The primary amino acid sequence of a polypeptide encodes all of the information necessary for folding and assembly pathways, as well as the native 3D structure Keywords acid and Gdm-HCl-
Trang 1from Escherichia coli
Damodara R Mendu1, Venkata R Dasari2, Mian Cai1and Kwang S Kim1
1 Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA
2 Department of Biomedical and Therapeutic Sciences, College of Medicine, University of Illinois, Peoria, IL, USA
Escherichia coli is the most common Gram-negative
organism that causes neonatal meningitis [1–4] E coli
has several virulence factors, including a 50 kDa IbeA
protein, which has been found to be unique to
cerebro-spinal fluid isolates from neonatal E coli meningitis
E coli invasin protein IbeA facilitates the E coli
penetration of human brain microvascular endothelial
cells (HBMEC) which constitute the blood–brain barrier (BBB) [5–7] The 8.2 kDa N-terminal IbeA protein was shown to inhibit E coli K1 invasion of HBMEC [5]
The primary amino acid sequence of a polypeptide encodes all of the information necessary for folding and assembly pathways, as well as the native 3D structure
Keywords
acid and Gdm-HCl-induced unfolding;
Escherichia coli; molten globule; protein
unfolding intermediates of IbeA
Correspondence
K S Kim, Division of Pediatric Infectious
Diseases, Johns Hopkins University School
of Medicine, 200 North Wolfe Street,
Room 3157, Baltimore, MD 21287, USA
Fax: +1 410 614 1491
Tel: +1 410 614 3917
E-mail: kwangkim@jhmi.edu
(Received 9 October 2007, revised
15 November 2007, accepted 28 November
2007)
doi:10.1111/j.1742-4658.2007.06213.x
IbeA of Escherichia coli K1 was cloned, expressed and purified as a His6 -tag fusion protein The purified fusion protein inhibited E coli K1 invasion
of human brain microvascular endothelial cells and was heat-modifiable The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution The far-UV CD spectrum of IbeA at
pH 7.0 has a strong negative peak at 215 nm, indicating the existence of b-sheet-like structure The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like struc-ture) with b-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding The pH dependent intrinsic fluorescence of IbeA was biphasic At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended b-sheet conformation and exhibits strong ANS binding Guanidine hydro-chloride denaturation of IbeA in the molten globule-like state is noncoop-erative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular struc-ture of IbeA, with differential unfolding stabilities Furthermore, trypto-phan quenching studies suggested the exposure of aromatic residues to solvent in this state Acid denatured unfolding of IbeA monitored by
far-UV CD is non-cooperative with two transitions at pH 3.0–1.5 and 1.5–0.5
At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state The presence of 0.5 m KCl in the solvent composition directs the transition to the A state
by bypassing the acid-unfolded state Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence
Abbreviations
ANS, 8-anilino-2-naphthyl sulfonic acid; BBB, blood–brain barrier; Gdm-HCl, guanidine hydrochloride; HBMEC, human brain microvascular endothelial cells; IMAC, immobilized metal affinity chromatography; LB, Luria-Bertani; oPOE, octylpolyoxyethylene; PVDF, poly(vinylidene difluoride); b-ME, b-mercaptoethanol.
Trang 2[8,9] Partially folded and denatured proteins can give
important insights into protein misfolding, and
aggre-gation It has been recognized that the structure
of non-native state of proteins can provide significant
insight into fundamental issues such as the relationship
between protein sequences and 3D structures, the
nat-ure of protein folding pathways, the stability of
pro-teins and the transport of propro-teins across membranes
[10] Even though a large amount of information is
available on protein folding intermediates [11–18],
there are no reports available for E coli invasin
pro-tein IbeA The process of unfolding and refolding is
useful for a complex unfolding transition, indicating
their presence of a partially folded intermediate with
one of the domains being ordered and disordered [19]
In the protein folding pathway, the identified
inter-mediates (aggregates) can be used to define the role of
the individual folding intermediaries in each pathway
and developing therapies against these intermediates
might be an attractive strategy Such delineation can
only be achieved by identifying partially unfolded
states formed during folding, and correlating their
populations by spectroscopy Using this approach,
IbeA protein folding intermediates (misfolded
aggre-gates) can be directly identified and attention focused
on defining the structural properties of these states To
date, no information about the structural properties of
IbeA has been available In the present study, we
char-acterized the biophysical properties of IbeA in solution
using spectroscopic techniques to identify protein
fold-ing intermediates
Results
The initial step in the action of IbeA for E coli
K1 traversal of the BBB is binding to a cell-surface
receptor, which induces the conformational changes of
the IbeA binding domains We hypothesize that the
resulting protein–receptor complexes are endocytosed
and delivered to an acidic compartment (endosome) of
the cell, forming a prepore-like structure, enabling the
internalization and traversal of E coli in HBMEC, but
the nature of this relationship is incompletely
under-stood No studies on E coli traversal mechanisms have
focused on the conformational changes occurring in
IbeA acidification, and no study has addressed the
acid-induced changes in the IbeA molecule We also
hypothesize that the HBMEC central lumen is too
small to accommodate native IbeA, necessitating some
degree of protein unfolding for efficient translocation
of E coli, and we have shown the existence of protein
folding intermediates and acidic unfolding
intermedi-ates in vitro
Purification of IbeA The expression level of IbeA was 6–8 mgÆL)1 of culture, and its molecular weight was 50 kDa by SDS⁄ PAGE The fractions eluted from an immobilized metal affinity chromatography (IMAC) column were analyzed by Coomassiee blue staining (Fig 1) The identity of the IbeA was analyzed by western blotting with monoclonal His6antibody (unpublished data) and with purified antibodies to IbeA (Fig 2) The correct refolding of IbeA was shown by invasion assays and heat modifiability experiments The purified recombi-nant IbeA blocked E coli K1 invasion in HBMEC
m (kDa)
250 150 100 75 50 37 25 20
A B C D E
30 40 50
m (kDa)
190 120 85 60 50 40 25
20
A
B
Fig 1 (A) Analysis of the purified IbeA by SDS ⁄ PAGE (4–20%) The alternate fractions from the TALON column were analyzed by SDS ⁄ PAGE Lane A, molecular weight markers (Invitrogen pre-stained markers); lane B, flow through; and lanes C–E, TALON fractions heated at 30, 40 and 50 C for 5 min, respectively (B) Purified IbeA (5 lg) was heated at 100 C for 5 min in SDS ⁄ PAGE sample buffer and analyzed by SDS ⁄ PAGE Lane A, molecular weight markers (Bio-Rad prestained markers) and lanes B–E, puri-fied protein.
Trang 3(25 lgÆmL)1 reduced the HBMEC invasion frequency
of E coli K1 strain RS218 by 73%) and it is assumed
that this blocking activity is due to its native structure,
and that there was no interference of the His6-tag On
the other hand, our His6-tag control protein and His6
-tag removed proteolytically from IbeA molecule did
not have any effect on HBMEC invasion of RS218 In
further studies, we used His6-tagged IbeA molecule
We have also shown the correct folding of purified
IbeA by invasion assays, heat modifiability
experi-ments and fluorescence spectroscopy (Fig 3) for both
neutral and acidic pH and denaturant The refolded
protein displays the heat shift typical of outer
mem-brane proteins in the correctly folded state The
puri-fied IbeA exists in three interconvertible forms,
distinguishable by SDS⁄ PAGE as 50, 53 and 55 kDa
when heated at 30, 40 and 50C, respectively
(Fig 1A) When the IbeA was heated at 100C for
5 min in SDS sample buffer, only the 50 kDa band
was observed (Fig 1B), suggesting the gel shift at high temperature The gel shift of 5 kDa is similar to the other modifiable membrane proteins [20–22] We assume that the 50 kDa protein is fully folded protein and that the 53 and 55 kDa proteins could be a fold-ing intermediate or off pathway species [23] A number
of membrane proteins differ in their migration veloci-ties in SDS⁄ PAGE depending on whether or not the protein was heated before electrophoresis [24–28] The fractions of TALON shown in Fig.1A were identified
by the polyclonal sera (Fig 2)
Characterization of IbeA The purified IbeA was equilibrated with denaturant up
to 24 h and no further spectroscopic changes were observed after 24 h, when the presented results were obtained, indicating that equilibrium was attained within this time Near-UV CD was employed to exam-ine the asymmetry of aromatic amino acids, and thereby to monitor the changes in the tertiary structure
of the protein [22] The CD spectrum of native IbeA exhibited a positive peak at 276–278 nm and a nega-tive peak at 297–299 nm, which is due to the presence
of tryptophan residues (Fig 4A) However, pH 2.0 and strong denaturant, such as 6 m guanidine hydro-chloride (Gdm-HCl), did not provide information due
to the disordered aromatic groups in the unfolded state The far-UV CD spectrum of a protein is a diag-nostic probe of secondary structure and facilitates determination of specific structural features that com-prise the native conformation The far-UV CD spec-trum of IbeA (Fig 4B) showed a negative peak at
215 nm, suggesting the presence of extended ß-sheet regions IbeA exhibited a negative peak at 200 nm, indicative of a strong contribution from disordered structural elements, characteristic of a protein in a ran-dom coil conformation
As can be seen, decreasing the pH below 2 changed the acid-induced unfolded state due to the formation
of the A-state The A-state of IbeA has a substantial non-native secondary structure, and little or no tertiary structure These data strongly indicate the presence of extended b-sheets and, in the presence of 6 m Gdm-HCl, IbeA lost all of the peaks, suggesting the loss of secondary structure The deconvolution spectrum obtained using the selcon program [29] provides the structural component of IbeA (Table 1)
The intrinsic fluorescence spectra of IbeA at pH 7.0 and 2.0 and in the presence of 6 m Gdm-HCl are shown in Fig 3 The lowering of pH from 7.0 to 2.0 drastically decreased fluorescence intensity by 70–75% with a blue shift of 16 nm in the emission maxima at
Fig 2 Western blot analysis of purified, refolded IbeA using
puri-fied sera raised against pure IbeA The pure IbeA (5 lg) was heated
at 30, 40 and 50 C for 5 min in SDS ⁄ PAGE sample buffer and
loaded on to the 12% SDS ⁄ PAGE gel.
0
50
100
150
200
250
300
350
400
450
Wavelength (nm)
pH 7.0
Gdm-HCl (6 M)
pH 2.0
Fig 3 Fluorescence spectroscopy analysis of denatured IbeA by
Gdm-HCl Purified IbeA (1 l M ) was denatured by titrating with
Gdm-HCl at room temperature (25 C) The denaturation mediated
changes in IbeA were monitored for tryptophan fluorescence;
exci-tation was 292 nm and emission was 300–420 nm at pH 7.0 and
2.0 and in the presence of 6 M Gdm-HCl.
Trang 4352–336 nm, indicating the non-polar environment of
tryptophan Although the fluorescence spectrum of
completely unfolded IbeA in 6 m Gdm-HCl remains
similar in shape, the emission maximum suffers a red
shift from 352 nm to 358 nm along with a decrease in
fluorescence intensity of 60–70% This red shift in the
wavelength maximum indicates that more tryptophan
residues of the protein are exposed to a polar
environ-ment, which is characteristic of unfolding, or could be
due to decreased distance between tryptophan and
quenching groups, resulting in tryptophan fluorescence
quenching
The far-UV CD spectrum of IbeA remains unchanged in the pH range of 3.0–10, and the spec-trum reveals two distinct peaks: one at 222 nm and the other at 208 nm (Data not shown) The unfolding of the IbeA, in the absence of added salt, followed by ellipticity at 222 nm, is noncooperative (Fig 5) A cooperative transition from the native state to an acid-unfolded state occurred at pH 3.0–1.5, and a second transition occurred on further lowering the pH from 1.5 to 0.5 The unfolded state at lower pH, exhibiting
a reduced secondary structure and loss of tertiary structure, represents the acid-unfolded state of the IbeA, indicating partial unfolding of the protein mole-cule Thus, IbeA at pH 1.5–1.0 exists in an acid-unfolded state Furthermore, addition of acid leads to
a second transition between pH 1.5 and 0.5 resulted in
an increase in secondary structure, leading to the
A state [30]
In the presence of 0.5 m KCl, pH-induced unfolding
of IbeA is cooperative, as manifested by a single tran-sition (Fig 5), in which the protein molecule passes from the native state to the A state directly without passing through the acid-unfolded state The secondary structural content of such a salt-induced A state is more ordered than that observed at pH 0.5 in the absence of added salt The CD spectrum of the protein
at pH 2.0–0.5, either in the presence or in the absence
of 0.5 m KCl, exhibits predominantly extended b-sheet structure and the negative peak at 215–217 nm at
pH 2.0 (Fig 4B) is a common characteristic feature of proteins having extended b-sheets At a higher concen-tration of KCl, aggregation or precipitation was observed
–30
0
30
60
90
120
150
Wavelength (nm)
Wavelength (nm)
2 d mo
pH 7.0
pH 2.0 Gdm-HCl (6 M)
–10
–7
–4
–1
2
5
8
11
180 190 200 210 220 230 240 250 260
–3 deg cm
2 d mo
pH 7.0
pH 2.0
6 M Gdm-HCl
A
B
Fig 4 (A) Near- and (B) far-UV CD of purified IbeA in the presence
of oPOE, as described in the Experimental procedures, were
analyzed at pH 7.0 and 2.0 and in the presence of Gdm-HCl The
protein concentrations were 3.25 l M and 1.5 l M in the near- and
far-UV CD, respectively.
Table 1 Secondary structure content of IbeA by SELCON
–8 –7 –6 –5 –4 –3 –2 –1 0
pH
–3 de
2 dm
presence of salt absence of salt
Fig 5 Effect of salt on the structure of IbeA Structural changes
of IbeA as a function of pH were monitored by studying ellipticity values at 222 nm in the presence and absence of 0.5 M KCl.
Trang 58-Anilino-2-naphthyl sulfonic acid (ANS)
fluorescence studies
The effect of pH on ANS binding shows that IbeA
binds more strongly at pH 2.0 rather than in its native
state (pH 7.0) and completely unfolded state (pH 0.5)
(Fig 6A,B) At pH 2.0, IbeA has maximum
hrdro-phobicity (480 nm) compared with native IbeA
(518 nm) because of the presence of more accessible
hydrophobic residues to ANS The ANS fluorescence
spectra between 10–0.5 pH (Fig 6B) strongly support
the acidic unfolding with a two state transition and the
formation of a molten globule state at pH 3.0–1.5 The
molten globule was formed at pH 2.0 (Fig 6B) with
high ANS binding capacity and significant secondary
structure with no tertiary structure At pH 0.5, ANS
binding capacity and secondary structure was minimal
as it reached the A state These data suggests the
pres-ence of a molten globule state at pH 2.0 with the
for-mation of the A state at pH 0.5, since the molten
globular state of IbeA molecule exposes hydrophobic residues All these data obtained at pH 2.0 support the definition of a molten globule with b-helical confirma-tion
The pH dependent intrinsic fluorescence of IbeA was carried out to evaluate its biphasic behavior Fig 7A,B demonstrates that the pH-induced transi-tions in IbeA molecule represent a two step process The first transition occurs between 4.0–6.0 with a mid-point of 4.8 and second transition occurs between 1.0– 3.0 with a midpoint of 2.0 The fluorescence decreases when the pH falls from 6.0 to 4.0 (blue shift) and,
in the latter transition, fluorescence intensity was increased (red shift) as the protein reached its acid unfolded state
Iodine-quenching studies The solvent accessibility of individual tryptophan resi-dues in the native, molten globule and unfolded states
0
100
200
300
400
500
600
700
A
B
pH
0
100
200
300
400
500
600
700
pH 7.4
pH 2.0
pH 3.0
pH 0.5
6 M Gdm-HCl
Wavelength (nm)
Fig 6 ANS binding to IbeA as a function of pH The samples were
incubated for 24 h at 25 C before the measurements were taken.
(A) ANS binding measurement was taken by excitation at 360 nm
and emission was collected between 400–600 nm (B) ANS
fluores-cence at different pH values.
330 335 340 345 350 355
360
A
B
pH
0 75 150 225 300 375 450
pH
Fig 7 Intrinsic fluorescence analysis of IbeA at varying pH values The effect of pH on the intrinsic fluorescence of IbeA at different
pH values was plotted The protein concentration was 1 l M in
20 m M PO4buffer pH 7.0, containing 5 m M oPOE (A) The wave-length maximum (B) The excitation wavewave-length was 292 nm with slit widths of 10 and 5 nm for excitation and emission, respec-tively.
Trang 6was investigated by iodine quenching studies The
quenching constants (KSV) and fraction of accessible
fluorophore (fa) at native pH and pH 2.0 in the
pres-ence of Gdm-HCl were 5.86, 8.64 and 9.28 m)1, and
0.36 ± 0.03, 0.89 ± 0.08 and 0.8 ± 0.07, respectively
The modified Stern–Volmer plot indicates that the
tryptophan residues in the IbeA at pH 2.0 are more
exposed to the solvent compared with native IbeA at
pH 7.0 (Fig 8) However 0–2 m Cs+was unable to
quench tryptophan fluorescence either at pH 2.0 or in
the presence of Gdm-HCl (data not shown) At neutral
pH, no noticeable changes were seen in the
fluores-cence spectra for both quenchers (data not shown)
These data indicate that no structural changes took place in the protein molecule
IbeA was denatured by Gdm-HCl at different
pH values and was monitored by near- and
far-UV CD and fluorescence spectroscopy to determine the secondary and tertiary structural changes (Table 2) The CD and intrinsic fluorescence spectrum
at pH 7.0 is sigmoidal and cooperative (Fig 9A) At
pH 3.0, the Gdm-HCl-induced unfolding curves of IbeA are cooperative (Fig 9B), with non-coincidental transition curves At this pH, IbeA loses its secondary, tertiary structure and fluorescence intensity, indicating the presence of intermediates in the unfolding process [31] The existence of intermediates was further con-firmed by ANS binding at 1.5 m Gdm-HCl (Fig 9C) However, at highly acidic pH < 2.0, IbeA lost its ter-tiary structure, as indicated by near UV-CD spectrum and, at pH 2.0, the Gdm-HCl-induced denaturation curve of IbeA was non-cooperative (Fig 9D) The ANS binding to IbeA (Fig 9E) was very strong after the first transition and gradually decreased with an increase in Gdm-HCl concentration, indicating the existence of hydrophobic domains at first unfolded
Discussion The biophysical analysis of IbeA provides much infor-mation about its conforinfor-mational states and protein folding intermediates In the present study, we have used multiple probes to investigate the structure of IbeA by pH and the denaturation process induced by Gdm-HCl These probes were used to study its solu-tion confirmasolu-tion and to identify protein unfolding intermediates We attempted to characterize the fold-ing intermediates in interaction with HBMEC, but low
pH (acidic) and denaturant damaged the HBMEC monolayer, precluding such experiments
IbeA was expressed, purified and refolded using octylpolyoxyethylene (oPOE) detergent and has no
0
0.5
1
1.5
2
2.5
3
3.5
4
[I-] –1
pH 7.0
pH 2.0
6 M Gdm-HCl
Fig 8 The modified Stern–Volmer’s of tryptophan fluorescence
quenching by iodide [I)1] Quenching of tryptophan fluorescence
intensity of IbeA at pH 7.0 and 2.0 and in the presence of
Gdm-HCl, was carried out with 0.0–0.2 M KI at 25 C KCl was added to
maintain the ionic strength constant The data was analyzed as per
modified Stern–Volmers’s equation.
Table 2 Unfolding parameters of IbeA RT, Room temperature –, unable to calculate.
Denatured by Gdm-HCl Method Transition mid point (Cm) ( M ) DG U-N (kcalÆmol)1) mU-NkcalÆmol)1Æ M )1
Trang 70.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
near UV-CD far UV-CD fluorescence
0 0.2 0.4 0.6 0.8
1 1.2 1.4
0 0.2 0.4 0.6 0.8
1 1.2 1.4
far UV-CD near UV-CD fluorescence
0
100
200
300
400
500
600
[Gdm-HCl] ( M )
[Gdm-HCl] ( M )
fluorescence far UV-CD near UV-CD
0
100
200
300
400
500
600
[Gdm-HCl] ( M )
C
E
D
Fig 9 Formation and identification of protein folding intermediates 1 l M IbeA in 20 m M PO 4 buffer pH 7.0, containing 5 m M oPOE was denatured as a function of Gdm-HCl Near-and far-UV CD and ANS fluorescence were measured (A) at pH 7.0, (B) 20 m M glycine buffer
pH 3.0 containing 5 m M oPOE, (C) 20 m M glycine buffer pH 2.0 containing 5 m M oPOE, (D) ANS fluorescence at pH 3.0 and (E) at pH 2.0.
Trang 8interference in invasion assays In principle, we cannot
discount the possibility that IbeA cannot refold in
solution is an in vitro artifact We strongly believe that
this is not the case because we performed an extensive
screening of refolding conditions
CD is a sensitive method for investigating the
pep-tide bond and has been widely used to elucidate the
structure of proteins [32–39] The far-UV CD of IbeA
(Fig 4B) is similar to the characteristic features of
porins [40–45] The membrane proteins exist as
a-helical and ß-barrel proteins and the transmembrane
ß-barrels are present in Gram-negative bacteria
because they could be easily detectable due to the
pres-ence of non-polar residues in their outer membranes
The strong ellipticities at 215 nm in far-UV CD
indi-cates that IbeA exists as an extended ß-sheet
confirm-ation in its native state and in the molten globular
state The negative ellipticity at 208 and 222 nm also
indicates the presence of a-helical confirmation
The fluorescence spectra of IbeA were identical
when excited at 278 and 295 nm (unpublished data)
and these data show that tyrosine fluorescence was
quenched by tryptophan The fluorescence spectra of
IbeA at 6 m Gdm-HCl and at pH 7 indicate that more
tryptophan residues are exposed to a polar
environ-ment It could be also possible that, at neutral pH, the
excitable chromophores are in a hydrophilic
environ-ment
Our acid unfolding studies indicate that the molten
globule state was formed at pH 2.0 in the unfolding
process from pH 3.0–1.5 and this was also confirmed
by the ANS data Thus, IbeA exhibits a two state
tran-sition in acidic denaturation, as the mechanisms of
acid-induced unfolding of proteins have been
eluci-dated in detail [30,46,47], and the pH-induced
unfold-ing of IbeA was explained accordunfold-ingly The decrease in
pH causes enhancement of protonation of the protein
At pH 2.0, the protonation becomes saturated and the
protein loses its structure and forms the A state Both
anions and cations will be present in the acidic
unfold-ing environment and addition of cations (K+) does
not have any impact on ionization At extreme acidic
pH, there will be repulsion between the charged groups
of the protein, and an even high concentration of the
salts (counter-ions) interacts with charged groups and
weakens the repulsions Thus, in the presence of salt
(KCl), the IbeA molecule directly reaches the A state
The pH-induced denaturation curves demonstrate
that the decrease in the fluorescence intensity, with a
blue shift (15–17 nm) between 6.0–4.0 pH, could be
due to either the microenvironmental changes in the
region of tryptophan residues protecting its overall
structure and the tertiary structure of the protein, or
to uncharged carboxylate groups causing the less polar environment near the tryptophan residues, resulting in
a blue shift of the tryptophan fluorescence [48] In the second transition, a red shift with an increase in fluo-rescence intensity in the pH range between 3.0–0.5 occurs as a result of loss in its secondary structure due
to the acid-induced unfolding state
The modified Stern–Volmer’s plot for the native IbeA indicates the limited accessibility of the aromatic residues but, at acidic pH, more aromatic groups are exposed to the solvent due to the presence of a molten globule These data indicate the high binding capacity
of quenchers at acidic pH due to the formation of molten globule compared to the native state We assume that the molten globule is a loosely packed intermediate with largely exposed tryptophan residues The Gdm-HCl-induced unfolding of IbeA from a molten globule to an unfolded state is noncooperative,
by contrast to the cooperative unfolding occurring at neutral pH This cooperative unfolding is due to the integrity of IbeA owing to side chain packing entailing the breaking of the tertiary structure required for non-cooperative transitions observed in the molten globule The Gdm-HCl-induced unfolding of the IbeA molten globule structure also denotes the presence of two domains that unfold independently of each other Our Gdm-HCl data also indicate the unfolding of one domain between 2.0–2.8 m Gdm-HCl whereas the other one is intact The ANS binding to the molten globule at pH 2.0 is also parallel with the first transi-tion because most of the hydrophobic residues are in the first unfolded domain
The Gdm-HCl and pH induced (3.0) unfolding curves were coincidental, and the mU-Nvalues at near –UV CD are considerably higher than the fluorescence and far-UV CD values These data indicate the presence of
an intermediate state between the native and denatured states Furthermore, the existence of an intermediate was demonstrated by ANS binding to IbeA at 1.5 m Gdm-HCl with a secondary structure The secondary structure of the intermediates at pH 3.0 and 1.5 m Gdm-HCl are almost similar, supporting the existence
of intermediates in the different conditions
The characteristic heat modifiability was mainly used
to study b-barrel outer membrane proteins The high content of b-strands reflected in the CD spectra reported in the present study suggests that a significant number of extracellular loops also adopt this second-ary structure We assume that IbeA molecule b-barrel strands traverse through the outer membrane into extracellular space IbeA had the characteristic features
of outer membrane proteins, with seven trans-membrane domains having extended b-sheets and two
Trang 9functional domains that unfold independently In the
IbeA unfolding process, an equilibrium intermediate
was found with 1.5 m Gdm-HCl and pH 2.0 The
unfolding pathway of IbeA could be divided into two
transition stages, namely an inactive intermediate and
a native state The proposed unfolding pathway of
IbeA is shown in Fig 10
Experimental procedures
Reagents
High purity grade Gdm-HCl, ANS, and KI were obtained
from Sigma Chemical Co (St Louis, MO, USA); oPOE was
from Bachem (Torrance, CA, USA); TALON IMAC
resin was from Clontech (Palo Alto, CA, USA);
poly(vinyli-dene difluoride) (PVDF) membrane was from Millipore
(Bedford, MA, USA); Novex gels and SDS⁄ PAGE markers
and monoclonal anti-His6-tag sera were from Invitrogen
(Carlsbad, CA, USA); Bradford reagent was from Bio-Rad
(Bio-Rad, Hercules, CA, USA); lysozyme was from Roche
Diagnostics (Indianapolis, IN, USA); horseradish
peroxi-dase conjugated anti-mouse sera and the PVDF membrane
ECL detection kit were from Amersham Biosciences
(Piscataway, NJ, USA); and ampicillin, isopropyl
thio-b-d-galactoside, EDTA, dithiothreitol, b-mercaptoethanol
and complete protease inhibitors, oPOE, were from Sigma
Chemical Co
Buffers and solutions
The buffers used for the spectroscopic measurements at
dif-ferent pH values were 20 mm KCl-HCl (0.5–1.5), 20 mm
glycine⁄ HCl (pH 2–3), 20 mm sodium acetate (pH 4–5),
20 mm sodium phosphate (pH 6–7.5), and 50 mm Tris–HCl
(pH 8.5–10.5); all the buffers contained 5 mm of oPOE
Unfolding conditions were provided by Gdm-HCl (0–6 m)
in 20 mm NaCl⁄ Pi, pH 7.0 ANS concentration was
calcu-lated spectrophotometrically using an extinction coefficient
of 5000 m)1Æcm)1at 350 nm All the solutions were prepared
in deionized water and filtered through a 0.22-lm filter
Expression of IbeA fusion protein
IbeA was cloned as described previously [6] as a 6· His6
-tag fusion protein E coli DH5a containing the
recombi-nant IbeA plasmid was grown overnight in 10 mL LB broth containing 100 lgÆmL)1 of ampicillin at 37C The overnight culture was inoculated to 1 L of fresh LB media containing 100 lgÆmL)1 of ampicillin at 37C until an at-tenuance of 0.4–0.6 at 600 nm was reached, after which recombinant protein expression was induced by 1 mm iso-propyl thio-b-d-galactoside for 3 h The cells were collected
by centrifugation at 6000 g for 15 min and were frozen at )20 C until further use Inclusion bodies were isolated as previously described [6] The cell pellet was suspended thor-oughly in 20 mm Tris pH 8.0 containing 1 mm EDTA, 5% glycerol, protease inhibitors (Roche Diagnostics), 100 mm NaCl, 1 mm dithiothreitol (buffer ratio = 3 mL⁄ 1 g of pel-let) After making an even suspension, 2 mg⁄ mL of lyso-zyme was added and the cells were lysed by sonication The unbroken cells were removed by centrifugation and the cell lysate was further centrifuged at 12 000 g for 1 h at 4C The pellet from the above step was washed with 2 m urea
in the lysis buffer, followed by centrifugation at 20 000 g for 30 min At this point, the white pellet was visible that contains partially purified inclusion bodies The partially purified inclusion bodies were suspended in 10 mL of freshly prepared denaturing buffer, 20 mm Tris pH 8.0 con-taining 8 m urea and centrifuged at 20 000 g for 2 h at room temperature and the clear supernatant was dialyzed
to a final concentration of 100 mm oPOE in the equilibra-tion buffer (50 mm Tris–HCl, pH 8.0 containing 0.2 m urea
150 mm NaCl, 1 mm b-mercaptoethanol, and complete pro-tease inhibitors, 5 mm imidazole for overnight with three regular changes every 4 h The dialysate was clarified by centrifugation 12 000 g for 30 min, loaded onto a 10 mL (15· 1 cm) of pre-equilibrated TALON IMAC column Then, the column was washed by 20 mL of the equilibra-tion buffer, eluted in the same buffer containing 50 mm imidazole and collected in 1 mL fractions The purity of the protein was detected by SDS⁄ PAGE The fractions having pure protein was pooled and stored at )70 C until further use
HBMEC invasion assays HBMEC invasion assays were carried out as described pre-viously [5–7] Briefly, confluent HBMEC in 24-well tissue culture plates were incubated with 107 colony forming units
of E coli K1 strain RS218 at a multiplicity of infection of
100 for 90 min at 37C The monolayers were washed once
Native
pH 3.0
U 1.5 M Gdm-HCl
at pH 3.0
1.5 M Gdm-HCl
at pH 2.0 Fig 10 Hypothesized unfolding pathway for IbeA N, native state at pH 7.0; N¢, non native state at acidic pH; MG, molten globule state at 1.5 M Gdm-HCl; U, unfolded state at pH < 2.0.
Trang 10and then incubated with experimental medium containing
gentamicin (100 lgÆmL)1) for 1 h to kill extracellular
bacte-ria The monolayers were washed three times with NaCl⁄ Pi,
lysed with sterile water, and released intracellular bacteria
were enumerated by plating on sheep blood agar plates
The results were calculated as a percent of the initial
inocu-lum The effect of exogenous IbeA protein on E coli K1
invasion of HBMEC was examined by pre-incubating
HBMEC with IbeA protein for 45 min at 37C, and then
followed by the above-mentioned invasion assays His6
-tagged AslA protein was shown not to interact with
HBMEC and used as a control for His6-tagged IbeA
Determination of correct folding:
heat-modifiability experiments
Samples were mixed 5 : 2 with SDS⁄ PAGE loading buffer
containing 100 mm SDS and either boiled for 5 min or
directly loaded onto the gel In all experiments, 4–20% gels
were used Protein was detected by staining with Safe
Coo-massie Protein Stain (Invitrogen)
Protein determination
The protein concentration was determined
spectrophoto-metrically using Bradford reagent
Western blot analysis
The purified protein was separated on 12% Novex
(tris-gly-cine gel) SDS⁄ PAGE gel and the protein was transferred
onto a PVDF membrane After transfer, the membrane was
blocked in 5% (w⁄ v) nonfat dried milk in NaCl ⁄ Pifor 1 h
at room temperature Monoclonal anti-His6-tagged sera
(1 : 2000) in the same blocking buffer was incubated at
room temperature for 1 h, followed by washing with
NaCl⁄ Pi containing Tween-20 (6· 5 min) and incubation
with horseradish peroxidase conjugated anti-mouse serum
for 1 h at room temperature Bound antibody was
visual-ized after six washings in NaCl⁄ Pi (6· 5 min), and
ana-lyzed using the ECL detection kit
CD studies
CD studies were performed on a Jasco Model J500A
spec-tropolarimeter (Jasco Inc., Easton, MD, USA) The
second-ary structure of the IbeA (1.5 lm) was monitored in the
far-UV region (190–260 nm) using a path length of 0.1 cm
The tertiary structure of the IbeA (3.25 lm) was monitored
in the near-UV (250–320 nm) region using a path length of
0.5 cm path Band widths were 1 nm in the far-UV and
0.4 nm in the near-UV CD Each spectrum was recorded as
the average of three scans The molar ellipticity (h) was
cal-culated using the formula:
h¼ ðhobserved molecular massÞ=ð10 l cÞ
Where l is the length (cm) of the light path and c is the concentration in gÆL)1[49] 20 mm NaCl⁄ PipH 7.0 contain-ing 5 mm oPOE was used as a blank under identical condi-tions to the sample, and the value of the blank was subtracted from the spectrum All measurements were made
at room temperature All data are the averages of three measures
Acidic denaturation of IbeA IbeA was denatured as a function of pH, as mentioned for the buffers above In all the experiments, the final concen-tration of the protein was 1 lm in 20 mm NaCl⁄ PipH 7.0 containing 5 mm oPOE
1-Anilino-8-naphthalene sulfonate binding measurements
The extrinsic fluorescence measurement was performed with
a Hitachi fluorimeter (Hitachi Corp., Tokyo, Japan) The protein concentration was 1 lm in 20 mm NaCl⁄ Pi buffer
pH 7.0 containing 5 mm oPOE and the concentration of ANS was 150 lm Solutions were left overnight for equilibra-tion The excitation wavelength was 380 nm and the emission fluorescence was monitored in the range 400–600 nm
Fluorescence quenching experiments Tryptophan quenching was performed by KI incubated at
pH 2.0 and 7.0 in the presence of 6 m Gdm-HCl at 25C for 1 h The samples of the protein with quencher were incubated at 25C in the dark for 30 min before fluores-cence measurements were taken Tryptophan residue was selectively excited at 292 nm The absorbance of the sample
at 292 nm was always kept below 0.06; thus, no correction
of an inner filter effect was necessary The intensity of the fluorescence at the emission maximum was monitored as a function of the increasing concentration of the quencher The quenching data were analyzed using the modified Stern–Volmer equation [50,51]:
Fo=ðFo FÞ ¼ 1=faþ 1=ðfaKsv½QÞ where Fo and F are the fluorescence intensities of the pro-tein in the absence and presence, respectively, of a given concentration of quencher [Q], Ksv is the Stern–Volmer quenching constant, and farefers to the fraction of trypto-phans accessible to the quencher
Denaturation of IbeA as a function of Gdm-HCl Gdm-HCl induced denaturation of IbeA at a given pH, was performed with increasing concentrations of the