax: (+ 81) 76-264-5739
E-mail: tando@kenroku.kanazawa-u.ac.jp[b] Dr. Y. Tsunaka , Dr. K. Mayanagi , Prof. Dr. K. Morikawa
The Takara-Bio Endowed Laboratory
Institute for Protein Research, Osaka University
6-2-3 Furuedai, Suita, Osaka 565-0874 (Japan)[c] Dr. Y. Tsunaka
JSPS, Ichiban-cho, Chiyoda-ku, Tokyo 102-8472 (Japan)[d] Dr. T. Uchihashi , Prof. Dr. K. Morikawa , Prof. Dr. T. Ando
CREST, JST, Sanban-cho, Chiyoda-ku, Tokyo 102-0075 (Japan)[e] Dr. K. Mayanagi
Nagahama Institute of Bio-Science and Technology
1266 Tamura-cho, NagahamaShiga 526-0829 (Japan)[f] Dr. K. Mayanagi
BIRD, JST, 5-3 Yonban-cho, Chiyoda-ku, Tokyo 102-8666 (Japan)[g] Prof. Dr. S. Hirose
Department of Development Genetics
National Institute of Genetics, Mishima
Shizuoka-ken 411-8540 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cphc.200800210.

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tion.[11, 12] For instance, the FACT (facilitates chromatin transcription) protein is predicted to contain large ID regions on thebasis of its amino acid sequences. Displacement of histone
H2A/H2B dimers from nucleosomes by FACT facilitates RNA
polymerase II transcription[13, 14] and chromatin remodeling.[15]
FACT is highly conserved in eukaryotes, and is a heterodimer
consisting of structure-specific recognition protein-1 (SSRP1)
and SPT16; the latter has a higher molecular mass than theformer. Intrinsically disordered regions are predicted to exist in
the charge-enriched termini of SSRP1 and SPT16. The predicted ID region of SPT16 is essential for mRNA transcriptional
elongation by FACT.[13] Herein, we use high-speed AFM to observe FACT and two of its deletion mutants. Successive AFM
images, captured at an imaging rate exceeding five frames per
second, clearly reveal two undulating tail-like structures of different lengths. The AFM analysis of the FACT deletion mutants
demonstrates that the two tail domains correspond to the predicted ID regions of SSRP1 and SPT16. In addition, the analysisof the AFM images reveals a unique mechanical property ofthe tail domains that suggests more relaxed conformations
than random coils.

minal region (residues 889–1044) of the SPT16 subunit wasalso predicted to be an ID region, indicated by a blue box in
Figure 1. Based on these predictions, we constructed two FACT
mutants. The first FACT mutant, termed M1, includes an intact
SPT16 subunit plus recombinant SSRP1 lacking 40 % of theamino acids from the C terminus (M1 in Figure 1). The second
FACT mutant, termed M2, includes an intact SSRP1 subunit
plus SPT16 lacking 15 % of the amino acids from the C terminus (M2 in Figure 1).

2.2. TEM Observation of Wild-Type FACT
We first obtained a TEM image of wild-type (WT) FACT
(Figure 2). The TEM image shows particles of various sizes,
most likely because FACT tends to form oligomers spontane-

2. Results and Discussion
2.1. Prediction of ID Regions in FACT
Primary-sequence analysis of FACT reveals that the smaller
SSRP1 subunit contains a structure-specific recognition (SSRC)
motif and a high-mobility group (HMG)-box domain, while
SPT16 has an amino peptidase-like domain (WT in Figure 1).
The solution structure of the Drosophila SSRP1-HMG-box
domain was previously determined by NMR analysis.[16] Two Cterminal segments around the HMG domain (residues 405–554
and 625–723), indicated by red boxes in Figure 1, were predicted to be the ID regions of the SSRP1 subunit. The acidic C-ter-Figure 2. Transmission electron micrograph of FACT on a carbon-coated EM
grid. Most of the FACT molecules form oligomers, but some remain monomers (indicated by arrows). The estimated size of the monomer is about
15 nm.

ously. These oligomers were also indicated by gel filtration(data not shown). Currently, the physiological significance of
FACT oligomerization is unclear. We observed that some particles have a minimum and similar size (arrows in Figure 2),
which suggests that these particles correspond to the non-oligomerized (i.e., monomeric) FACT protein. The monomericmolecule is oval-shaped and about 15 nm in length along the
major axis. The TEM images show solid and globular structures,
but not tail-like structures.
2.3. AFM Observation of WT FACT

Figure 1. Schematic representation of the domain organization of the FACT
proteins analyzed in this study. WT: Domain structure of wild type Drosophila
SSRP1 and SPT16 proteins. The ID regions of SSRP1 and SPT 16 are predictedfrom their amino acid sequences and are highlighted in red (residues
405–554 and 625–723) for SSRP1 and blue (residues 889–1044) for SPT16.
M1: Domain structure of the FACT mutant termed M1 in which SSRP1 residues 420–723 are deleted. Most of the SSRP1 ID regions, as well as the HMG
domain, are missing. M2: Domain structure of the FACT mutant termed M2
that lacks the predicted SPT16 ID regions (residues 891–1044).

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High-speed AFM imaging also shows that WT FACT forms oligomers similar to those seen in the TEM images. We obtained
successive AFM images of oligomeric FACT at an imaging rateof six frames per second (Figure 3 and Supporting Information,
Video 1). Two distinct structures are clear in the images. The
first one is a lumpy structure that appears to be strongly adsorbed onto the mica surface; its shape is almost unchanged
over time. The second is a tail-like structure with a height ofless than 1 nm. In the case of the oligomer shown in Figure 3,
four tail-like structures are visible. We frequently detected several tail-like structures anchored to larger oligomers. These taillike structures exhibit rapid fluctuations in position due to

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Visualizing Intrinsically Disordered Regions of Proteins
the major axis of the globular structure are about 4 and25 nm, respectively. Taking into account the tip radius of about
4 nm, estimated with an electron microscope, the true widthof the solid structure is approximately 17 nm.[17] This size is
close to the size of a FACT monomer estimated from the TEM
image. We therefore conclude that the image of the FACT protein shown in Figure 4 represents a monomer.

Figure 3. Successive AFM images of oligomerized FACT captured at an imaging rate of six frames per second. The scan range is 150150 nm. A lumpy
shape and four tail-like structures can be seen (see inset schematic). The position of the tail-like structures fluctuate markedly over time. The images
were processed by using brightness-equalizing software.

Brownian motion, indicative of flexibility and relatively weakadsorption on the mica surface. We sometimes observed smaller particles with a globular shape and two tail-like structuresof different lengths; an example is shown in Figure 4 and theSupporting Information (Video 2). In Figure 4 a , the longer taillike structure is labeled “i”, and the shorter “ii”. In the cross-section profile shown in Figure 4 b , the height and full width of

Figure 5. Successive AFM images of a FACT monomer taken every 156 ms.
The scan size is 150150 nm. The two tail domains appeared to wobble on
the mica surface.

Figure 5 shows successive AFM images of a FACT monomer.
This gallery of images clearly reveals the dynamic behavior of
the two distinct tails that are anchored to the globular structure and undulate in the solution. Figure 6 shows a histogram

Figure 6. Histogram of the macroscopic contour length estimated for the
tail domains. This shows two small peaks at 17.5 nm and 27.5 nm, although
they are not statistically distinctive.

Figure 4. a) AFM image of a FACT monomer captured at an imaging rate of
6.4 frames per second. The scan size is 150150 nm. One globular and two
tail-like structures can be seen. The longer tail-like structure is indicated by‘i’, and the other by “ii”. b) Cross-sectional profile at the position indicatedby the white line in a). The height and full width are about 4 and 25 nm, respectively. The size of the globular shape is consistent with that observedby TEM when the broadening effect of the tip radius is taken into account.

ChemPhysChem 2008, 9, 1859 – 1866of the apparent contour lengths measured for the two tails.
One can see two small peaks around 17.5 and 27.5 nm, although they are not so statistically distinctive. These observations suggest that the two observed tails correspond to thetwo large ID regions predicted from the amino acid sequences
in both subunits (SSRP1 and STP16) of FACT. The estimatedheight of the tails is 0.6 nm. Slow AFM imaging of dried FACT
samples do not show the tail structures (data not shown). This

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is probably because these regions cannot remain extended
under the conditions we used, or because the drying process
flattens these regions so much that they are no longer detectable by AFM. In fact, under dried conditions, even the globular
structure of FACT has a height of only about 0.6 nm (versus
4 nm by AFM in solution). Slow in-liquid imaging of FACT fixedon aminosilane- or spermine-coated surfaces also do notreveal the tail structures, and high-speed imaging of thesesamples shows that the ID regions are not firmly attached tothe surfaces. Glutaraldehyde fixation of the samples on theaminosilane-treated surfaces results in lump-shaped structuresthat makes identification of the ID regions impossible. In addition, TEM combined with an averaging process cannot be applied because of the smeared images. We conclude that visualization of the thin unstructured ID regions of FACT is possible
only by using high-speed AFM imaging in aqueous solutions.

2.4. Identification of the ID Regions of FACT by Means ofDeletion Mutants
To determine definitively whether the predicted ID regions of
FACT correspond to the tail structures observed with highspeed AFM, we constructed two FACT deletion mutants. In
mutant M1, the predicted ID regions and the HMG-box
domain of SSRP1 were deleted, and in M2 the predicted ID
region of SPT16 was deleted. Representative high-speed AFM
images of mutants M1 and M2 are shown in Figure 7 (also see

Figure 8. Histograms of the apparent contour lengths estimated for the tailsegments of a) M1 and b) M2. The contour lengths at the peaks are 17.5 and27.5 nm for M1 and M2, respectively, while their average values are 17.8 and26.2 nm, respectively.

peak values of the histogram obtained for the WT-FACT shownin Figure 6. Assuming that the distance between two aminoacid residues ranges from 0.15 to 0.36 nm, the stretchedlengths of the predicted ID regions of SSRP1 and SPT16 are estimated to be 37–90 and 23–56 nm, respectively. The observed
average lengths of the tails are approximately one-third of thetheoretical maximum lengths. The observed maximum lengths
are 50–60 % of the theoretical maximum lengths. These differences are not due to an insufficient AFM imaging rate, as indicated in Section 2.5. Even for a completely loose polypeptide
chain, its macroscopic contour length is shorter than the fully
stretched length due to an entropic effect. For a polymer chain
containing N identical segments which are connected throughfree joints, the entropic effect results in a smallerpﬃﬃﬃﬃ molecularsize than the fully stretched form by a factor of 1/ N.[18]2.5. Imaging-Rate Dependence of ID Region Detection
Figure 7. AFM images of the a) M1 and b) M2 mutants. The domain structures of the M1 and M2 mutants are depicted in Figure 1. Both images wereacquired at an imaging rate of six frames per second. The scan range is200200 nm.

the Supporting Information, Video 3 and Video 4). The M1
mutant has only one short tail segment, as shown in Figure 7 a,and the M2 mutant has only one longer tail segment. Therefore, the tail domains observed in the M1 and M2 mutants are
assigned to the ID regions of SPT16 and SSRP1, respectively.
The ID regions of SSRP1 and SPT16 contain 249 and 156 aminoacid residues, respectively. It is thus reasonable that the tailstructure observed in images of M2 is longer than that of M1.
We measured the apparent contour lengths of both tails fromthe AFM images, and found that the averaged contour lengthsof the observed tails of M1 and M2 are 17.8 and 26.2 nm, respectively (see histograms in Figure 8). In the most extended
forms, the contours reach about 35 and 42 nm for the M1 andM2 tails, respectively. The wide distribution of apparent contour lengths is a fingerprint of the flexible structures. Theseaverage contour lengths approximately coincide with the two1862

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We investigated the effect of the AFM imaging rate on the appearance of the tail-like structure in M2 (Figure 9). We used theM2 mutant for this study, since M2 has a longer ID region (i.e.
a longer tail). At an imaging rate of 12.5 frames per second(Figure 9 a), the tail-like ID region is clearly seen. Even at fiveframes per second (Figure 9 b), the ID region is still observed
as a string. However, the free end of the ID region seems
broader in both these images, presumably due to its rapidchanges in position. At two frames per second (Figure 9 c), theID region is still observable, but is blurry. Finally, at one frame
per second (Figure 9 d), the ID region is no longer discernible
as a string, but appears hazy; even the ordered globular
region of M2 has an irregular shape, probably because diffusion of M2 on the mica surface is faster than the imaging
speed. These experiments suggest that an imaging rate greaterthan about five frames per second is required to visualize the
ID regions of FACT clearly. Therefore, the imaging rate (sixframes per second or higher) used for imaging M1 and M2
does not significantly affect the measurements of the apparent
contour lengths of the ID regions.

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Visualizing Intrinsically Disordered Regions of Proteins

Figure 9. AFM images of mutant M2 acquired at different imaging rates.
a) 12.5, b) 5, c) 2, and d) 1 frame per second. The images shown are
6262 nm clipouts from 10062 nm images obtained for the same single
molecule of the M2 mutant.

2.6. Mechanical Properties of ID Regions
Here we analyze mechanical properties (stiffness and flexural
relaxation time) of the observed tail-like structures. The stiffness of a polymer chain is described by the persistence length
p of the chain, that is, the length over which the average correlation in the tangent direction decays by 1/e. In two dimensions, the mean square point-to-point distance of the chain isgiven by Equation (1)[19]



2p 
hr2 ðlÞi2D ¼ 4pl 1 
1  el=2plð1Þwhere l is the contour length between two points on the
chain. For l!1, hr2(l)i2D = 4pl. The persistence length depends
on the hierarchy of the polymer structure. For example, thepersistence length of a double-helical DNA strand is different
from those of thicker strings formed by supercoiling of a DNA
strand. In the AFM images obtained for the tail-like structuresof FACT, polypeptide chains are not resolved. Therefore, thepersistence lengths that can be obtained from the images arethose for overall (macroscopic) string structures (see inset
image in Figure 10 b showing the relationship between the microscopic and macroscopic structures). We analyzed the taillike structures of mutants M1 and M2. The mean-square pointto-point distance r2 as a function of l is well-fitted by Equation (1) in both cases, and gives persistence lengths of 10.5
and 12.1 nm for the tail-likes structures of M1 and M2, respectively (Figure 10). These similar values indicate that the two ID
regions have physically similar structures.
To estimate the persistence length of the microscopic structures of the ID regions, we assume that their C-terminal ends
are located near the free ends of the tail-like structures. Under
this assumption, the two-dimensional end-to-end distances R
were obtained. The average distance hRi of the longer tail contained in M2 is 21.7 nm, while that of the shorter tail contained
ChemPhysChem 2008, 9, 1859 – 1866

Figure 10. Persistence-length determination of the macroscopic structuresof the tail segments of mutants a) M1 and b) M2. The mean-square point-topoint distance is plotted as a function of the macroscopic contour length
between the corresponding two points. The solid lines indicate the best-fit
curves of Equation (1) with a) p = 10.5 nm and b) p = 12.1 nm. The inset
image indicates the relationship between the microscopic contour and themacroscopic structure. The microscopic contour is schematically drawn as athin white line.

in M1 is 15.5 nm. The microscopic persistence lengths Lp were
estimated by using these average values and the relationship
hR2i = 4 LpL, where L is the polypeptide chain length. The ID regions and HMG-box domain of SSRP1 contain a total of 319
amino acid residues, while the ID region of SPT16 contains 156
amino acid residues. Supposing that the distance between adjacent amino acid residues is 0.36 nm for each adjacentcouple, the microscopic contour lengths of the tail-like structures of M1 and M2 are 56 and 114 nm, respectively. Thus, themicroscopic persistence lengths are estimated to be 1.2 and
1.1 nm for the tail-like structures of M1 and M2, respectively.
The shorter persistence length of the M2 tail is likely due tothe presence of the HMG-box domain. Note that these values
are minimum estimates, since the employed values for thecontour length are those at the maximum limit. Microscopic
measurements of persistence length have previously been performed by measuring force–extension curves by AFM,[20, 21] optical tweezers,[22] and magnetic tweezers.[23] The microscopic
persistence length of the PEVK region (186 amino acid residues), which is responsible for the elastic property of titin andis thought to form a random coil, is reported to show a wide

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range of Lp (0.4–2.5 nm, on average ca. 0.93 nm).[21] On theother hand, for proteins with ordered structures, the reported
values for the microscopic persistence length are within arange of 0.36–0.5 nm.[20, 24] Thus, the long microscopic persistence lengths of the ID regions of FACT are fingerprints of theirvery loose structures, which are slightly more relaxed than the
random coil of the PEVK segment.
The Young modulus E is a material property that represents
its stiffness and is related to the macroscopic persistence
length by Equation (2)[25]
E¼

pkB T
H

ð2Þ

where kB is the Boltzmann constant, T the absolute temperature, and H the momentum of inertia of the cross section of
the object. Assuming that the tail-like structures of FACT arethin rods with radius 1, H is given by H = p14/4. We estimated
1 (1–1.6 nm) from the width of the tail-like structures in several
AFM images obtained by using different cantilever tips, taking
into account the tip radii. The width is less affected by the substrate surface than the height, and its estimation is reliable
when the sample height is similar to or smaller than the tip
radius. Thus, the estimated Young modulus of the two tail-likestructures is 9–58 MPa from their similar macroscopic persistence lengths. This estimated value of the macroscopic stiffness of the ID regions is 1–2 orders of magnitude smaller thanthose reported for globular proteins such as an actin monomer(ca. 2 GPa)[26] and lysozyme (0.5–2 GPa).[27, 28] The value for an
actin monomer was estimated from the force–extension relationship measured for single actin filaments.[26] The value of
0.5 GPa for lysozyme was estimated by AFM force–indentation
experiments with a tip with a radius of about 20 nm,[27] andthe value of 2 GPa for lysozyme by measuring the speed of ultrasound propagation through a lysozyme solution.[28] Therefore, these values represent the macroscopic stiffness of these
proteins. Interestingly, the Young modulus as an estimate of
the macroscopic stiffness of bovine carbonate anhydrase II isreported to be in the range of 2–6 MPa when the sample is incubated in the presence of 2–6 m guanidine·HCl.[29] Althoughthese values of Young’s modulus are somewhat smaller thanthose of the ID regions of FACT, the structures of the ID regions are considered to be roughly similar to those of denatured proteins.
A tethered polymer chain exhibits cyclic dynamics, andtherefore its complex motion cannot be described simply withdiffusion constants. Polymer dynamics is usually considered byusing the Rouse model,[30] in which the polymer chain is modeled as a series of blobs (Rouse segments) connected bysprings. The polymer motion contains a series of normal
modes. However, in practical situations, the polymer dynamics
is dominated by the slowest fundamental mode. In a small extension regime, the dynamics is similar in the longitudinal andtransverse directions.[31] To estimate the relaxation time, wemeasured the end-to-end distance of the M2 tail as a functionof time using images captured at 16.7 frames per second.
Then,
wecalculated
anautocorrelation
function

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G(t) = hR(t)hRi)[RACHTUNGRE(t+t)hRi]i. The autocorrelation function isnearly zero (data not shown), irrespective of the t values(except for t = 0), that is, the structural relaxation occurs fasterthan the imaging rate in spite of the presence of a drag effectof the mica surface.

2.7. Charged Residues in ID Regions
It was previously reported that a combination of low mean hydrophobicity and relatively high net charge is a prerequisite
for the absence of ordered structures of proteins under physiological conditions.[32] In fact, the acidic and basic clusters areenriched in the ID regions of transcriptional factors and DNA
binding proteins. These ID regions play important functionalroles in transcriptional regulation.[33] The ID regions of FACT
indeed have low hydrophobicity and both positively and negatively charged clusters (Figure 11). In our AFM observations,Figure 11. Distribution of charged amino acids in the ID regions of a) SPT16
and b) SSRP1. Positive and negative charges are indicated in blue and red,respectively. The HMG-box domain of SSRP1 is shaded green.

FACT and its mutants are placed on a negatively charged mica
surface. In principle, it is possible that the electrostatic interaction between the basic clusters of FACT and the negative
charges of a mica surface alter the native structures of the ID
regions to form more extended forms. It is difficult to exclude
this possibility, although we think that such alteration is unlikely, as the interaction is weak. However, the results are still interesting even were this to be the case, because DNA is also richin negative charges and may act on FACT similarly to the mica
surface.

3. Conclusion
This is the first study to use high-speed AFM to observe ID regions directly. The AFM images provide a direct view of FACT
in solution on a mica surface; in this view, the two distinct taillike structures of different lengths of FACT undulate and

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Visualizing Intrinsically Disordered Regions of Proteins
wobble. This method for visualizing ID regions can be applied
to other proteins, including mammalian nuclear proteins that
contain many long unstructured segments. High-speed AFM
can also be used to assist in successful protein crystallization,
since predicted ID regions can be confirmed and deleted
before crystallization to facilitate crystal growth. Herein, slow
imaging of FACT samples that were dried or attached to amica surface through adhesive chemicals did not reveal thetail structures. We found that imaging rates higher than about
five frames per second are required to visualize the flexible ID
regions clearly in physiological solutions. Our results indicatethat high-speed AFM is a new imaging methodology that canbe used to characterize flexible, unstructured segments of proteins at the single-molecule level.

Experimental Section
Prediction of ID Regions: ID regions of FACT were predicted byusing the genomes-to-protein (GTOP) structures and functions database. The GTOP is a database of protein structures predictedfrom genome sequences.[34] Predictions were mainly carried out by
using the homology search program PSI-BLAST.[35] Similar results
were also predicted by other programs such as Disprot.[36]Construction, Expression, and Purification of FACT Proteins: We
used the same Drosophila melanogaster FACT cDNA as in our previous study.[15] The SSRP1 and SPT16 cDNAs were cloned into thepCold I and pCold III vectors, respectively.[37] The molecular massesof SSRP1 and SPT16 are 84 and 122 kDa, respectively. The expression plasmids were designed to co-express the SSRP1 and SPT16
proteins by using two inducible promoters in the pCold vectors.[38]The FACT proteins were simultaneously overexpressed in Escherichia coli strain BL21 (DE3). Protein co-expression was induced at
OD600 = 0.6 by cold shock from 37 to 16 8C and by the addition of
isopropyl-b-d-thiogalactopyranoside (IPTG, 1 mm). Cells were harvested, resuspended in buffer A containing Tris–HCl (20 mm)
pH 8.5, NaCl (0.5 m), glycerol (10 %), and 2-mercaptoethanol(5 mm), and lysed by sonication. After pelleting, the supernatant of
the cell lysate was loaded onto a Histrap column (GE Healthcare).
After washing the column with buffer A containing imidazole(20 mm), bound proteins were eluted with buffer A containing imidazole (500 mm). Finally, the proteins were purified on a Hitrap Q
anion-exchange column (GE Healthcare) by elution with a concentration gradient of sodium chloride (0.25–1 m).
TEM Observations: To prepare specimens for TEM observation, analiquot (3 mL) of a solution containing FACT was applied to eachcarbon-coated grid and negatively stained with uranyl acetate(2 %). The specimens were examined with a JEM 1010 electron microscope (JEOL), operated at an accelerating voltage of 100 kV.
Images were taken with a Bioscan CCD camera (GATAN). The step
size of a pixel of the image was calibrated to be 5.1 R by using
TMV as a reference sample. A minimum-dose system (MDS) wasused to reduce electron radiation damage to the sample.
High-Speed AFM Imaging: We used a laboratory-built high-speed
AFM, details of which were described previously.[7, 8] AFM images
were acquired in tapping mode. We used cantilevers that were
specially designed for high-speed imaging and provided by Olympus; the resonant frequency was about 1 MHz in water, and thespring constant was about 0.1 N m1. The quality factor of the cantilevers in water was about 2. As an AFM probe, we used an amorphous carbon tip grown on the cantilever by electron beam depoChemPhysChem 2008, 9, 1859 – 1866

sition. For high-speed AFM observation, a droplet (2 mL) of buffer A
containing FACT (or the mutants M1 or M2) was deposited on a
freshly cleaved mica surface. After incubation for 5 min, the sample
was washed with buffer B, which contained Tris-HCl (20 mm)
pH 7.5, MgCl2 (10 mm), and KCl (50 mm). AFM observation wasthen performed under buffer B solution.
Slow AFM Imaging: AFM imaging of dried FACT samples was carried out in tapping mode at 0.004 frames per second on a Nanoscope IV (Veeco). The cantilevers with a thickness of 3.5–4.5 mm, a
length of 115–135 mm, and a width of 30–40 mm were obtained
from Veeco; the resonance frequency in air was 253–306 kHz, andthe spring constant was 20–80 N m1. A drop (20 mL) of buffer A solution containing WT FACT was deposited on a freshly cleaved
mica surface and incubated for 5 min. After washing the sample
with buffer B and then with milli-Q water, the sample was dried
with an air blower. FACT samples attached to modified mica surfaces in solution were imaged at 0.004 frames per second by using
cantilevers (Bio-Lever mini, Olympus) with a thickness of 0.2 mm, a
length of 37 mm, and a width of 16 mm; the resonance frequency
was 25 kHz in water, and the spring constant was 0.1 N m1. Mica
surfaces treated with aminosilane were prepared by placing adroplet (2 mL) of aminopropyltriethoxysilane (0.01 vol %) on a freshly cleaved mica surface for 5 min followed by washing with milli-Q
water. For spermine-coated mica surfaces, a droplet (2 mL) of buffer B solution containing spermine (1 mg mL1) was used. For covalent attachment of FACT to a surface, the aminosilane-treated mica
surface was further treated with a droplet (2 mL) of glutaraldehyde
solution (0.01 vol %) for 5 min followed by rinsing with milli-Q
water. Immediately after this treatment, FACT samples were placedon the prepared mica. However, glutaraldehyde activation resulted
in surface roughness that was inappropriate for observing the tailstructures of the FACT ID regions. In some cases, FACT samples
placed on the aminosilane-treated mica surfaces were further treated with glutaraldehyde (0.01 vol %) for 5 min. The other proceduresfor slow AFM observation were the same as those for high-speed
imaging, except that an imidazole buffer solution was used insteadof Tris–HCl buffer.

Acknowledgements
This work was supported in part by the Core Research for Evolutional Science and Technology (CREST) of the Japan Science andTechnology Agency (JST), by the Japan Society for the Promotionof Science (JSPS), by the Mitsubishi Foundation, and by a Grantin-Aid for Basic Scientific Research (S) from MEXT Japan.
Keywords: atomic force microscopy · biophysics · protein
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Received: April 7, 2008
Revised: July 4, 2008
Published online on August 12, 2008

C 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemPhysChem 2008, 9, 1859 – 1866