A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system Tomoaki Kato1,2, Daizo Hamada2, Takashi Fukui2, Makoto Hayashi1, Takeshi Honda3, Yoshikatsu Murooka1 and Itaru Yanagihara2 Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Japan Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Japan Keywords ANS binding; CD; FT-IR; partially unfolded; sedimentation equilibrium Correspondence D Hamada, Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo, Izumi, Osaka 594-1011, Japan Fax: +81 725 57 3021 Tel: +81 725 56 1220 E-mail: daizo@mch.pref.osaka.jp (Received 21 September 2004, revised March 2005, accepted April 2005) doi:10.1111/j.1742-4658.2005.04697.x pH-Dependent structural changes for Escherichia coli O157:H7 EspA were characterized by CD, 8-anilino-2-naphthyl sulfonic acid (ANS) fluorescence, and sedimentation equilibrium ultracentrifugation Far- and nearUV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures An increase in ANS fluorescence intensity (in the presence of EspA) was observed at acidic pH; whereas, no increased ANS fluorescence was observed at pH 7.0 These results suggest the presence of a partially unfolded state Interestingly, urea-induced unfolding transitions, monitored by far-UV CD spectroscopy, showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH Although increased ANS fluorescence was observed at pH 3.0, the urea-induced unfolding curve is similar to that found at pH 7.0 This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule The results of analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing a-helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular b-sheets at pH 2.0 Our experiments indicate that EspA has the potential to spontaneously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) cause outbreaks of serious diarrhoea These bacteria express type III secretion systems [1], which consist of various protein components encoded at the locus of enterocyte effacement, LEE [2–5] To date, type III secretion systems have been identified in more than 20 pathogenic bacterial species [6] The type III secretion system is a filamentous multiprotein complex that assembles across the bacterial and host cell surfaces For EHEC and EPEC, such complex structures, which include the proteins, EspA, EspB, EspD [7,8], probably permit direct delivery of effector proteins, such as, Tir [9–11], EspF [12,13], EspG [14] and Orf19 [15], into the host cell [16] EspA is a major component of this large, transiently expressed, filamentous surface organelle [17,18] EspA oligomerization may be mediated by interactions between coiled-coil regions of individual EspA molecules [19] in a manner similar to that of falgellin Abbreviations ANS, 8-anilinonaphthalene-1-sulfonic acid; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; FT-IR, Fourier transform infrared; LB, Luria–Bertani FEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS 2773 pH-Dependent EspA conformational change molecules, which assemble to form flagella filaments [20] The EspA-containing filamentous apparatus may form a conduit for translocation of bacterial proteins into host cells [21] Recently, a model of EspA filaments has been built based on negative-stain EspA electron micrographs [18] Interestingly, the model is a ˚ helical tube with a diameter of 120 A, enclosing a cen˚ diameter, and has an axial rise of tral channel of 25 A ˚ 4.6 A per subunit EspA filaments may attach to host cells via an EspB ⁄ D pore-forming complex [22] and the EspB ⁄ D complex may also specifically interact with the host-target protein, a-catenin [23] Such a superstructure, formed by EspA ⁄ B ⁄ D and a-catenin, facilitates the delivery of effector proteins into host cells [16] Although there is information available concerning the roles and structural properties of EspA filaments, to date, the conformation and the thermodynamic properties of EspA have not been characterized In this study, we characterized certain conformational and thermodynamic properties of EspA in solution using spectroscopic and physicochemical techniques Far-UV CD shows that the protein has a substantial amount of secondary structure throughout the pH range of 2.0–7.0 However, an analysis of 8-anilino2-naphthyl sulfonic acid (ANS) fluorescence (in the presence of EspA) suggests that a conformational transition occurs between pH 3.0 and 5.0, with exposure of hydrophobic protein surfaces Consistent with this observation, urea-induced EspA unfolding transitions, as followed by far-UV CD, indicate that the folded structure is less stable at pH 2.0 A sedimentation equilibrium study shows that EspA forms oligomers at pH 7.0, indicating an ability by EspA to form filamentous structures The data now reported suggest that EspA, at near physiological conditions, assumes short filamentous oligomers, but dissociates into partially unfolded species at acidic pH T Kato et al Fig Secondary structure prediction for EspA based on its amino acid sequence H and E refer to a-helical and b-strand propensities, respectively Recombinant EspA prepared from soluble fractions without unfolding the protein, at pH 7.0 and 20 °C, showed a CD spectrum typical of a protein with a significant amount of secondary structure (Fig 2A) Importantly, the spectrum for the sample prepared by urea-solubilized cells was almost completely superimposable on the above spectrum This observation Results CD The secondary structure prediction for EspA, based on its amino acid sequence, suggests that the protein is predominantly a-helical in conformation (Fig 1) The secondary structures of the recombinant EspA prepared here were analysed by far-UV CD spectra In this study, the recombinant EspA protein was prepared under either native or denaturing conditions To clarify whether both preparations yielded protein with similar propertiees, we first compared the CD spectra of EspA prepared under the different conditions 2774 Fig CD spectra of EspA at various pH values (A) Far- and (B) near-UV CD spectra at pH 2.0 (broken line), pH 3.0 (dotted line), and pH 7.0 (continuous line) Circles indicate the spectrum at pH 7.0 for recombinant EspA prepared from soluble fraction of cell lysate (C) Plot of ellipticity at 222 nm vs pH FEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS T Kato et al pH-Dependent EspA conformational change suggests that our preparation of recombinant EspA using urea, which included unfolding and refolding steps was successful, and that this protein reversibly unfolds and refolds, at least under the controlled conditions used here The ellipticity at 222 nm for EspA at pH 7.0 was )13 600 degỈcm2Ỉdmol)1 This ellipticity value yields an a-helical content of 37.2% when used in the equation: fH ẳ ẵh222 ỵ 2340ị=30300 where fH and [h]222 are the a-helical fraction and the ellipticity at 222 nm, respectively [24] This value, derived from the pH 7.0 CD spectrum, is smaller than that estimated from the secondary structure prediction using the amino acid sequence (62.0% and 8.3% for a-helices and b-sheets, respectively, Table 1) The secondary structure contents, estimated by the program CDPro, are 39.6% and 13.6% for a-helices and b-sheets, respectively This a-helical value is also smaller than that predicted using the amino acid sequence (62.0% and 8.3% for a-helices and b-sheets, respectively, Table 1) Although the intensity is significantly low, the nearUV CD spectrum of EspA at pH 7.0 and 20 °C, showed a minimum and a maximum around 280 and 290 nm As in the case of far-UV CD, the near-UV CD spectrum at pH 2.0 was similar to the spectrum at pH 7.0, although the intensity of each peak bacome slightly smaller It is in the near-UV region that aromatic residues display optical activity EspA contains five tyrosines at positions 22, 51, 53, 110 and 182, and no tryptophans Therefore, the shape of the near-UV CD spectra of EspA suggests the presence of some tertiary contacts around at least one of the tyrosines both at pH 7.0 and 2.0 (Fig 2B) To gain further insight into the conformational properties of EspA, we recorded far-UV CD spectra for solutions with the pH adjusted between 2.0 and 7.0 Interestingly, when the solution pH was between 3.0 and 7.0, the spectra are almost identical and the derived secondary structure estimates are similar to each other (Fig 2C and Table 1) The pH 2.0 spectrum also indicates a significant amount of secondary structure, although the spectral intensity is smaller than those obtained at higher pH This observation suggests that at pH 2.0 EspA is less ordered than at pH 3.0–7.0 ANS binding ANS binds to solvent-accessible hydrophobic surfaces and when bound its fluorescence intensity at % 500 nm increases This property of ANS is often used to detect partially unfolded protein intermediates [25], e.g molten globules, which are compact intermediates with significant amounts of native-like secondary structure, but with disordered tertiary contacts and solventexposed hydrophobic clusters [26–34] To determine if partially unfolded EspA species are present as a result of solution conditions, we recorded ANS fluorescence spectra, with EspA present at various pH conditions Between pH 6.0 and 9.0 the ANS fluorescence was insignificant (Fig 3), suggesting that negligible amounts of hydrophobic surfaces are solvent-accessible However, ANS fluorescence increased when the pH decreased from 6.0 to 2.0 (Fig 3) This observation suggests that hydrophobic surfaces become exposed upon decreasing the pH Since the protein maintains a significant amount of secondary structure (as estimated Table EspA Secondary structure composition at various pH values as estimated using the far-UV CD spectral data Values were calculated using CDPro [44,45] Conditions a-Helix (%) b-Sheet (%) Turn (%) Others (%) pH 2.0 pH 3.0 pH 5.0 pH 7.0 Predicted valuesa 32.6 ± 3.0 40.9 ± 6.9 39.8 ± 8.1 39.6 ± 7.1 62.0 29.9 ± 0.4 29.1 ± 2.0 28.5 ± 1.0 27.6 ± 0.4 16.1 ± 2.4 12.0 ± 4.1 13.0 ± 1.1 13.6 ± 5.2 8.3 a 21.9 ± 0.9 19.6 ± 2.1 19.0 ± 2.0 19.7 ± 1.7 29.7b Estimated from the secondary structure prediction (Fig 1) using the PHDSEC algorithm available at the PREDICTPROTEIN server [46–48] b The value is for non-a-helical and non-b-strand regions FEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS Fig ANS fluorescence at 460 nm as a function of pH Circles indicate the raw data The line is drawn only to assist the reader and has no theoretical relevance The approximated baselines for NII and NI (see text in detail) are shown by dotted and broken lines, respectively 2775 pH-Dependent EspA conformational change from far-UV CD spectra between pH 2.0 and 5.0, Fig 2), the results of the ANS study suggest formation of a partially unfolded state, possibly similar to the a-lactalbumin molten globule characterized at acidic pH [34] Sedimentation equilibrium Under physiological conditions, during EHEC or EPEC infection, EspA is associated with filamentous structures We therefore tested, using sedimentation equilibrium ultracentrifugation, whether recombinant EspA has the potential to form oligomers Figure shows the results for the EspA sedimentation equilibrium experiments at pH 7.0 and 20 °C If a protein solution contains only a single molecular weight species, then a plot of the natural logarithm of the protein absorption at 280 nm [ln(A280)] vs the square of the radial distance (r2) shows a linear T Kato et al correlation between ln(A280) and r2 However, the data (Fig 4A) indicate that ln(A280) exponentially increases with increased r2 This sedimentation equilibrium profile indicates either the presence of large protein oligomers or a contribution to the plot by nonideal solution behaviour Probably, the solution behaves as an ideal system under the experimental conditions, i.e., 10 mm sodium phosphate, pH 7.0, 100 mm NaCl Thus, it is unlikely that the curvature shown in Fig 4A is caused by nonideal behaviour Figure 4B shows that Mapp increases with an increase in the concentration of EspA The data of Fig 4B suggest that the size distribution of EspA ranges from that of the monomer to approximately that of a 30-mer when the protein concentration is mgỈmL)1, i.e 44 lm We also attempted to analyse the sedimentation profile of the partially unfolded state that exists at pH 2.0 However, it was extremely difficult to obtain the exact size of protein at pH 2.0 probably due to the formation of irreversible aggregates during the long period of centrifugation Although no visible precipitates were found at the beginning, protein absorption started to decrease after about 24 h, and become almost undetectable after 48 h This may be caused by the requirement of high protein concentration (> mgỈmL)1) due to the lack of tryptophan residues in EspA for reliable detection as well as the need for a long equilibration period (> 24 h) essential for the sedimentation equilibrium study The result is, however, consistent with the idea that the protein is partially unfolded at pH 2.0, because partially unfolded species are generally prone to form nonspecific aggregates (see below) Thus, compared with other simple spectroscopic measurements such as CD, ultracentrifugation may generally not be suitable for the analysis of partially folded proteins which are prone to aggregate FT-IR spectroscopy Fig Sedimentation equilibrium (A) Plot of the logarithm of the absorbance at 280 nm, A280, as a function of the square of the radial distance, r2 Data were collected at pH 7.0 with 1.0 mgỈmL)1 EspA (B) Plot of Mapp vs protein concentration 2776 The previous sedimentation equilibrium study indicated that after a long incubation at pH 2.0 the EspA solution contains aggregates, although these are invisible just after preparation of the sample FT-IR spectroscopy also confirmed the presence of molecular species containing intermolecular b-strands typical for nonspecific aggregates As indicated by CD, the soluble EspA at pH 7.0 shows an IR spectrum suggestive of the formation of a-helical structures with a peak around 1650 cm)1 (Fig 5) However, the spectrum taken for the solution at pH 2.0 has an additional maximum peak around 1620 cm)1 which is characteristic for the intermoleFEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS T Kato et al Fig FT-IR spectroscopy of EspA The spectra at pH 2.0 (broken line), aggregates formed at pH 7.0 (dotted line), and soluble fraction at pH 7.0 (continuous line) cular b-sheets usually formed in the nonspecific aggregates Importantly, precipitates are also formed at pH 7.0 in the presence of EspA > mgỈmL)1 The IR spectrum for these aggregates, however, is significantly similar to the spectrum taken for the soluble EspA (Fig 5) This observation suggests that EspA has an intrinsic potential to self-associate into oligomeric structures, which consist of a-helical secondary structures Urea-induced unfolding The stability of EspA at various pH values was analysed using far-UV CD spectroscopy By plotting the ellipticity at 222 nm as a function of urea concentration, cooperative unfolding transitions were obtained at all pH values (Fig 6) Between pH 3.0 and 7.0, the transitions occurred between 3.0 and 6.0 m urea However, the transition region shifted towards lower urea concentrations of about 0.0–3.0 m at pH 2.0 This observation is qualitatively consistent with the ANS binding results, which show that the protein, at pH 2.0, assumes a partially unfolded conformation with exposed hydrophobic surfaces Interestingly, the stability of the protein at pH 3.0 seems comparable to that at pH 7.0 This observation would seem to be inconsistent with the pH 3.0 ANS binding experiment as that experiment indicates a degree of unfolding resulting in hydrophobic surface solvent-exposure Therefore, between pH and 5, a partially unfolded state with highly ordered native-like tertiary contacts, but also with fluctuating regions, may exist This FEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS pH-Dependent EspA conformational change Fig Urea-induced EspA unfolding transitions at various pH values, 20 °C The transition curves are obtained from far-UV CD spectra at pH 2.0 (triangles), pH 3.0 (squares) and pH 7.0 (circles) The approximated baselines for folded (NI or NII) and unfolded states are drawn by dotted and broken lines, respectively, The ideal ellipticity for 50% of folded or unfolded species is shown by a thin line conformational state is clearly distinguishable from the typical molten globule formed by EspA at pH 2.0 Discussion pH-dependence of EspA conformations The present analysis suggests that the amount of EspA secondary structure, at various pH conditions, is highly conserved, even at pH 2.0 However, ANS binding experiments indicate that a conformational change occurred upon decreasing pH The characteristics of this conformational change are consistent with the formation of a partially unfolded species, probably similar to a molten globule Molten globules are compact denatured states with significant amounts of native-like secondary structure, but with disrupted tertiary interactions [26–34] Although peak intensity is slightly different, the near-UV CD spectrum at pH 7.0 is closely similar to that at pH 2.0 (Fig 2B) This is apparently inconsistent with the idea that the conformational species of EspA at pH 2.0 is in a typical molten globule state In this sense, the partially unfolded structure at pH 2.0, which exposes hydrophobic clusters to the solvent, may contain rather rigid tertiary conformation compared with the classical molten globule state It should be noted that the urea-induced denaturation data indicated a decreased stability and cooperativity against urea-induced unfolding for EspA at pH 2.0 compared with that at pH 7.0 This suggests that some conformational transitions may occur around 2777 pH-Dependent EspA conformational change pH 2.0–3.0 Importantly, at pH 2.0, the ellipticity at 222 nm decreased compared with the value at pH 3.0– 7.0 Thus, some of the a-helical structure formed at pH 3.0–7.0 may be disrupted at pH 2.0, whereas tertiary contacts, at least, around one of the tyrosine residues are conserved Recently, the three-dimensional structure of EspA complexed with its chaperone, CesA, has been solved by X-ray crystallography [35] In this model, only the N-terminal 29 and C-terminal 43 residues (amino acid positions at 31–59 and 148–190) of EspA corresponding to the binding interface of CesA could be clearly solved The other regions corresponding to the amino acid positions between 60 and 147 could not be solved, possibly due to the conformational disorder or multiple conformations If the unsolved regions in the EspA–CesA complex structure are disordered, the a-helical content of EspA should be 37.5% This value is highly consistent with the a-helical content estimated here from far-UV CD spectra of EspA at pH 7.0 (39.6%) It is generally considered that the native-like secondary structures are present in the partially folded state of a protein Thus, it would be natural to assume that the two a-helices of EspA shown in the EspA– CesA complex structures may be also formed in the partially folded state of EspA at pH 2.0 According to the EspA–CesA complex structure, only Y53 forms tertiary contacts with the C-terminal a-helix of EspA and CesA, and other tyrosines located in these a-helices are exposed to the solvent Therefore, the near-UV CD signals observed at pH 7.0 and 2.0 in Fig 2B might be responsible for the formation of tertiary contacts around Y53 The formation of nonspecific aggregates which occurred at pH 2.0 in the presence of high concentration of EspA indicate that the oligomeric EspA at pH 7.0 can tend to dissociate into monomers at pH 2.0 since the oligomerization into native structure should prevent the formation of nonspecific aggregates In this sense, the near-UV CD signal observed at pH 2.0 can be responsible for the intramolecular tertiary contacts around Y53, whereas the signal at pH 7.0 might reflect the intermolecular tertiary contacts However, the information on three-dimensional structure of EspA at different pH, particularly around the amino acids between 60 and 147, which could not be resolved by X-ray crystallography of EspA–CesA complex, is critical to evaluate such a possibility Importantly, the pH 3.0, urea-induced unfolding transition is almost superimposable onto the pH 7.0 transition curve This observation suggests that the protein, at pH 3.0, is as stable as that at pH 7.0 However, the ANS binding data indicate exposure of hydrophobic surfaces at pH 3.0, probably due to 2778 T Kato et al partial unfolding One possible explanation, reconciling this discrepancy, is that, unlike the traditional molten globule, EspA maintains a well-ordered native-like domain, but also has less structured regions with exposed hydrophobic patches at pH 3.0 We designate this conformational state, NII, the native structure at acidic pH, which has a distinctive character compared with the native conformation at neutral pH (NI) Thus, the conformational change of EspA, associated with changing pH, can be schematically represented as in Scheme 1: pH 2:0 IA … 3:0 NII … 5:0–7:0 NI The evidence suggests that the partially folded state at pH 2.0 may have native-like tertiary contacts but a lower a-helical structures content compared with NI and NII It is now designated as IA, i.e acid-induced intermediate structure In an attempt to understand how pH and urea concentration affect the conformations of EspA, we constructed an EspA pseudo phase diagram with urea concentration as a function of pH (Fig 7), according to Scheme For ANS binding (Fig 3), the ANS transition midpoint can be considered to be the apparent NI to NII transition midpoint, assuming that the maximum ANS intensity in Fig corresponds to the ANS fluorescence for NI The urea-induced unfolding transitions, between pH 3.0 and 5.0 (Fig 6), provide apparent midpoints for the transitions from either NII or NI to the unfolded state (U); whereas, the transition Fig Pseudo phase diagram for EspA: urea concentration vs pH at 20 °C The boundaries are defined by the ANS binding and the urea-induced unfolding curves shown in Figs and U, Unfolded state; IA, acid-induced intermediate state; NI, native state at neutral pH; NII, native state at acidic pH The transition midpoints for NI (or NII) to U (circles), IA to U (squares) and NI to NII (triangles) are shown by lines FEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS T Kato et al midpoint for IA to U is found using the pH 2.0 ureainduced unfolding data Importantly, since we have no clear information on the transition between NII and NI by the addition of urea due to the spectral similarity between these species, the boundary between NI and U shown around neutral pH may actually correspond to the boundary between NII and U Also, unfortunately, the experiments reported herein not provide the boundary between NII and IA Additional experimentation using, for example, NMR or calorimetry is needed to construct a more complete EspA phase diagram Although the phase diagram of Fig is incomplete, it contains sufficient information such that, for a given set of solution conditions, the existing conformational state(s) can probably be identified The C-terminal regions (Val138 to Gln181) of two EspA molecules may associate to form coiled-coil structures [19] These coiled-coils may then associate further, forming oligomers Based on our data, we propose that the oligomeric native state, found at neutral pH, dissociates at pH into a monomeric native-like state with an ordered N-terminal domain and less structured hydrophobic C-terminal tail The dissociation of oligomers into monomers upon decreasing pH was previously observed for Salmonella strain SJ25 flagellin [36] In that case, the protein, at acidic pH, assumes a conformation with an associated ellipticity at 222 nm of )3800 deg °CỈm-2Ỉdmol)1 Thus, some residual conformation may be present in monomeric flagellin at acidic pH It is possible that the structural properties of monomeric flagellin, at acidic pH, are similar to those of molten globules Oligomerization The EspA filamentous superstructure has been analysed by electron microscopy [17,18] It was suggested that other factors, such as molecular chaperones, are required to form an ordered EspA filamentous assembly [18] However, based on our sedimentation equilibrium data, we suggest that recombinant EspA spontaneously forms oligomers For flagellin, several additives, e.g salts or polyethylenglycoles, are required to induce formation of long, ordered filaments [37–41] Unfortunately, we were unable to produce long EspA filaments even when such additives were present (data not shown) The results of the sedimentation equilibrium experiment indicate that the largest oligomer formed by the recombinant protein is approximately a 30-mer According to the model derived from electron ˚ microscopy, an axial rise for one filament is 4.6 A per subunit [18] Thus, a 30-mer, formed by recombinant EspA, corresponds to a filament with a length of FEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS pH-Dependent EspA conformational change approximately 14 nm This is significantly shorter than the length of EspA filaments formed on EHEC and EPEC cell surfaces Therefore, the assistance of additional factors, such as molecular chaperones, may be needed to form longer EspA filaments, or the additional residues present at the N-terminal region of our recombinant protein can destabilize the filaments Alternatively, time scales longer than those used in our experiments may be necessary for the formation of sufficiently long filaments In summary, we provide, herein, the first study concerning the properties of the secondary structure of EspA EspA is shown to spontaneously associate into oligomeric structures at neutral pH However, two distinctive partially unfolded species occur at lower pH Based on these results, a phase diagram, illustrating potential EspA conformational transitions, was constructed Additional studies are necessary to characterize the EspA filamentous structure at the atomic level and to elucidate the thermodynamic requirements for filament formation Such information should clarify the role of EspA during host cell infection by EPEC and EHEC Experimental procedures Expression and purification of recombinant EspA The espA gene was amplified from an E coli O157:H7 cosmid library (RIMD 0509890, Sakai strain) [42,43] by PCR and PCR product was cloned into pT7 vector (Novagen, Madison, WI, USA) The650 bp NdeI–SacI fragment containing the espA gene was then inserted into the expression vector, pET28a (Novagen) The recombinant EspA has an additional 20 amino acids with the sequence MGSSHHHHHHSSGLVPRGSH on the N-terminal side of the native sequence The plasmid pET28a-EspA was transformed into E coli BL21 (DE3) Luria–Bertani (LB) broth, supplemented with 50 lgỈmL)1 kanamycin, was inoculated with E coli BL21 colonies and incubated overnight at 37 °C with shaking A portion of the overnight culture was diluted 100-fold into fresh LB medium and incubated at 37 °C with shaking Protein expression was induced by addition of IPTG (at concentrations up to mm) when the cultures reached an optical density of 0.5 at 600 nm After h of further shaking at 37 °C, the cells were harvested by centrifugation at 6000 g for 20 at °C and the pellet was placed on ice for 15 Most expressed EspA are located in insoluble fractions However, some EspA are also present in the soluble fraction Therefore, we prepared the recombinant EspA from total cell solubilized by urea or from only soluble fractions 2779 pH-Dependent EspA conformational change For preparation of EspA from urea-solubilized total cells, the cells were resuspended in 100 mm sodium phosphate, pH 8.0, 10 mm Tris ⁄ HCl, 8.0 m urea and lysed by sonication The solution was centrifuged at 10 000 g for 30 at °C to separate the soluble and pellet fractions The soluble fraction was diluted drop-wise 100-fold into 50 mm sodium phosphate, pH 8.0, 300 mm NaCl, 10 mm imidazole at °C The solution was loaded onto Ni–NTA agarose (Qiagen, Valencia, CA, USA) and eluted using a 0–0.5-m imidazole gradient The eluted EspA was dialysed against 50 mm sodium phosphate pH 8.0, 300 mm NaCl, 10 mm imidazole and rechromatographed over Ni–NTA agarose Eluted EspA was concentrated by ultrafiltration using a YM10 filter (Millipore, Billerica, MA, USA) and then dialysed against 10 mm sodium phosphate pH 7.0 Protein solutions were stored at )20 °C For purification from the soluble fraction, cells collected by centrifugation were resuspended in 50 mm sodium phosphate pH 8.0, 300 mm NaCl, 10 mm imidazole Lysozyme (1 mgỈmL)1 final concentration) was added, and the solution was incubated at °C for 30 RibonucleaseA and dideoxynuclease I (10 and lgỈmL)1 final concentrations, respectively) were then added Incubation was continued at °C for a further 15 The solution was cleared by centrifugation at 10 000 g at °C for 30 The supernatant was applied to Ni–NTA agarose equilibrated with 50 mm sodium phosphate pH 8.0, 300 mm NaCl, 20 mm imidazole, and washed with the same buffer The recombinant EspA was eluted with 50 mm sodium phosphate pH 8.0, 300 mm NaCl, 250 mm imidazole The eluted protein was dialysed against, 50 mm sodium phosphate pH 8.0, 300 mm NaCl, 10 mm imidazole, and purified again by Ni–NTA agarose The purity of the recombinant protein was checked by SDS ⁄ PAGE, which provided a single band around molecular weight of 20 kDa, a value consistent with calculated molecular weight of recombinant EspA About mg of EspA were purified from L culture by urea-solubilization procedure, whereas only 0.1 mg of protein could purified without solubilization by urea CD spectroscopy CD spectra were recorded using a J-600 spectropolarimeter (Jasco, Tokyo, Japan) The temperature was adjusted to 20 °C using a thermostatically controlled cell holder connected to a circulating water bath For far- and nearUV CD spectra, cells of mm and cm path length were used, respectively Protein concentrations were 0.1 and mgỈmL)1 for far- and near-UV CD measurements, respectively The samples were prepared about 12 h before the measurements and the measurements were completed within 24 h after preparation of samples The sample pH was checked by pH electrode, Horiba compact pH meter, B-212 (Horiba, Kyoto, Japan) after each measurement The 2780 T Kato et al data were expressed as mean residue ellipticity, [h], where [h] is defined as [h] ¼ 100 hobs (c · l))1, hobs is the observed intensity, c is the concentration in residue moles per litre, and l is the path length in cm The secondary structure composition of EspA was estimated using the program package CDPro [44,45] Reported values are the average of the results obtained from three independent programs: continll, selcon3 and cdsstr, according to the instruction of cdpro program package The [h] values between 200 and 250 nm with an interval of 0.2 nm taken at different pH were directly used for input data The urea-induced unfolding curves were obtained by plotting the ellipticity at 222 nm against urea concentration To estimate the urea concentration of midpoint of the unfolding reaction (Cm), the baseline for folded and unfolded species are approximated from the plateau regions of pre- and post-transition, respectively The data were analysed according to the assumption of two-state transition between a native and an unfolded state However, we should stress here that this analysis should be incorrect because various oligomeric forms are present among native conformers However, without any data about the proportion of each native oligmer, this treatment is the only the probable and most conventional method to estimate Cm values without any bias The details in the analysis and the parameters for unfolding are available as supplementary material in Table S1 Fluorescence spectrum ANS fluorescence spectra were recorded using a FP-777 fluorimeter (Jasco) The excitation wavelength was 350 nm and fluorescence emission spectra were recorded between 400 and 650 nm The protein concentration was 0.1 mgỈmL)1 (4.4 lm) and the ANS concentration was lm The temperature was kept at 20 °C using a Peltier-type thermostatically controlled cell holder Sedimentation equilibrium Sedimentation equilibrium experiments were performed using a Beckman Optima XL-I analytical ultracentrifuge (Fullerton, CA, USA) operated at 15 000 r.p.m., 20 °C Various amounts of protein were dissolved in 20 mm sodium phosphate pH 7.0, 100 mm NaCl Using the program, AA comp (RASMB web site: http://www.rasmb bbri.org/rasmb/mac/aa_comp-stafford), in conjunction with the EspA amino acid composition, the partial specific volume of EspA was calculated as 0.731 The apparent molecular weight (Mapp) was estimated according to the following equation: Mapp ẳ 2RT d lnCị ð1 À tqÞx2 dðr2 Þ ð1Þ FEBS Journal 272 (2005) 2773–2783 ª 2005 FEBS T Kato et al where R is the gas constant, T is the absolute temperature, x is the angular velocity, q is the solvent density and c is the protein concentration at the radial distance r pH-Dependent EspA conformational change FT-IR Infrared spectra were recorded using Avatar 370 (Thermo Nicolet Co., Madison, WI, USA) under continuous purge with dry nitrogen gas Normal spectral resolution used was cm)1 The spectra of 128 scans were averaged A Happ–Genzel apodization function was applied before Fourier transformation The samples were transferred to an IR sample cell consisting of a pair of CaF2 windows separated by a 15-lm spacer FT-IR measurements were carried out at room temperature Recombinant protein (5 mg) dissolved in mL 10 mm sodium phosphate was lyophilized and resuspended in 200 lL 10 mm sodium phosphate ⁄ 2H2O at pH 7.0 or 2H2O at pH 2.0 At pH 7.0, visible precipitates were found in the solution The spectra of soluble and insoluble fractions were individually taken after separating each fraction by centrifugation at 13 000 g at °C for 20 The concentration of soluble EspA at pH 7.0 was mgỈmL)1 estimated by UV absorption spectrum At pH 2.0, no visible precipitates were found Thus, the concentration of EspA is considered to be 10 mgỈmL)1 However, the presence of some aggregated species was obvious from the FT-IR spectrum as discussed in the text Acknowledgements We thank Prof Yuji Goto for the use of the CD spectrometer and Miyo Sakai for performing the ultracentrifugation experiments This work was supported in part by grants-in-aid for scientific research from the Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT) References Galan JE & Collmer A (1999) Type III secretion machines: bacterial devices for protein delivery into host cells Science 284, 1322–1328 McDaniel TK, Jarvis KG, Donnenberg MS & Kaper JB (1995) A genetic locus of enterocyte effacement conserved among diverse enterobactrial pathogens Proc Natl Acad Sci USA 92, 1664–1668 McDaniel TK & Kaper JB (1997) A cloned pathogenicity island from enteropathogenic Escherichia coli confers the 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Kurokawa K, Ishii K, Yokoyama K, Han CG, Ohtsubo E, Nakayama K, Murata T, Tanaka M, Tobe T, Iida T, Takami H, Honda T, Sasakawa C, Ogasawara N, Yasunaga T, Kuhara S, Shiba T, Hattori M & Shinagawa... species, then a plot of the natural logarithm of the protein absorption at 280 nm [ln (A2 80)] vs the square of the radial distance (r2) shows a linear T Kato et al correlation between ln (A2 80) and... even at pH 2.0 However, ANS binding experiments indicate that a conformational change occurred upon decreasing pH The characteristics of this conformational change are consistent with the formation