Protein folding intermediates of invasin protein IbeA from Escherichia coli Damodara R. Mendu 1 , Venkata R. Dasari 2 , Mian Cai 1 and Kwang S. Kim 1 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 His 6 - 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. 458 FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS [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 proteins 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 His 6 antibody (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 C D E A B Fig. 1. (A) Analysis of the purified IbeA by SDS ⁄ PAGE (4–20%). The alternate fractions from the TALONÒ column were analyzed by SDS ⁄ PAGE. Lane A, molecular weight markers (Invitrogen pre- stained markers); lane B, flow through; and lanes C–E, TALONÒ 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. D. R. Mendu et al. Protein folding intermediates FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 459 (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 His 6 -tag. On the other hand, our His 6 -tag control protein and His 6 - tag removed proteolytically from IbeA molecule did not have any effect on HBMEC invasion of RS218.In further studies, we used His 6 -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 50 °C, respectively (Fig. 1A). When the IbeA was heated at 100 °C 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 TALONÒ 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 AB C 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 50 100 150 200 250 300 350 400 450 300 320 340 360 380 400 Wavelength (nm) Fluorescence intensity 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. Protein folding intermediates D. R. Mendu et al. 460 FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 352–336 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 90 120 150 250 270 290 310 Wavelength (nm) Wavelength (nm) [ ] deg cm 2 d mo l –1 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 [ ] × 10 –3 deg cm 2 d mol –1 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 lM in the near- and far-UV CD, respectively. Table 1. Secondary structure content of IbeA by SELCON. State a (%) b (%) Other (%) Native 30 34 36 Acid unfolded 5 40 55 Gdm-HCl unfolded 10 50 40 –8 –7 –6 –5 –4 –3 –2 –1 0 0246810 pH [θ] 222 × 10 –3 deg cm 2 dmol –1 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. D. R. Mendu et al. Protein folding intermediates FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 461 8-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 02468 10 pH Fluorescence intensity 0 100 200 300 400 500 600 700 400 450 500 550 600 Fluorescence intensity 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 Wavelength maxima (nm) 0 75 150 225 300 375 450 0246810 012345678910 pH Fluorescence intensity 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 PO 4 buffer pH 7.0, containing 5 mM oPOE. (A) The wave- length maximum. (B) The excitation wavelength was 292 nm with slit widths of 10 and 5 nm for excitation and emission, respec- tively. Protein folding intermediates D. R. Mendu et al. 462 FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS was investigated by iodine quenching studies. The quenching constants (K SV ) and fraction of accessible fluorophore (f a ) 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 conformational 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 confirmation 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 F0/ (F0 – F) 024681012 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 (C m )(M) DG U-N (kcalÆmol )1 ) m U-N kcalÆmol )1 ÆM )1 pH 7.0 (RT) CD [h] 222 4.5 ± 0.1 )12.9 ± 0.5 )2.8 ± 0.1 CD [h] 278 4.4 ± 0.1 )12.8 ± 0.5 )2.6 ± 0.1 Fluorescence 4.5 ± 0.1 )13.0 ± 0.5 )2.7 ± 0.1 pH 3.0 (RT) CD [h] 222 2.4 ± 0.1 )4.9 ± 0.2 )1.8 ± 0.1 CD [h] 278 1.1 ± 0.1 )3.4 ± 0.2 )3.4 ± 0.1 Fluorescence 2.5 ± 0.1 )4.8 ± 0.2 )1.8 ± 0.1 pH 2.0 (RT) CD [h] 222 1.8 ± 0.1 (C m1 )– – 3.2 ± 0.1 (C m2 )– – Fluorescence 1.8 ± 0.1 (C m1 )– – 3.2 ± 0.1 (C m2 )– – D. R. Mendu et al. Protein folding intermediates FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 463 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 [Gdm-HCl] ( M ) [Gdm-HCl] ( M ) Fraction unfolded 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 Fraction unfolded far UV-CD near UV-CD fluorescence 0 100 200 300 400 500 600 [Gdm-HCl] (M) ANS fluorescence [Gdm-HCl] (M) Fraction unfolded fluorescence far UV-CD near UV-CD 0 100 200 300 400 500 600 [Gdm-HCl] ( M ) ANS fluorescence 3.5 4.5 5.5 6.5 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 02468 A B C E D Fig. 9. Formation and identification of protein folding intermediates. 1 lM IbeA in 20 mM PO 4 buffer pH 7.0, containing 5 mM 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 mM glycine buffer pH 2.0 containing 5 mM oPOE, (D) ANS fluorescence at pH 3.0 and (E) at pH 2.0. Protein folding intermediates D. R. Mendu et al. 464 FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS interference 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 accordingly. 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 m U-N values 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 D. R. Mendu et al. Protein folding intermediates FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 465 functional 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); TALONÒ 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-His 6 -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 ⁄ P i , pH 7.0. ANS concentration was calcu- lated spectrophotometrically using an extinction coefficient of 5000 m )1 Æcm )1 at 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 · His 6 - tag fusion protein. E. coli DH5a containing the recombi- nant IbeA plasmid was grown overnight in 10 mL LB broth containing 100 lg Æ mL )1 of ampicillin at 37 °C. The overnight culture was inoculated to 1 L of fresh LB media containing 100 l g ÆmL )1 of ampicillin at 37 °C 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 4 °C. 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 TALONÒ 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 37 °C. The monolayers were washed once Native IbeA N´ MG pH 2.0 < pH 2.0 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. Protein folding intermediates D. R. Mendu et al. 466 FEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS and 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 ⁄ P i , 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 37 °C, and then followed by the above-mentioned invasion assays. His 6 - tagged AslA protein was shown not to interact with HBMEC and used as a control for His 6 -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- massieÒ 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 ⁄ P i for 1 h at room temperature. Monoclonal anti-His 6 -tagged sera (1 : 2000) in the same blocking buffer was incubated at room temperature for 1 h, followed by washing with NaCl ⁄ P i 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 ⁄ P i (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: h ¼ðh observed  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 ⁄ P i pH 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 ⁄ P i pH 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 ⁄ P i 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 25 °C for 1 h. The samples of the protein with quencher were incubated at 25 °C 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]: F o =ðF o À FÞ¼1=f a þ 1=ðf a ÁK sv Á½QÞ where F o and F are the fluorescence intensities of the pro- tein in the absence and presence, respectively, of a given concentration of quencher [Q], K sv is the Stern–Volmer quenching constant, and f a refers 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 D. R. Mendu et al. 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Protein folding intermediates of invasin protein IbeA from Escherichia coli Damodara R. Mendu 1 , Venkata R. Dasari 2 ,. 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