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Protein folding intermediates of invasin protein IbeAfrom Escherichia coliDamodara R. Mendu1, Venkata R. Dasari2, Mian Cai1and Kwang S. Kim11 Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA2 Department of Biomedical and Therapeutic Sciences, College of Medicine, University of Illinois, Peoria, IL, USAEscherichia coli is the most common Gram-negativeorganism that causes neonatal meningitis [1–4]. E. colihas several virulence factors, including a 50 kDa IbeAprotein, 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. colipenetration of human brain microvascular endothelialcells (HBMEC) which constitute the blood–brainbarrier (BBB) [5–7]. The 8.2 kDa N-terminal IbeAprotein was shown to inhibit E. coli K1 invasion ofHBMEC [5].The primary amino acid sequence of a polypeptideencodes all of the information necessary for folding andassembly pathways, as well as the native 3D structureKeywordsacid and Gdm-HCl-induced unfolding;Escherichia coli; molten globule; proteinunfolding intermediates of IbeACorrespondenceK. S. Kim, Division of Pediatric InfectiousDiseases, Johns Hopkins University Schoolof Medicine, 200 North Wolfe Street,Room 3157, Baltimore, MD 21287, USAFax: +1 410 614 1491Tel: +1 410 614 3917E-mail: kwangkim@jhmi.edu(Received 9 October 2007, revised15 November 2007, accepted 28 November2007)doi:10.1111/j.1742-4658.2007.06213.xIbeA of Escherichia coli K1 was cloned, expressed and purified as a His6-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasionof human brain microvascular endothelial cells and was heat-modifiable.The structural and functional aspects, along with equilibrium unfolding ofIbeA, were studied in solution. The far-UV CD spectrum of IbeA atpH 7.0 has a strong negative peak at 215 nm, indicating the existence ofb-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 showsthe existence of a partially unfolded molecule (molten globule-like struc-ture) with b-sheet-like structure and displays strong 8-anilino-2-naphthylsulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence ofIbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state withcharacteristics of a molten globule-like state, and the protein is in extendedb-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 tosolvent 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 furtherdecrease in pH to 2.0 drives the protein to the A state. The presence of0.5 m KCl in the solvent composition directs the transition to the A stateby bypassing the acid-unfolded state. Additional guanidine hydrochlorideinduced conformational changes in IbeA from the native to the A-state, asmonitored by near- and far-UV CD and ANS-fluorescence.AbbreviationsANS, 8-anilino-2-naphthyl sulfonic acid; BBB, blood–brain barrier; Gdm-HCl, guanidine hydrochloride; HBMEC, human brain microvascularendothelial cells; IMAC, immobilized metal affinity chromatography; LB, Luria-Bertani; oPOE, octylpolyoxyethylene; PVDF, poly(vinylidenedifluoride); 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 giveimportant insights into protein misfolding, and aggre-gation. It has been recognized that the structureof non-native state of proteins can provide significantinsight into fundamental issues such as the relationshipbetween 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 isavailable on protein folding intermediates [11–18],there are no reports available for E. coli invasin pro-tein IbeA. The process of unfolding and refolding isuseful for a complex unfolding transition, indicatingtheir presence of a partially folded intermediate withone 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 ofthe individual folding intermediaries in each pathwayand developing therapies against these intermediatesmight be an attractive strategy. Such delineation canonly be achieved by identifying partially unfoldedstates formed during folding, and correlating theirpopulations by spectroscopy. Using this approach,IbeA protein folding intermediates (misfolded aggre-gates) can be directly identified and attention focusedon defining the structural properties of these states. Todate, no information about the structural properties ofIbeA has been available. In the present study, we char-acterized the biophysical properties of IbeA in solutionusing spectroscopic techniques to identify protein fold-ing intermediates.ResultsThe initial step in the action of IbeA for E. coliK1 traversal of the BBB is binding to a cell-surfacereceptor, which induces the conformational changes ofthe IbeA binding domains. We hypothesize that theresulting protein–receptor complexes are endocytosedand delivered to an acidic compartment (endosome) ofthe cell, forming a prepore-like structure, enabling theinternalization and traversal of E. coli in HBMEC, butthe nature of this relationship is incompletely under-stood. No studies on E. coli traversal mechanisms havefocused on the conformational changes occurring inIbeA acidification, and no study has addressed theacid-induced changes in the IbeA molecule. We alsohypothesize that the HBMEC central lumen is toosmall to accommodate native IbeA, necessitating somedegree of protein unfolding for efficient translocationof E. coli, and we have shown the existence of proteinfolding intermediates and acidic unfolding intermedi-ates in vitro.Purification of IbeAThe expression level of IbeA was 6–8 mgÆL)1ofculture, and its molecular weight was 50 kDa bySDS ⁄ PAGE. The fractions eluted from an immobilizedmetal affinity chromatography (IMAC) column wereanalyzed by Coomassiee blue staining (Fig. 1). Theidentity of the IbeA was analyzed by western blottingwith monoclonal His6antibody (unpublished data) andwith purified antibodies to IbeA (Fig. 2). The correctrefolding of IbeA was shown by invasion assays andheat modifiability experiments. The purified recombi-nant IbeA blocked E. coli K1 invasion in HBMECm (kDa)2501501007550372520A B CD E 30 40 50 m (kDa)190120856050402520A B C D E ABFig. 1. (A) Analysis of the purified IbeA by SDS ⁄ PAGE (4–20%).The alternate fractions from the TALONÒ column were analyzed bySDS ⁄ PAGE. Lane A, molecular weight markers (Invitrogen pre-stained markers); lane B, flow through; and lanes C–E, TALONÒfractions 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 ⁄ PAGEsample buffer and analyzed by SDS ⁄ PAGE. Lane A, molecularweight markers (Bio-Rad prestained markers) and lanes B–E, puri-fied protein.D. R. Mendu et al. Protein folding intermediatesFEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 459(25 lgÆmL)1reduced the HBMEC invasion frequencyof E. coli K1 strain RS218 by 73%) and it is assumedthat this blocking activity is due to its native structure,and that there was no interference of the His6-tag. Onthe other hand, our His6-tag control protein and His6-tag removed proteolytically from IbeA molecule didnot have any effect on HBMEC invasion of RS218.Infurther studies, we used His6-tagged IbeA molecule.We have also shown the correct folding of purifiedIbeA by invasion assays, heat modifiability experi-ments and fluorescence spectroscopy (Fig. 3) for bothneutral and acidic pH and denaturant. The refoldedprotein 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 kDawhen heated at 30, 40 and 50 °C, respectively(Fig. 1A). When the IbeA was heated at 100 °C for5 min in SDS sample buffer, only the 50 kDa bandwas observed (Fig. 1B), suggesting the gel shift at hightemperature. The gel shift of 5 kDa is similar to theother modifiable membrane proteins [20–22]. Weassume that the 50 kDa protein is fully folded proteinand that the 53 and 55 kDa proteins could be a fold-ing intermediate or off pathway species [23]. A numberof membrane proteins differ in their migration veloci-ties in SDS ⁄ PAGE depending on whether or not theprotein was heated before electrophoresis [24–28]. Thefractions of TALONÒ shown in Fig.1A were identifiedby the polyclonal sera (Fig. 2).Characterization of IbeAThe purified IbeA was equilibrated with denaturant upto 24 h and no further spectroscopic changes wereobserved after 24 h, when the presented results wereobtained, indicating that equilibrium was attainedwithin this time. Near-UV CD was employed to exam-ine the asymmetry of aromatic amino acids, andthereby to monitor the changes in the tertiary structureof the protein [22]. The CD spectrum of native IbeAexhibited a positive peak at 276–278 nm and a nega-tive peak at 297–299 nm, which is due to the presenceof tryptophan residues (Fig. 4A). However, pH 2.0and strong denaturant, such as 6 m guanidine hydro-chloride (Gdm-HCl), did not provide information dueto the disordered aromatic groups in the unfoldedstate. The far-UV CD spectrum of a protein is a diag-nostic probe of secondary structure and facilitatesdetermination of specific structural features that com-prise the native conformation. The far-UV CD spec-trum of IbeA (Fig. 4B) showed a negative peak at215 nm, suggesting the presence of extended ß-sheetregions. IbeA exhibited a negative peak at 200 nm,indicative of a strong contribution from disorderedstructural elements, characteristic of a protein in a ran-dom coil conformation.As can be seen, decreasing the pH below 2 changedthe acid-induced unfolded state due to the formationof the A-state. The A-state of IbeA has a substantialnon-native secondary structure, and little or no tertiarystructure. These data strongly indicate the presence ofextended b-sheets and, in the presence of 6 m Gdm-HCl, IbeA lost all of the peaks, suggesting the loss ofsecondary structure. The deconvolution spectrumobtained using the selcon program [29] provides thestructural component of IbeA (Table 1).The intrinsic fluorescence spectra of IbeA at pH 7.0and 2.0 and in the presence of 6 m Gdm-HCl areshown in Fig. 3. The lowering of pH from 7.0 to 2.0drastically decreased fluorescence intensity by 70–75%with a blue shift of 16 nm in the emission maxima atAB CFig. 2. Western blot analysis of purified, refolded IbeA using puri-fied sera raised against pure IbeA. The pure IbeA (5 lg) was heatedat 30, 40 and 50 °C for 5 min in SDS ⁄ PAGE sample buffer andloaded on to the 12% SDS ⁄ PAGE gel.050100150200250300350400450300 320 340 360 380 400Wavelength (nm)Fluorescence intensitypH 7.0Gdm-HCl (6 M)pH 2.0Fig. 3. Fluorescence spectroscopy analysis of denatured IbeA byGdm-HCl. Purified IbeA (1 lM) was denatured by titrating withGdm-HCl at room temperature (25 °C). The denaturation mediatedchanges in IbeA were monitored for tryptophan fluorescence; exci-tation was 292 nm and emission was 300–420 nm at pH 7.0 and2.0 and in the presence of 6M 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 FEBS352–336 nm, indicating the non-polar environment oftryptophan. Although the fluorescence spectrum ofcompletely unfolded IbeA in 6 m Gdm-HCl remainssimilar in shape, the emission maximum suffers a redshift from 352 nm to 358 nm along with a decrease influorescence intensity of 60–70%. This red shift in thewavelength maximum indicates that more tryptophanresidues of the protein are exposed to a polar environ-ment, which is characteristic of unfolding, or could bedue to decreased distance between tryptophan andquenching groups, resulting in tryptophan fluorescencequenching.The far-UV CD spectrum of IbeA remainsunchanged in the pH range of 3.0–10, and the spec-trum reveals two distinct peaks: one at 222 nm and theother at 208 nm (Data not shown). The unfolding ofthe IbeA, in the absence of added salt, followed byellipticity at 222 nm, is noncooperative (Fig. 5). Acooperative transition from the native state to an acid-unfolded state occurred at pH 3.0–1.5, and a secondtransition occurred on further lowering the pH from1.5 to 0.5. The unfolded state at lower pH, exhibitinga reduced secondary structure and loss of tertiarystructure, represents the acid-unfolded state of theIbeA, 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 toa second transition between pH 1.5 and 0.5 resulted inan increase in secondary structure, leading to theA state [30].In the presence of 0.5 m KCl, pH-induced unfoldingof IbeA is cooperative, as manifested by a single tran-sition (Fig. 5), in which the protein molecule passesfrom the native state to the A state directly withoutpassing through the acid-unfolded state. The secondarystructural content of such a salt-induced A state ismore ordered than that observed at pH 0.5 in theabsence of added salt. The CD spectrum of the proteinat pH 2.0–0.5, either in the presence or in the absenceof 0.5 m KCl, exhibits predominantly extended b-sheetstructure and the negative peak at 215–217 nm atpH 2.0 (Fig. 4B) is a common characteristic feature ofproteins having extended b-sheets. At a higher concen-tration of KCl, aggregation or precipitation wasobserved.–300306090120150250 270 290 310Wavelength (nm)Wavelength (nm)[ ] deg cm2d mo l–1pH 7.0pH 2.0Gdm-HCl (6 M)–10–7–4–125811180 190 200 210 220 230 240 250 260[ ] × 10–3 deg cm2 d mol–1pH 7.0pH 2.06 M Gdm-HClABFig. 4. (A) Near- and (B) far-UV CD of purified IbeA in the presenceof oPOE, as described in the Experimental procedures, wereanalyzed at pH 7.0 and 2.0 and in the presence of Gdm-HCl. Theprotein concentrations were 3.25 lM and 1.5 lM in the near- andfar-UV CD, respectively.Table 1. Secondary structure content of IbeA by SELCON.State a (%) b (%) Other (%)Native 30 34 36Acid unfolded 5 40 55Gdm-HCl unfolded 10 50 40–8–7–6–5–4–3–2–100246810pH[θ] 222 × 10–3 deg cm2 dmol–1presence of saltabsence of saltFig. 5. Effect of salt on the structure of IbeA. Structural changesof IbeA as a function of pH were monitored by studying ellipticityvalues at 222 nm in the presence and absence of 0.5M KCl.D. R. Mendu et al. Protein folding intermediatesFEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 4618-Anilino-2-naphthyl sulfonic acid (ANS)fluorescence studiesThe effect of pH on ANS binding shows that IbeAbinds more strongly at pH 2.0 rather than in its nativestate (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 accessiblehydrophobic residues to ANS. The ANS fluorescencespectra between 10–0.5 pH (Fig. 6B) strongly supportthe acidic unfolding with a two state transition and theformation of a molten globule state at pH 3.0–1.5. Themolten globule was formed at pH 2.0 (Fig. 6B) withhigh ANS binding capacity and significant secondarystructure with no tertiary structure. At pH 0.5, ANSbinding capacity and secondary structure was minimalas 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 moltenglobular state of IbeA molecule exposes hydrophobicresidues. All these data obtained at pH 2.0 support thedefinition of a molten globule with b-helical confirma-tion.The pH dependent intrinsic fluorescence of IbeAwas 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 decreaseswhen the pH falls from 6.0 to 4.0 (blue shift) and,in the latter transition, fluorescence intensity wasincreased (red shift) as the protein reached its acidunfolded state.Iodine-quenching studiesThe solvent accessibility of individual tryptophan resi-dues in the native, molten globule and unfolded states0100200300400500600700AB0246810pHFluorescence intensity0100200300400500600700400 450 500 550 600Fluorescence intensitypH 7.4pH 2.0pH 3.0pH 0.56 M Gdm-HCl Wavelength (nm)Fig. 6. ANS binding to IbeA as a function of pH. The samples wereincubated for 24 h at 25 °C before the measurements were taken.(A) ANS binding measurement was taken by excitation at 360 nmand emission was collected between 400–600 nm. (B) ANS fluores-cence at different pH values.330335340345350355360ABpHWavelength maxima (nm)0751502253003754500246810012345678910pHFluorescence intensityFig. 7. Intrinsic fluorescence analysis of IbeA at varying pH values.The effect of pH on the intrinsic fluorescence of IbeA at differentpH values was plotted. The protein concentration was 1 lM in20 mM PO4buffer pH 7.0, containing 5 mM oPOE. (A) The wave-length maximum. (B) The excitation wavelength was 292 nm withslit 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 FEBSwas investigated by iodine quenching studies. Thequenching constants (KSV) and fraction of accessiblefluorophore (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, and0.36 ± 0.03, 0.89 ± 0.08 and 0.8 ± 0.07, respectively.The modified Stern–Volmer plot indicates that thetryptophan residues in the IbeA at pH 2.0 are moreexposed to the solvent compared with native IbeA atpH 7.0 (Fig. 8). However 0–2 m Cs+was unable toquench tryptophan fluorescence either at pH 2.0 or inthe presence of Gdm-HCl (data not shown). At neutralpH, no noticeable changes were seen in the fluores-cence spectra for both quenchers (data not shown).These data indicate that no structural changes tookplace in the protein molecule.IbeA was denatured by Gdm-HCl at differentpH values and was monitored by near- and far-UV CD and fluorescence spectroscopy to determinethe secondary and tertiary structural changes(Table 2). The CD and intrinsic fluorescence spectrumat pH 7.0 is sigmoidal and cooperative (Fig. 9A). AtpH 3.0, the Gdm-HCl-induced unfolding curves ofIbeA are cooperative (Fig. 9B), with non-coincidentaltransition curves. At this pH, IbeA loses its secondary,tertiary structure and fluorescence intensity, indicatingthe 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 spectrumand, at pH 2.0, the Gdm-HCl-induced denaturationcurve of IbeA was non-cooperative (Fig. 9D). TheANS binding to IbeA (Fig. 9E) was very strong afterthe first transition and gradually decreased with anincrease in Gdm-HCl concentration, indicating theexistence of hydrophobic domains at first unfolded.DiscussionThe biophysical analysis of IbeA provides much infor-mation about its conformational states and proteinfolding intermediates. In the present study, we haveused multiple probes to investigate the structure ofIbeA by pH and the denaturation process induced byGdm-HCl. These probes were used to study its solu-tion confirmation and to identify protein unfoldingintermediates. We attempted to characterize the fold-ing intermediates in interaction with HBMEC, but lowpH (acidic) and denaturant damaged the HBMECmonolayer, precluding such experiments.IbeA was expressed, purified and refolded usingoctylpolyoxyethylene (oPOE) detergent and has no0 0.5 1 1.5 2 2.5 3 3.5 4 [I-]–1F0/ (F0 – F)024681012pH 7.0 pH 2.0 6 M Gdm-HCl Fig. 8. The modified Stern–Volmer’s of tryptophan fluorescencequenching by iodide [I)1]. Quenching of tryptophan fluorescenceintensity of IbeA at pH 7.0 and 2.0 and in the presence of Gdm-HCl, was carried out with 0.0–0.2M KI at 25 °C. KCl was added tomaintain the ionic strength constant. The data was analyzed as permodified 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) DGU-N(kcalÆmol)1) mU-NkcalÆmol)1ÆM)1pH 7.0 (RT) CD [h]2224.5 ± 0.1 )12.9 ± 0.5 )2.8 ± 0.1CD [h]2784.4 ± 0.1 )12.8 ± 0.5 )2.6 ± 0.1Fluorescence 4.5 ± 0.1 )13.0 ± 0.5 )2.7 ± 0.1pH 3.0 (RT) CD [h]2222.4 ± 0.1 )4.9 ± 0.2 )1.8 ± 0.1CD [h]2781.1 ± 0.1 )3.4 ± 0.2 )3.4 ± 0.1Fluorescence 2.5 ± 0.1 )4.8 ± 0.2 )1.8 ± 0.1pH 2.0 (RT) CD [h]2221.8 ± 0.1 (Cm1)– –3.2 ± 0.1 (Cm2)– –Fluorescence 1.8 ± 0.1 (Cm1)– –3.2 ± 0.1 (Cm2)– –D. R. Mendu et al. Protein folding intermediatesFEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 46300.[Gdm-HCl] (M) [Gdm-HCl] (M) Fraction unfolded near UV-CDfar 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 0100200300400500600[Gdm-HCl] (M) ANS fluorescence [Gdm-HCl] (M) Fraction unfolded fluorescence far UV-CD near UV-CD 0100200300400500600[Gdm-HCl] (M) ANS fluorescence 3.5 4.5 5.5 6.5 0 1 2 3 4 5 6 70 1 2 3 4 5 6 7 0 1 2 3 4 5 6 02468A B C E D Fig. 9. Formation and identification of protein folding intermediates. 1 lM IbeA in 20 mM PO4buffer pH 7.0, containing 5 mM oPOE wasdenatured as a function of Gdm-HCl. Near-and far-UV CD and ANS fluorescence were measured (A) at pH 7.0, (B) 20 mM glycine bufferpH 3.0 containing 5 mM 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 FEBSinterference in invasion assays. In principle, we cannotdiscount the possibility that IbeA cannot refold insolution is an in vitro artifact. We strongly believe thatthis is not the case because we performed an extensivescreening of refolding conditions.CD is a sensitive method for investigating the pep-tide bond and has been widely used to elucidate thestructure of proteins [32–39]. The far-UV CD of IbeA(Fig. 4B) is similar to the characteristic features ofporins [40–45]. The membrane proteins exist asa-helical and ß-barrel proteins and the transmembraneß-barrels are present in Gram-negative bacteriabecause 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 globularstate. The negative ellipticity at 208 and 222 nm alsoindicates the presence of a-helical confirmation.The fluorescence spectra of IbeA were identicalwhen excited at 278 and 295 nm (unpublished data)and these data show that tyrosine fluorescence wasquenched by tryptophan. The fluorescence spectra ofIbeA at 6 m Gdm-HCl and at pH 7 indicate that moretryptophan residues are exposed to a polar environ-ment. It could be also possible that, at neutral pH, theexcitable chromophores are in a hydrophilic environ-ment.Our acid unfolding studies indicate that the moltenglobule state was formed at pH 2.0 in the unfoldingprocess from pH 3.0–1.5 and this was also confirmedby the ANS data. Thus, IbeA exhibits a two state tran-sition in acidic denaturation, as the mechanisms ofacid-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 inpH causes enhancement of protonation of the protein.At pH 2.0, the protonation becomes saturated and theprotein loses its structure and forms the A state. Bothanions and cations will be present in the acidic unfold-ing environment and addition of cations (K+) doesnot have any impact on ionization. At extreme acidicpH, there will be repulsion between the charged groupsof the protein, and an even high concentration of thesalts (counter-ions) interacts with charged groups andweakens the repulsions. Thus, in the presence of salt(KCl), the IbeA molecule directly reaches the A state.The pH-induced denaturation curves demonstratethat the decrease in the fluorescence intensity, with ablue shift (15–17 nm) between 6.0–4.0 pH, could bedue to either the microenvironmental changes in theregion of tryptophan residues protecting its overallstructure and the tertiary structure of the protein, orto uncharged carboxylate groups causing the less polarenvironment near the tryptophan residues, resulting ina blue shift of the tryptophan fluorescence [48]. In thesecond transition, a red shift with an increase in fluo-rescence intensity in the pH range between 3.0–0.5occurs as a result of loss in its secondary structure dueto the acid-induced unfolding state.The modified Stern–Volmer’s plot for the nativeIbeA indicates the limited accessibility of the aromaticresidues but, at acidic pH, more aromatic groups areexposed to the solvent due to the presence of a moltenglobule. These data indicate the high binding capacityof quenchers at acidic pH due to the formation ofmolten globule compared to the native state. Weassume that the molten globule is a loosely packedintermediate with largely exposed tryptophan residues.The Gdm-HCl-induced unfolding of IbeA from amolten globule to an unfolded state is noncooperative,by contrast to the cooperative unfolding occurring atneutral pH. This cooperative unfolding is due to theintegrity of IbeA owing to side chain packing entailingthe breaking of the tertiary structure required for non-cooperative transitions observed in the molten globule.The Gdm-HCl-induced unfolding of the IbeA moltenglobule structure also denotes the presence of twodomains that unfold independently of each other. OurGdm-HCl data also indicate the unfolding of onedomain between 2.0–2.8 m Gdm-HCl whereas theother one is intact. The ANS binding to the moltenglobule at pH 2.0 is also parallel with the first transi-tion because most of the hydrophobic residues are inthe first unfolded domain.The Gdm-HCl and pH induced (3.0) unfolding curveswere coincidental, and the mU-Nvalues at near –UV CDare considerably higher than the fluorescence andfar-UV CD values. These data indicate the presence ofan intermediate state between the native and denaturedstates. Furthermore, the existence of an intermediatewas demonstrated by ANS binding to IbeA at 1.5 mGdm-HCl with a secondary structure. The secondarystructure of the intermediates at pH 3.0 and 1.5 mGdm-HCl are almost similar, supporting the existenceof intermediates in the different conditions.The characteristic heat modifiability was mainly usedto study b-barrel outer membrane proteins. The highcontent of b-strands reflected in the CD spectrareported in the present study suggests that a significantnumber of extracellular loops also adopt this second-ary structure. We assume that IbeA molecule b-barrelstrands traverse through the outer membrane intoextracellular space. IbeA had the characteristic featuresof outer membrane proteins, with seven trans-membrane domains having extended b-sheets and twoD. R. Mendu et al. Protein folding intermediatesFEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 465functional domains that unfold independently. In theIbeA unfolding process, an equilibrium intermediatewas found with 1.5 m Gdm-HCl and pH 2.0. Theunfolding pathway of IbeA could be divided into twotransition stages, namely an inactive intermediate anda native state. The proposed unfolding pathway ofIbeA is shown in Fig. 10.Experimental proceduresReagentsHigh purity grade Gdm-HCl, ANS, and KI were obtainedfrom Sigma Chemical Co (St Louis, MO, USA); oPOE wasfrom Bachem (Torrance, CA, USA); TALONÒ IMACresin was from Clontech (Palo Alto, CA, USA); poly(vinyli-dene difluoride) (PVDF) membrane was from Millipore(Bedford, MA, USA); Novex gels and SDS ⁄ PAGE markersand 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 RocheDiagnostics (Indianapolis, IN, USA); horseradish peroxi-dase conjugated anti-mouse sera and the PVDF membraneECL detection kit were from Amersham Biosciences(Piscataway, NJ, USA); and ampicillin, isopropyl thio-b-d-galactoside, EDTA, dithiothreitol, b-mercaptoethanoland complete protease inhibitors, oPOE, were from SigmaChemical Co.Buffers and solutionsThe buffers used for the spectroscopic measurements at dif-ferent pH values were 20 mm KCl-HCl (0.5–1.5), 20 mmglycine ⁄ 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 coefficientof 5000 m)1Æcm)1at 350 nm. All the solutions were preparedin deionized water and filtered through a 0.22-lm filter.Expression of IbeA fusion proteinIbeA 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 LBbroth containing 100 lg Æ mL)1of ampicillin at 37 °C. Theovernight culture was inoculated to 1 L of fresh LB mediacontaining 100 l g ÆmL)1of ampicillin at 37 °C until an at-tenuance of 0.4–0.6 at 600 nm was reached, after whichrecombinant protein expression was induced by 1 mm iso-propyl thio-b-d-galactoside for 3 h. The cells were collectedby centrifugation at 6000 g for 15 min and were frozen at)20 °C until further use. Inclusion bodies were isolated aspreviously 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 mmNaCl, 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. Theunbroken cells were removed by centrifugation and the celllysate was further centrifuged at 12 000 g for 1 h at 4 °C.The pellet from the above step was washed with 2 m ureain the lysis buffer, followed by centrifugation at 20 000 gfor 30 min. At this point, the white pellet was visible thatcontains partially purified inclusion bodies. The partiallypurified inclusion bodies were suspended in 10 mL offreshly prepared denaturing buffer, 20 mm Tris pH 8.0 con-taining 8 m urea and centrifuged at 20 000 g for 2 h atroom temperature and the clear supernatant was dialyzedto a final concentration of 100 mm oPOE in the equilibra-tion buffer (50 mm Tris–HCl, pH 8.0 containing 0.2 m urea150 mm NaCl, 1 mm b-mercaptoethanol, and complete pro-tease inhibitors, 5 mm imidazole for overnight with threeregular changes every 4 h. The dialysate was clarified bycentrifugation 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 mmimidazole and collected in 1 mL fractions. The purity ofthe protein was detected by SDS ⁄ PAGE. The fractionshaving pure protein was pooled and stored at )70 °C untilfurther use.HBMEC invasion assaysHBMEC invasion assays were carried out as described pre-viously [5–7]. Briefly, confluent HBMEC in 24-well tissueculture plates were incubated with 107 colony forming unitsof E. coli K1 strain RS218 at a multiplicity of infection of100 for 90 min at 37 °C. The monolayers were washed onceNativeIbeAN´MGpH 2.0 < pH 2.0pH 3.0U1.5 M Gdm-HClat pH 3.01.5 M Gdm-HClat pH 2.0Fig. 10. Hypothesized unfolding pathway for IbeA. N, native state at pH 7.0; N¢, non native state at acidic pH; MG, molten globule state at1.5M 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 FEBSand then incubated with experimental medium containinggentamicin (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 bacteriawere 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 K1invasion of HBMEC was examined by pre-incubatingHBMEC with IbeA protein for 45 min at 37 °C, and thenfollowed by the above-mentioned invasion assays. His6-tagged AslA protein was shown not to interact withHBMEC and used as a control for His6-tagged IbeA.Determination of correct folding: heat-modifiability experimentsSamples were mixed 5 : 2 with SDS ⁄ PAGE loading buffercontaining 100 mm SDS and either boiled for 5 min ordirectly loaded onto the gel. In all experiments, 4–20% gelswere used. Protein was detected by staining with Safe Coo-massieÒ Protein Stain (Invitrogen).Protein determinationThe protein concentration was determined spectrophoto-metrically using Bradford reagent.Western blot analysisThe purified protein was separated on 12% Novex (tris-gly-cine gel) SDS ⁄ PAGE gel and the protein was transferredonto a PVDF membrane. After transfer, the membrane wasblocked in 5% (w ⁄ v) nonfat dried milk in NaCl ⁄ Pifor 1 hat room temperature. Monoclonal anti-His6-tagged sera(1 : 2000) in the same blocking buffer was incubated atroom temperature for 1 h, followed by washing withNaCl ⁄ Picontaining Tween-20 (6 · 5 min) and incubationwith horseradish peroxidase conjugated anti-mouse serumfor 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 studiesCD 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 thefar-UV region (190–260 nm) using a path length of 0.1 cm.The tertiary structure of the IbeA (3.25 lm) was monitoredin the near-UV (250–320 nm) region using a path length of0.5 cm path. Band widths were 1 nm in the far-UV and0.4 nm in the near-UV CD. Each spectrum was recorded asthe average of three scans. The molar ellipticity (h) was cal-culated using the formula:h ¼ðhobserved molecular massÞ=ð10  l  cÞWhere l is the length (cm) of the light path and c is theconcentration 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 wassubtracted from the spectrum. All measurements were madeat room temperature. All data are the averages of threemeasures.Acidic denaturation of IbeAIbeA was denatured as a function of pH, as mentioned forthe buffers above. In all the experiments, the final concen-tration of the protein was 1 lm in 20 mm NaCl ⁄ PipH 7.0containing 5 mm oPOE.1-Anilino-8-naphthalene sulfonate bindingmeasurementsThe extrinsic fluorescence measurement was performed witha Hitachi fluorimeter (Hitachi Corp., Tokyo, Japan). Theprotein concentration was 1 lm in 20 mm NaCl ⁄ PibufferpH 7.0 containing 5 mm oPOE and the concentration ofANS was 150 lm. Solutions were left overnight for equilibra-tion. The excitation wavelength was 380 nm and the emissionfluorescence was monitored in the range 400–600 nm.Fluorescence quenching experimentsTryptophan quenching was performed by KI incubated atpH 2.0 and 7.0 in the presence of 6 m Gdm-HCl at 25 °Cfor 1 h. The samples of the protein with quencher wereincubated at 25 °C in the dark for 30 min before fluores-cence measurements were taken. Tryptophan residue wasselectively excited at 292 nm. The absorbance of the sampleat 292 nm was always kept below 0.06; thus, no correctionof an inner filter effect was necessary. The intensity of thefluorescence at the emission maximum was monitored as afunction of the increasing concentration of the quencher.The quenching data were analyzed using the modifiedStern–Volmer equation [50,51]:Fo=ðFoÀ FÞ¼1=faþ 1=ðfaÁKsvÁ½QÞwhere Foand F are the fluorescence intensities of the pro-tein in the absence and presence, respectively, of a givenconcentration of quencher [Q], Ksvis the Stern–Volmerquenching constant, and farefers to the fraction of trypto-phans accessible to the quencher.Denaturation of IbeA as a function of Gdm-HClGdm-HCl induced denaturation of IbeA at a given pH,was performed with increasing concentrations of theD. R. Mendu et al. Protein folding intermediatesFEBS Journal 275 (2008) 458–469 ª 2007 The Authors Journal compilation ª 2007 FEBS 467[...]... 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Protein folding intermediates of invasin protein IbeA from Escherichia coli Damodara R. Mendu1, Venkata R. Dasari2,. 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
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