EspB from enterohaemorrhagic Escherichia coli
is a natively partially folded protein
Daizo Hamada
1
, Tomoaki Kato
1,2
, Takahisa Ikegami
3
, Kayo N. Suzuki
1
, Makoto Hayashi
2
,
Yoshikatsu Murooka
2
, Takeshi Honda
4
and Itaru Yanagihara
1
1 Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Japan
2 Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
3 Laboratory of Structural Proteomics, Institute for Protein Research, Osaka University, Japan
4 Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Japan
Several bacteria, including enterohaemorrhagic and
enteropathogenic Escherichia coli (EHEC and EPEC,
respectively), express type III secretion systems [1]
consisting of various proteins encoded at the genetic
locus of enterocyte effacement [2–5]. To date, type III
secretion systems have been identified in more than
20 pathogenic bacterial species [6]. Type III secretion
systems are multiprotein complexes that span the

exist. All tyrosine side-chains are exposed to water, as determined by acryl-
amide fluorescence quenching spectroscopy. An increase in the fluorescence
intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the
presence of EspB, whereas no such increase in fluorescence was observed at
pH 7.0. These data suggest the formation of a molten globule state at
pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding
transitions, monitored by far-UV CD spectroscopy. The result from a sedi-
mentation equilibrium study indicated that EspB assumes a monomeric
form at pH 7.0, although its Stokes radius (estimated by multiangle laser
light scattering) was twice as large as expected for a monomeric globular
structure of EspB. These data suggest that EspB, at pH 7.0, assumes a
relatively expanded conformation. The chemical shift patterns of EspB
15
N-
1
H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0
are qualitatively similar to that of urea-unfolded EspB. Taken together, the
properties of EspB reported here provide evidence that EspB is a natively
partially folded protein, but with less exposed hydrophobic surface than
traditional molten globules. This structural feature of EspB may be advan-
tageous when EspB interacts with various biomolecules during the bacterial
infection of host cells.
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; HSQC,
heteronuclear single quantum correlation; LB, Luria–Bertani.
756 FEBS Journal 272 (2005) 756–768 ª 2005 FEBS
EspB is an E. coli type III system protein that inter-
acts with various biomolecules. For example, EspB
binds to EspD, forming a pore complex of 3–5 nm
diameter in the host cell membrane [17]. The N-ter-

heteronuclear NMR. The results of our analyses allow
us to understand the conformational property of EspB
and predict its role in bacterial infection to the host cell.
Results
CD
The secondary structure of EspB, predicted from its
amino acid sequence by using the PredictProtein server
[24–26], indicates that the protein is predominantly
a-helical (Fig. 1). As stated in the Experimental proce-
dures, the recombinant EspB was purified from both
soluble and insoluble fractions of cell lysates. At
pH 7.0 and at a temperature of 20 °C, recombinant
EspB prepared from the insoluble fraction showed a
far-UV CD spectrum equivalent to EspB prepared
from the soluble fraction. This suggests that both
purification procedures adequately yielded the native
conformation of EspB. The CD spectra are typical for
the presence of a-helices (Fig. 2). However, the a-heli-
cal content estimated from far-UV CD data is % 23%,
Fig. 1. Secondary structure prediction of EspB derived from its
amino acid sequence. H and E refer to a-helical and b-strand struc-
tures, respectively. The data were obtained by using the Predict-
Protein server [24,25].
Fig. 2. CD spectra of EspB. (A) Far-UV and (B) near-UV CD spectra
of recombinant EspB purified from the insoluble fraction at pH 2.0
(dashed lines) and 7.0 (solid lines), and from the soluble fraction at
pH 7.0 (s). (C) The dependence of the ellipticity, at 222 nm, on pH.
D. Hamada et al. EspB is a natively partially folded protein
FEBS Journal 272 (2005) 756–768 ª 2005 FEBS 757
which is substantially less than the predicted amount

tyrosine residues in proteins can be a good conforma-
tional probe. In particular, the fluorescence quenching
effect by small chemicals such as acrylamide provides
information on the solvent-exposure of aromatic side-
chains in proteins.
As mentioned above, EspB contains only three tyro-
sines and no tryptophan. The quenching effect of acryl-
amide on the fluorescence of these EspB tyrosine
side-chains at pH 7.0 was analyzed. Interestingly, a
plot of F
0
⁄ F
obs
vs. [Q] (Stern–Volmer plot [27]), where
F
0
and F
obs
are the fluorescence intensities in the
absence and presence of quencher, respectively, and [Q]
is the concentration of quencher, shows a positive devi-
ation from linearity at high acrylamide concentrations
(Fig. 3). Therefore, the quenching behavior does not
follow the simple Stern–Volmer equation (F
0
⁄ F
obs
¼
1+K
sv

0
À F
obs
Þ¼1=ðf
a
K
sv
½QÞ þ 1=f
a
ð1Þ
where f
a
is the fraction of accessible tyrosines. The plot
of F
0
⁄ (F
0
– F
obs
) vs. 1 ⁄ [Q] (Fig. 3) shows a linear cor-
relation between F
0
⁄ (F
0
– F
obs
) and 1 ⁄ [Q]. The values
for K
sv
and f

globule structure for EspB at acidic pH 2.0.
Below pH 2.0, the ANS fluorescence decreased. For
these experiments, the pH of the solution was adjusted
by the addition of HCl. The decreased fluorescence
intensity may be caused by the quenching effect of
chloride ions on ANS fluorescence, rather than reflect-
ing additional conformational changes in EspB.
Urea unfolding
Urea-induced unfolding transitions of EspB were
monitored by far-UV CD spectroscopy. Plots of [h]
at 222 nm vs. urea concentration show co-operative
unfolding transitions throughout the pH range of
1.0–7.3 (Fig. 5). Between pH 3.0 and 7.3, unfolding
Fig. 4. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence at
500 nm as a function of pH. Data were taken at 20 °C in the pres-
ence of 0.1 mgÆmL
)1
EspB. Circles represent the raw data. The line
is drawn only for visual assistance and is not a mathematical fit.
Fig. 5. Urea unfolding of EspB at various pH values and at 20 °C.
(A) The far-UV CD spectra obtained in the presence and absence
of urea. The numbers refer to the concentration of added urea.
(B) The urea-unfolding transition curves obtained at pH 2.0 (s),
pH 5.4 (h) and pH 7.3 (n). Continuous lines are theoretical curves.
The dotted and dashed lines correspond to the baselines for
unfolded and folded states.
D. Hamada et al. EspB is a natively partially folded protein
FEBS Journal 272 (2005) 756–768 ª 2005 FEBS 759
transitions occur between 2.5 and 4.5 m urea, but shift
to lower urea concentrations of 1.0–3.5 m at pH 2.0.

a single species with a Stokes radius of 3.7 and
3.1 nm, respectively (Fig. 6). A similar value was
obtained at pH 4.0 and pH 6.0 (3.4 and 3.5 nm,
respectively). This size is larger than the expected
value for a globular protein of 32 kDa molecular
mass, and corresponds to the value of globular pro-
teins, of % 70 kDa. From its amino acid sequence,
the molecular mass of the recombinant EspB is calcu-
Table 2. Values of DG
water
and m for urea-induced unfolding of
EspB.
pH
(kJÆmol
)1
)
DG
water
(kJÆmol
)1
ÆM
)1
) m
2.0 6.5 ± 2.1 3.9 ± 1.1
3.0 13.2 ± 1.7 3.9 ± 0.5
4.1 14.7 ± 1.9 4.1 ± 0.5
5.4 16.5 ± 1.6 3.8 ± 3.4
6.6 11.5 ± 2.3 3.4 ± 0.6
7.3 9.5 ± 1.3 3.0 ± 0.4
Fig. 6. Hydrodynamic property of EspB at

To further probe the structural properties of EspB, we
recorded its
15
N-
1
H heteronuclear single quantum cor-
relation (HSQC) spectra at pH 2.0 in the absence of
urea and at pH 7.0 in the presence and absence of
urea.
At pH 2.0, in the absence of urea, the
15
N-
1
H
HSQC spectrum shows little chemical shift dispersion
(Fig. 7). Although the resolution is poor owing to the
overlapping of peaks, the number of peaks that cor-
respond to the main-chain
1
H-
15
N crosspeaks was
estimated to be % 120. These peaks are relatively
sharp and may reflect the amino acid residues that
rapidly fluctuate with a timescale of nanosecond
order. The recombinant EspB used in this study
contains 333 amino acid residues. Thus, % 64% of
main-chain
1
H-

about 140. This implies the significant overlapping of
crosspeaks or the presence of some residual struc-
tures, even in the presence of 8.0 m urea.
The result of little chemical shift dispersions with the
small number of observable crosspeaks in the
15
N-
1
H
HSQC spectrum of EspB at pH 7.0 in the absence of
urea is inconsistent with the previous data obtained by
CD and fluorescence spectroscopies showing the pres-
ence of well-ordered conformations. This discrepancy
suggests that, at neutral pH, EspB assumes a natively
partially folded conformation without exposed hydro-
phobic clusters accessible to ANS molecules.
Fig. 7.
15
N-
1
H Heteronuclear single quantum correlation (HSQC)
spectra of EspB taken at 15 °C. (A) pH 7.0 in the absence of urea.
(B) pH 2.0 in the absence of urea. (C) pH 7.0 in the presence of
8.0
M urea.
D. Hamada et al. EspB is a natively partially folded protein
FEBS Journal 272 (2005) 756–768 ª 2005 FEBS 761
Discussion
Conformational properties of EspB
We analyzed the conformational properties of EspB by

result because partially folded proteins generally have
exposed hydrophobic clusters that are detected by
increases of ANS fluorescence intensity. One possible
explanation for the discrepancy could be that the
hydrophobic clusters, found for the EspB molten glob-
ule at pH 2.0, are disrupted in the structure found at
pH 7.0. A similar situation is, indeed, often found
for a-helical polypeptides in alcohol ⁄ water solvents
[45–51]. However, this explanation can be ruled out as
EspB is more stable at higher pH, which would prob-
ably be inconsistent with the loss of intramolecular
hydrophobic contacts. Thus, most EspB hydrophobic
clusters should be buried at neutral pH. Variations in
the conformational and thermodynamic properties of
molten globules have been characterized. For example,
the thermal unfolding experiments on the molten glob-
ule state of a-lactalbumin shows a gradual transition,
which suggests less organized hydrophobic contacts
[34]. However, the cytochrome c molten globule state
is highly ordered and the thermal unfolding transition
of this species is co-operative with a clear enthalpy
change upon unfolding [52,53]. This indicates that
some organized hydrophobic contacts exist in the mol-
ten globule state of cytochrome c. Furthermore, the
presence of tertiary contacts in the molten globule
states are shown by apomyoglobin and cytochrome c
[54,55], and EspB also showed the presence of weak,
but distinctive, peaks in the near-UV CD spectrum.
Therefore, EspB, at neutral pH, may have the charac-
ter of a highly ordered molten globule [56] with dis-

at a temperature of 20 °C. ANS, 8-anilinonaphthalene-1-sulfonate;
HSQC, heteronuclear single quantum correlation.
pH
Far-UV
CD
Near-UV
CD
Hydrophobic
exposure
by ANS
15
N-
1
H
HSQC
Urea
unfolding
7.0 Folded Folded Less exposed Unfolded Co-operative
2.0 Folded Partially
folded
Highly exposed Unfolded Co-operative
EspB is a natively partially folded protein D. Hamada et al.
762 FEBS Journal 272 (2005) 756–768 ª 2005 FEBS
disordered. The results from PONDRÒ suggest that
the amino acid residues at 1–53, 128–230 and 247–287
may be disordered. This is relatively consistent with the
prediction of DisEMBL. If the prediction from
PONDR is correct, only the amino acid sequences at
residues 54–127 and 288–325 of EspB, i.e. one-third of
the EspB sequence, assume ordered conformations.

folded or completely unfolded conformations in an
aqueous environment at neutral pH and, ideally, under
near-physiological conditions.
Our results clearly indicate that the structural char-
acteristics of EspB are those of a natively partially
folded protein. The far-UV CD spectra of IpaC, a
homolog of EspB from Shigella flexneri, revealed an
absence of significant amounts of secondary structure
at neutral pH [62]. Thus, the intrinsically less organ-
ized conformations of EspB and IpaC may be a com-
mon property for this class of proteins.
Importantly, some proteins that are natively unfol-
ded show dramatic conformational changes into well-
ordered structures when bound to their target
molecules [40–44]. Therefore, it will be important to
characterize the conformational state of EspB when
bound to its target molecules, e.g. EspA, EspD, a-cate-
nin and a1-antitrypsin.
Using the genomic sequence of E. coli, Dunker and
co-workers predicted that 8% of all proteins will have
intrinsically disordered segments of greater than 50 res-
idues in length [62]. Interestingly, the same predictions
indicated that this percentage increases to 41% for
Drosophila melanogaster proteins. Thus, intrinsically
structural protein disorder is probably a common
occurrence in vivo. It is unclear why structural disorder
would confer a physiological advantage to the function
of a protein function. Several possible reasons have
been proposed to answer this question [39–44]. For
example, if a protein is highly flexible, its association

Expression and purification of EspB
The cDNA, encoding EspB, was amplified from an EHEC
E. coli O157:H7 cosmid library (RIMD 0509890, Sakai
strain) [66,67] by PCR and cloned into a pT7 vector
(Novagen). The full-length espB gene was subcloned into
the expression vector pET28a (Novagen, Madison, WI,
D. Hamada et al. EspB is a natively partially folded protein
FEBS Journal 272 (2005) 756–768 ª 2005 FEBS 763
USA). The recombinant EspB has 20 amino acids, MGSS
HHHHHHSSGLVPRGSH, added at the N terminus of the
original sequence.
Recombinant EspB was expressed in E. coli BL21(DE3)
transformed with the afore mentioned plasmid. Cultures of
Luria–Bertani (LB) broth, supplemented with 50 lgÆmL
)1
of
kanamycin, were inoculated with colonies and grown over-
night at 37 °C with shaking. Then, a portion of each culture
was diluted 100-fold into 1 L of fresh LB medium and incu-
bated at 37 °C with shaking. Protein expression was induced
by the addition of isopropyl thio-b-d-galactoside (at a final
concentration of 1 mm) when cultures reached an attenuance
(D)of% 0.6 at 600 nm. For the expression of protein uni-
formly labeled with
15
N, M9 medium supplemented with
15
NH
4
Cl (Nippon Sanso Co., Kanagawa, Japan) was used

20 mm sodium phosphate, pH 7.0, containing 8.0 m urea.
This solution was clarified by centrifugation and diluted
100-fold by dropwise addition into 20 mm sodium phos-
phate, pH 7.0, at 4 °C. The solution was then purified by
Chelating Sepharose Fast Flow supplemented with NiCl
2
and further purified by size-exclusion chromatography
(S-300) as in the case of preparation from the soluble frac-
tions. The purification yields from soluble and insoluble
fractions were 15 and 30 mg from 1 L of culture in LB
medium, respectively. As judged by SDS ⁄ PAGE, the purity
of recombinant EspB prepared from the insoluble fraction is
relatively higher than that of EspB purified from the soluble
fraction. According to the CD spectrum, both purifications
yielded the same conformational state of EspB (see text for
details). Owing to the higher yields of purification,
15
N pro-
tein was prepared from insoluble fractions.
The protein concentration was determined by absorption
at 276 nm with the extinction coefficient of 4350 mÆcm
)1
calculated from amino acid composition [68]. The protein
solution was stored at )20 °C.
CD spectroscopy
CD spectra were measured by using a J-600 spectropola-
rimeter (Jasco, Tokyo, Japan). The temperature was held at
20 °C by using a thermostatically controlled cell holder in
conjunction with a circulating waterbath. For far-UV and
near-UV CD spectra, cells of 1 mm and 1 cm path length

)1
and the ANS concentration
was 5 lm. The temperature was maintained at 20 °C with a
peltier-type thermostatically controlled cell holder.
Fluorescence quenching of EspB tyrosines was measured
in the presence of various concentrations of acrylamide,
with spectra acquired as described above.
Urea-induced unfolding measurements
Urea-unfolding curves were plotted with [h] at 222 nm vs.
the urea concentration. The data were analyzed assuming
a two-state unfolding mechanism and assuming that the
change in free energy of unfolding (DG), is linearly depend-
ent on urea concentration:
DG ¼ DG
water
À m½ureað2Þ
Here, DG
water
corresponds to DG of unfolding in the
absence of urea; m is a measure of the co-operativity
EspB is a natively partially folded protein D. Hamada et al.
764 FEBS Journal 272 (2005) 756–768 ª 2005 FEBS
of the unfolding transition; and [urea] is the urea concen-
tration.
The fractions of unfolded (f
U
) and folded (f
F
) species at
various urea concentrations can be expressed as:

ð5Þ
Here, [h ]
F
and [h]
U
are the [h]
222
of the folded and unfolded
species, respectively.
The values for DG
water
and m were obtained by nonlinear
curve fitting to the transition curves, according to
Eqns (2–5), by using the program igorpro (WaveMetrics
Inc., Lake Oswego, OR, USA). The linear dependences of
[h]
F
and [h]
U
on urea concentrations were also considered
in the fitting analysis. The same baselines for folded and
unfolded species were used for the fitting of data obtained
at pH 2.0–7.0.
Multiangle laser light scattering
Multiangle laser light scattering data were obtained by
using a dynapro Molecular Sizing Instrument (Protein
Solutions Inc., Milton Keynes, UK) at 20 °C. Various con-
centrations of protein solution at pH 2.0, 4.0, 6.0 and 7.0
(400 lL) were passed through 0.22 lm of centrifugal filter
unit, ultrafree-MC from Millipore (Billerica, MA, USA),

) and 64, 1168 Hz
(
15
N, F
1
), and for those using the DRX800 spectrometer,
the parameters were 1024, 12821 Hz (
1
H, F
2
) and 64,
1866 Hz (
15
N, F
1
). The
1
H carrier was set at 4.7 p.p.m.,
and the
15
N carrier at 120 p.p.m. The
15
N-
1
H HSQC
experiments included the WATERGATE and Water-flip-
back techniques. The data were processed by using nmrpipe
[72] and visualized by using sparky (TD Goddard & DG
Kneller, University of California, San Francisco, CA, USA;
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