Báo cáo khoa học: The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pH ppt

The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pH Gregorio Ferna ´ ndez-Ballester 1 , Javier Maya 1 , Alejandro Martı ´ n 1 , Stephan Parche 2, *, Javier Go ´ mez 1 , Fritz Titgemeyer 2 and Jose ´ L. Neira 1,3 1 Instituto de Biologı ´ a Molecular y Celular, Universidad Miguel Herna ´ ndez, Elche (Alicante), Spain; 2 Lehrstuhl fu ¨ r Mikrobiologie, Friedrich-Alexander-Universita ¨ t Erlangen-Nu ¨ rnberg, Germany; 3 Instituto de Biocomputacion y Fisica de los sistemas complejos, Zaragoza, Spain The folding of a 93-residue protein, the histidine-phospho- carrier protein of Streptomyces coelicolor,HPr,hasbeen studied using several biophysical techniques, namely fluo- rescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calori- metry. The chemical-denaturation behaviour of HPr, fol- lowed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those con- ditions is DG ¼ 4.0 ± 0.2 kcalÆmol )1 (1 kcal ¼ 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7–9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5–4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumu- lates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pK a ¼ 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluores- cence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilino- naphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-fil- tration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family. Keywords: folding; molten-globule; protein stability; PTS; structure. The phosphoenolpyruvate phosphotransferase system (PTS) catalyzes the uptake and phosphorylation of carbo- hydrates in most bacterial species, via a cascade of several proteins [1]. Enzyme I (EI), the first protein in the cascade, is autophosphorylated by phosphoenolpyruvate, yielding phosphorylated EI (P-EI). P-EI acts as a phosphoryl donor to the histidine-phosphocarrier protein (HPr). Phosphoryl- ated HPr, in turn, donates the phosphoryl moiety to a group of specific sugar-transporter proteins, known as enzymes II (EII). The transfer of the phosphoryl moiety from EII to the sugar occurs concomitantly to its transport into the cell. HPr, the smallest protein in the cascade, is thought to be the key component in that cascade, because it phosphorylates all sugar-specific EII proteins. EI phosphorylates HPr at the imidazole ring of the highly conserved histidine [1]. This phosphorylation is common to both Gram-positive and Gram-negative bacteria. However, in Gram-positive bac- teria there is also an additional regulatory phosphorylation site in HPr at the conserved Ser46. This regulatory site is thought to be involved in carbon catabolite repression of several genes, and as a transcription regulator of several operons in Gram-positive bacteria [2]. Streptomyces are soil-dwelling, high GC Gram-positive actinomycetes which grow on a variety of carbon sources, such as cellulose and many monosaccharides and disaccha- rides. They are the source of approximately two-thirds of all natural antibiotics currently produced by the pharmaceu- tical industry. Despite their importance, our knowledge on nutrient sensing, carbohydrate transport and regulation is very poor. The complete sequence of Streptomyces coeli- color has been sequenced, showing the largest number of genes found in any bacteria [3]. The presence of the different components of the PTS in S. coelicolor has been reported, and the corresponding proteins cloned and expressed [4–9]. EI and HPr proteins from S. coelicolor are similar, in Correspondence to J. L. Neira, Instituto de Biologı ´ a Molecular y Celular, Edificio Torregaita ´ n, Universidad Miguel Herna ´ ndez, Avda del Ferrocarril s/n, 03202, Elche (Alicante), Spain. Fax: +34 966658758, Tel.: +34 966658459, E-mail: jlneira@umh.es Abbreviations: ANS, 8-anilinonaphthalene-1-sulfonate ammonium salt; DSC, differential scanning calorimetry; DC p , heat capacity change; DH cal , the calorimetric enthalpy change; DH VH , the van’t Hoff enthalpy change; EI, enzyme I; EII, enzyme II; FTIR, Fourier trans- form infrared spectroscopy; GdmCl, guanidine hydrochloride; HPr, histidine-phosphocarrier protein; PTS, the phosphoenolpyruvate- dependent sugar phosphotransferase system; T m ,thermal denaturation midpoint. *Present address:Nestle ´ Research Center, Vers-chez-les-Blanc, CH-100, Lausanne 26, Switzerland. (Received 7 January 2003, revised 5 March 2003, accepted 26 March 2003) Eur. J. Biochem. 270, 2254–2267 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03594.x molecular size and activity, to those corresponding proteins of other microorganisms previously characterized [4]. Because of its key role in the PTS and its small size, we are studying HPr from S. coelicolor as a model for: (a) advancing our knowledge of the PTS enzymatic activity, and then, its involvement in antibiotic production; and (b) research in protein folding. The structures of HPrs from several species have been studied by NMR spectroscopy [10–12 and references therein] and X-ray diffraction [13,14,12 and references therein]. HPrs from those species show a classical open-face b-sandwich fold consisting of three a helices packed against a four- stranded antiparallel b sheet; this fold has been related to other proteins with no apparent relationship in function, such as ferredoxin and diphosphate kinase [15,16]. HPr from S. coelicolor contains 93 amino acid residues; it lacks cysteine and tyrosine, but it has a large number of alanine, valine and leucine residues. Furthermore, the protein only contains one tryptophan residue and one phenylalanine residue, which makes it a good model to follow its folding mechanism and other biochemical features by using fluor- escence spectroscopy. Assignment and preliminary NMR studies of the HPr from S. coelicolor indicate that its structure is similar to that observed in other members of the HPr family (J. L. Neira, unpublished results). As the HPr from S. coelicolor has a similar structure, but a completely different amino acid sequence to those HPrs from Escheri- chia coli or Bacillus subtilis, whose structure and folding properties have been described, it is important to understand if the structure, the sequence or both determine the conformational stability and biochemical properties in the HPr family. There is much current interest in determining the extent to which related proteins share stability and folding features [17]. The exploration of the folding and stability among the different HPr members will allow us to decide whether there is a common thermodynamic equili- brium behaviour in this important family. In this study, we use several biophysical techniques (CD, fluorescence, 8-anilinonaphthalene-1-sulfate (ANS) binding, differential scanning calorimetry (DSC), thermal CD, FTIR and gel filtration chromatography) to follow the folding of HPr from S. coelicolor. Our findings indicate that the folding of HPr can be adequately described as a two-state process without the accumulation of intermediates at neutral and moderately basic conditions (pH 7–9) at 25 °C. The stability of the protein, at pH 7.5 and 25 °C, as obtained by chemical and thermal denaturation experiments, is low: DG ¼ 4.0±0.3kcalÆmol )1 . At moderately acid pH values (pH 2.5–4), in the absence of cosmotropic agents, the protein undergoes noncooperative thermal denaturations and it accumulates partially folded species. Although extensive folding studies have been carried out with the E. coli HPr [16], no such species have been previously observed nor characterized. Thus, this is the first member of the HPr where partially folded species have been found. Experimental procedures Materials Urea and guanidine hydrochloride (GdmCl) ultra-pure were from ICN Biochemicals. Urea and GdmCl molecular biology grade, imidazole, trizma base, NaCl and ANS were from Sigma. 2-Mercaptoethanol was from Bio-Rad, and the Ni 2+ -resin was from Invitrogen. Glutaraldehyde was from Fluka. Dialysis tubing was from Spectrapore with a molecular mass cut-off of 3500 Da. Standard suppliers were used for all other chemicals. Water was deionized and purified on a Millipore system. Urea and GdmCl stock solutions were prepared gravimetrically and filtered using 0.22-lm syringe driven filters from Millipore. Exact con- centrations of urea stock solutions were calculated from the refractive index of the solution using an Abbe 325 refractometer [18]. Protein expression and purification The HPr clone comprises residues 1–93 (with the extra methionine at the N terminus) and the His 6 -tag at the N terminus. We have carried out all the studies with this construction as its structure, as observed by NMR (unpub- lished results), is similar to that found in other members of the HPr family and the His 6 -tag is disordered in solution, making no contacts with the rest of the protein. Recom- binant protein was expressed in E. coli C43 strain [19], and purified using Ni 2+ -chromatography. To eliminate any protein or DNA bound to the resin, coeluting with the protein, an additional gel filtration chromatography step was carried out by using a Superdex 75 16/60 gel filtration column, running on an AKTA-FPLC (Amersham Bio- sciences) system. The yield was 25–30 mg of protein per litre of culture. Protein was more than 99% pure as judged by SDS protein-denaturing gels. Also, mass spectrometry analysis was carried out in a MALDI-TOF instrument; only one peak was observed. The samples were dialyzed exten- sively against water and stored at )80 °C. Samples were prepared by dissolving the lyophilized protein in deionized water (unfolding) or in 8 M urea (in the refolding experi- ments) and adding the proper buffer solution. Protein concentration was calculated from the absorbance of stock solutions measured at 280 nm, using the extinction coeffi- cients of model compounds [20]. Protein without the His 6 -tag was obtained using the thrombin-cleavage capture kit from Novagen (Germany). This protein was used as a control experiment to address the importance of the His 6 -tag in the biophysical properties of the protein under different pH conditions. Cross-linking experiments Cross-linking reactions were performed at 25 °Cinthe corresponding buffers at different protein concentrations by addition of glutaraldehyde to a final concentration of 4 m M . Reactions were stopped after 15 min by addition of SDS- buffer. Fluorescence measurements All fluorescence spectra were collected on a SLM 8000 spectrofluorometer (Spectronics Instruments, Urbana, IL), interfaced with a Haake water bath, at 25 °C. All measure- ments were corrected for wavelength dependence on the exciting-light intensity through the use of the quantum counter rhodamine B in the reference channel [21]. Sample Ó FEBS 2003 Stability of Streptomyces coelicolor HPr (Eur. J. Biochem. 270) 2255 concentration was in the range 6–20 l M , and the final concentration of the buffer was, in all cases, 10 m M .A 0.5-cm path-length quartz cell (Hellma) was used. Steady state fluorescence measurements. All protein sam- ples were excited at 280 nm, as excitation at 295 nm yielded the same spectrum with smaller intensity (data not shown). The slit width was typically equal to 4 nm for the excitation light, and 8 nm for the emission light. The fluorescence experiments were recorded between 300 and 400 nm. The signal was acquired for 1 s and the increment of wavelength was set to 1 nm. Blank corrections were made in all spectra. The urea titrations, followed either by fluorescence or CD, were carried out by two different procedures: (a) dilution of the proper amount of the 8 M denaturant stock solution and leaving the samples at 25 °C, for at least 8 h prior to performing the experiments; or (b) by directly titrating the protein with urea. No differences were observed between the procedures. As the concentration of urea was increased, the fluorescence spectra were red-shifted and their intensities decreased (data not shown). Experiments carried out at different protein concentrations (6–20 l M ) did not show any difference in the thermodynamic parameters obtained (data not shown). In the pH-induced unfolding experiments, followed either by fluorescence or CD, the pH was measured after comple- tion of the experiments with an ultra-thin Aldrich electrode in a Radiometer (Copenhagen) pH meter. The pH range explored using both techniques was 1.5–12. The buffers were: pH 1.5–3.0, phosphoric acid; pH 3.0–4.0, formic acid; pH 4.0–5.5, acetic acid; pH 6.0–7.0, NaH 2 PO 4 ;pH7.5– 9.0, Tris acid; pH 9.5–11.0, Na 2 CO 3 ; pH 11.5–12, Na 3 PO 4 . Fluorescence quenching experiments. Quenching of intrin- sic tryptophan fluorescence by iodide or acrylamide [21] was examined at different pH values. Excitation was at 280 nm, and emission was measured from 300 to 400 nm. In experiments employing KI as a quencher, ionic strength was kept constant by addition of KCl; also, Na 2 S 2 O 3 was added to a final concentration of 0.1 M to avoid formation of I 3 – . The slit width was set at 8 nm for both excitation and emission. The dynamic and static quenching constants for acrylamide were obtained by fitting the data from different wavelengths (in the range 330–340 nm) to the Stern–Volmer equation, which includes an exponential term to account for static quenching [21]: F 0 F ¼ 1 þ K sv ½Xe ðv½XÞ ð1Þ where K sv is the Stern–Volmer constant for collisional quenching and v is the static quenching constant. Iodide quenching did not show a significant static component, and then the exponential term was not included in the fitting of Eqn (1). The range of concentrations used in both quenc- hers was 0–0.7 M . Experiments carried out at different protein concentrations did not show any difference in the parameters obtained (data not shown). ANS binding. ANS binding was measured by collecting fluorescence spectra at different pH values in the presence of 50 l M dye. Excitation wavelength was 380 nm, and emis- sion was measured from 400 to 600 nm. Slit widths were 4 nm for excitation and 8 nm for emission. Stock solutions ofANSwerepreparedinwateranddilutedintothesamples to the above final concentration. In all cases, the blank solutions were subtracted from the corresponding spectra. Experiments carried out a different protein concentrations did not show any difference (data not shown). Circular dichroism Circular dichroism spectra were collected on a Jasco J810 spectropolarimeter fitted with a thermostated cell holder and interfaced with a Neslab RTE-111 water bath. The instrument was periodically calibrated with (+)10-cam- phorsulfonic acid. Isothermal wavelength spectra at differ- ent pH values were acquired with a scan speed of 50 nmÆmin )1 , and a response time of 2 s and averaged over four scans at 25 °C. Far-UV measurements were performed using 14–295 l M of protein in 10 m M buffer, using 0.1- or 0.2-cm pathlength cells (Hellma). During the pH titration experiments no significant changes either in the shape or in the molar ellipticity were observed as the concentration of protein was increased; thus, we can rule out the presence of concentration-dependence at those pH values. Near-UV spectra were acquired using 30–40 l M of protein in a 0.5-cm pathlength cell (Hellma). All spectra were corrected by subtracting the proper baseline. To allow for comparison at different pH values and different urea concentrations, raw ellipticity was converted to molar ellipticity [22]. Thermal-denaturation experiments were performed at constant heating rates of 60 °CÆh )1 and 30 °CÆh )1 ;the response time was 8 s. Thermal scans were collected in the far-UV region at 222 nm from 25 °C(or5°C) to 90 °C(or 95 °C) in 0.1-cm pathlength cells (Hellma) with a total protein concentration of 40–100 l M . Conditions were the same as those reported in the steady-state far-UV experi- ments. The reversibility of thermal transitions was tested by recording a new scan after cooling down to 5 °Cthe thermally denatured samples. To check also for reversibility, we carried out the reheating experiments at different speeds to the heating measurements; no differences among the scans acquired at different speeds were observed at those pH values where HPr unfolds reversibly. Every thermal dena- turation experiment was repeated at least three times with fresh new samples at different concentrations. The measured thermodynamic parameters did not change when experi- ments were acquired at different protein concentrations. In all cases, after the reheating experiment, the samples were transparent and no precipitation was observed. The possi- bility of drifting of the CD spectropolarimeter was tested by running two samples containing buffer, before and after the thermal experiments. No difference was observed between the scans. In the urea-denaturation experiments, far-UV CD spec- tra were acquired at a scan speed of 50 nmÆmin )1 and four scans were recorded and averaged at 25 °C. The response time was 2 s. The pathlength cell was 0.1 cm, with protein ranging in 10–30 l M . Spectra were corrected by subtracting the proper baseline in all cases. The chemical denaturation reaction was fully reversible, as demonstrated by the agreement between the folding and unfolding curves (data 2256 G. Ferna ´ ndez-Ballester et al.(Eur. J. Biochem. 270) Ó FEBS 2003 not shown). Each chemical denaturation experiment was repeated at least three times with new samples. Experiments carried out at different protein concentrations did not show any difference. Analysis of the pH- and chemical-denaturation curves, and free energy determination The average energy of emission (or the intensity weighted average of the inverse wavelengths) in the fluorescence spectra, <k>,wascalculatedasdefinedin[23]. The pH-denaturation experiments were analyzed assu- ming that both species, protonated and deprotonated, contributed to the fluorescence (or CD) spectrum: X ¼ ðX a þ X b 10 ðpHÀpK a Þ Þ ð1 þ 10 ðpHÀpK a Þ Þ ð2Þ where X is the physical property being observed (ellipticity or fluorescence), X a is the physical property being observed at low pH values (that is, the fluorescence or ellipticity of the acid form), X b is the physical property observed at high pH values, and pK a is the apparent pK of the titrating group. The apparent pK a reported was obtained from three different measurements, prepared with new samples. In the fluorescence experiments, the determinations were carried out using either the <k> or the maximum wavelength in fitting the Eqn (2). In the CD experiments, the ellipticity at 222 nm was the chosen parameter, either in the pH-denaturation or chemical-denaturation experiments. To facilitate comparison among the different biophysical techniques, data were converted to the fraction of folded and unfolded molecules [24]. The denaturation data obtained by fluorescence or CD were fit to the two-state equation: X ¼ ðX N þ X D e ðÀDG=RTÞ Þ ð1 þ e ðÀDG=RTÞ Þ ð3Þ where X N and X D are the corresponding fractions of the folded (N) and unfolded states (U), respectively, which were allowed to change linearly with either the denaturant (X N ¼ a N + b N [D], and X D ¼ a D + b D [D]), or the tem- perature (that is, X N ¼ a N + b N T and X D ¼ a D + b D T ), R is the gas constant and T is the temperature in K. Chemical-denaturation curves were analyzed using a two-state unfolding mechanism, according to the linear extrapolation model: DG ¼ m([D] 50% ) [D]) [20], where DG is the free energy of denaturation, and [D] 50% is the denat- urant concentration at the midpoint of the transition. The chemical-denaturation-binding model [25,26] was also used for the fitting of the chemical denaturation data, but no reliable parameters were obtained either for DG or m (data not shown). Thus, the linear extrapolation method was used in all the conformational stability calculations. The change in free energy upon temperature in Eqn (3) is given by the Gibbs–Helmholtz equation: DGðT Þ¼DH VH 1 À T T m  À DC p ðT m À TÞþT ln T T m  ð4Þ where DH VH is the van’t Hoff enthalpy change. By substitution of this expression in Eqn (3), we obtain DH VH , T m and DC p of the thermal experiments. Fittings by nonlinear least-squares analysis to Eqns (1, 2 and 3) were carried out by using the general curve fit option of KALEIDAGRAPH (Abelbeck software). Gel filtration chromatography Analytical gel filtration experiments were carried out by using an analytical gel filtration Superdex 75 HR 16/60 (Amersham Biosciences) running on an AKTA FPLC system at 25 °C. Flow rates of 0.8 mLÆmin )1 (at high urea concentrations) or 1 mLÆmin )1 were used. The elution buffers for the pH experiments were those described above with 150 m M NaCl added to avoid non-specific inter- actions with the column. To check for the presence of aggregated species at low pH values, protein concentra- tions ranged from 20 to 60 l M . No differences in the elution volumes were observed among the different concentrations used. The chemical denaturation experiments were acquired at pH 7.5, 10 m M phosphate buffer and 150 m M NaCl. Protein concentration was 20–60 l M and absorbance was monitored at 280 nm. No differences in the elution volumes were observed when the protein concentration was increased. The column was calibrated using the gel filtration low relative molecular mass calibration kit (Amersham Biosciences). The standards used and their corresponding Stokes radii were: ribonuclease A (16.4 A ˚ ); chymotrypsi- nogen (20.9 A ˚ ); ovoalbumin (30.5 A ˚ ), and bovine serum albumin (35.5 A ˚ )[27]. The elution of a macromolecule in gel filtration experi- ments is usually given by the partition coefficient, r,which is defined as the fraction of solvent volume within the gel matrix accessible to the macromolecule [28]. The r of protein standards and HPr were calculated by: r ¼ ðV e À V o Þ V i ð5Þ where V e is the elution volume of the protein, and, V o and V i are the void and internal volumes of the column, respect- ively. The values of those volumes are, respectively, 8.13 ± 0.06 mL and 28.43 ± 0.03 mL. The V o and V i volumes were, respectively, determined using Blue dextran (5 mgÆmL )1 ,in10m M phosphate buffer plus 150 m M NaCl) and L -tryptophan (0.5 mgÆmL )1 , in the above buffer) by averaging four measurements for each agent. There is a linear relationship between the molecular Stokes radius, R s , and the inverse error function comple- ment of r (erfc )1 (r)), given by [28,29]: R s ¼ a þ bðerfc À1 ðrÞÞ ð6Þ where a and b are the calibration constants for the column. Fitting of the calculated erfc )1 (r) to the above equation by linear least-squares analysis was carried out with KALEIDA- GRAPH (Abelbeck software) working on a PC computer. Once the calibration parameters are obtained, the Stokes radius of any macromolecule can be obtained by using Eqn (6). Ó FEBS 2003 Stability of Streptomyces coelicolor HPr (Eur. J. Biochem. 270) 2257 Fourier transform infrared spectroscopy The protein was lyophilized and dissolved in deuterated buffer at different pH values. The buffer was composed of 0.1 M NaCl, 0.1 m M ethylenediaminetetracetate, 0.02% NaN 3 ,10m M sodium acetate, 10 m M N-(1-morpholino)- propane-sulfonic acid, and 10 m M 3-(cyclohexylamino)- 1-propane-sulfonic acid. No corrections were done for the isotope effects in the measured pH. Samples of HPr at a final concentration of 5–6 mgÆmL )1 were placed between a pair of CaF 2 windows separated by a 50-lm thick spacer in a Harrick Ossining demountable cell. Spectra were acquired on a Nicolet 520 instrument equipped with a deuterated triglycine sulfate detector and thermostated with a Braun water bath at 25 °C. The cell container was continuously filled with dry air. Usually 600 scans per sample were taken, averaged, apodized with a Happ– Genzel function, and Fourier transformed to give a final resolution of 2 cm )1 . The contributions of buffer spectra were subtracted, and the resulting spectra used for analysis after smoothing. The spectra smoothing was carried out by using the maximum entropy method [30]. Derivation of FTIR spectra was performed using a power of 3 and a breakpoint of 0.3. Fourier self-deconvolution was per- formed using a Lorentzian bandwidth of 18 cm )1 and a resolution enhancement factor of 2 [30]. The prediction of protein secondary structure was quantified by deconvolu- tion of the amide I band, as described elsewhere [31], yielded essentially the same percentages of a helix, bturns and b sheet, which have been observed in the NMR structure (unpublished results). Thermal denaturation experiments followed by FTIR were performed at a protein concentration of 6 mgÆmL )1 , with a scanning rate of 50 °CÆh )1 , and acquired every 5 °C. Differential scanning calorimetry DSC experiments [32] were performed with a MicroCal MC-2 differential scanning calorimeter interfaced to a computer equipped with a Data Translation DT- 2801 A/D converter board for instrument control and automatic data collection. Lyophilized protein was dis- solved in 10 m M phosphate buffer, pH 7.5 and dialyzed extensively against 2 L of the same buffer (twice) at 4 °C. Protein concentration was calculated from the absorbance of the solution at 280 nm [20]. Samples were degassed under vacuum for 10–15 min with gentle stirring prior to being loaded into the calorimetric cell. DSC experiments were performed under a constant external pressure of 1 bar in order to avoid bubble formation, and samples were heated at a constant scan rate of 60 °CÆh )1 . Once the first scan was completed, the samples were cooled in situ downto10°C for 40 min and rescanned under the same experimental conditions in order to check the reversibility of the heat-induced denaturation reaction. Experimental data were corrected from small mismatches between the two cells by subtracting a buffer vs. buffer baseline prior to data analysis. After normalizing to concentration, a chemical baseline calculated from the progress of the unfolding transition was subtracted. The excess heat capacity functions were then analyzed using the software package ORIGIN (Microcal Software, Inc.). Results pH-induced unfolding of HPr To examine how the secondary and tertiary structure of HPr changes with the pH, we used multiple spectroscopic techniques, which give complementary information about the melting of the secondary and/or tertiary structure of the protein. Fluorescence experiments. We used fluorescence to map any change in the tertiary structure of the protein upon pH changes [33]. HPr has one tryptophan residue, which is at the C-terminal region of the first b strand. The emission fluorescence spectrum of native HPr showed a maximum at 337 nm at neutral pH (Fig. 1A), indicating Trp burial. As the pH decreased, the maxima wavelength were red-shifted towards 350 nm, and the fluorescence intensities increased. Fig. 1. pH-induced unfolding of HPr followed by fluorescence and ANS binding. (A) Steady-state fluorescence: the average energy of emission (filled circles) and the maxima wavelength (open circles) are repre- sented vs. the pH. (B) ANS-binding experiments: the average energy (filled circles) and the maxima wavelength (open circles) are repre- sented vs. the pH. (C) The low-pH region of the steady-state fluores- cence experiments (the symbols are the same as in A). The conditions were: 6 l M of protein, and 40 l M ANS, when required, at 25 (± 0.1) °C; buffer concentration was 10 m M in all cases; spectra were acquired in 0.5-cm pathlength cells. The lines are fittings to Eqn (2). 2258 G. Ferna ´ ndez-Ballester et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Conversely, as the pH increased, the maxima wavelength and the spectral intensities remained constant up to high pH (Fig. 1A). Theprofileof<k> vs. pH (Fig. 1A) showed a sigmoidal behaviour at low pH, and a plateau region above pH 4. The maxima wavelength followed the same pattern than the <k>. The apparent pK a was 3.2 ± 0.3 (Fig. 1A). The protein without the His 6 -tag showed the same behaviour at the different pH values than that observed for the His 6 -tagged protein (data not shown). Examination of tryptophan exposure by fluorescence quenching. To further check whether the tertiary structure around the sole Trp residue changes upon pH, we examined iodide and acrylamide quenching by excitation at 280 nm, which provided us with information about burial of the indole moiety. Acrylamide-quenching experiments, carried out at pH 3.3 and 7.5, yielded exponential Stern–Volmer plots. At pH 7.5, where as judged by fluorescence, CD and FTIR measurements the protein was folded, the K sv and v were small, indicating burial of the tryptophan moiety (Table 1). These results are in agreement with the observa- tion that the maxima wavelength of spectra appeared at 337 nm at that pH. Conversely, at low pH, both quenching parameters were larger, indicating solvent-exposure of the aromatic ring. This is also in agreement with previous observations on the shift of the maxima wavelength at low pH values. Larger values of the quenching parameters were also observed at pH 7.5 in the presence of 6 M urea, where the protein was completely unfolded. The fact that the values of K sv in the presence and in the absence of urea, as measured by acrylamide, are very similar within the error (Table 1) is not fully understood, but it could be due to the larger size of acrylamide when compared to that of KI. Therefore, we can conclude that as the pH decreases, the Trp is more solvent-exposed. The use of KI as a quenching agent yielded similar results, but experiments at very low pH values could not be carried out because of precipitation. We do not know why HPr precipitates, but it might be due to the presence of the negative charge of the I – and the large amount of positive charges at low pH, as happens in other molten-globule-like species [34]. Here, conversely to that observed in acrylamide experiments, only linear plots were found. A small K sv value was found when the protein is folded, and larger values were observed when the protein was completely unfolded (6 M urea). It is interesting to note here, that the K sv (in acrylamide and KI) and v (in acrylamide) values for folded (and unfolded) HPr are similar to those found in other folded (an unfolded) proteins [35,36]. ANS binding fluorescence. ANS binding is used to monitor the extent of exposure of protein hydrophobic regions, and to detect the existence of non-native partially folded conformations. When ANS is bound to solvent- exposed hydrophobic patches of proteins, its quantum yield is enhanced and the maxima of the emission spectra is shifted from 520 nm to 480 nm [37]. At low pH values, the intensity of the ANS in the presence of HPr was largely enhanced and the maxima wavelength appeared at 482 nm (Fig. 1B). As the pH was increased, the spectra intensity was reduced and the maxima wavelength shifted towards 528 nm. These results suggest that: (a) ANS was bound to HPr at low pH values, probably because of the presence of solvent-exposed hydrophobic regions and (b) those hydro- phobic patches were probably buried in the pH range 4–7, as concluded from the titration curve measured (Fig. 1B). The apparent pK a was 5.3 ± 0.5. The ANS-binding experiments carried out with the protein without the His 6 -tag showed the same behaviour (data not shown). Far-UV and near-UV CD. We used far-UV CD in the analysis of the unfolding of HPr as a spectroscopic probe that is sensitive to the presence of protein secondary structure [22]. Its CD spectrum was intense and showed the typical features of an a-helical protein, with intense minima at 222 and 208 nm (Fig. 2A), although interference from the absorbance of tryptophan and histidine residues could not be ruled out at 222 nm [22]. The shape of the CD spectrum of S. coelicolor HPrwassimilartothatobserved for E. coli HPr [16]. This shape did not change substantially over the pH range explored (Fig. 2A), but the intensity at low pH values was smaller than at neutral pH values. However, the ellipticity at low pH values (pH 3.0) was not as small as that observed at high urea concentrations, where the protein was completely unfolded suggesting that the protein has residual structure at low pH values. The apparent pK a was 3.5 ± 0.3 (Fig. 2B), which is, within the error, the same as that determined by fluorescence. We used near-UV CD to detect possible changes in the asymmetric environment of aromatic residues [22]. The near-UV of HPr was very weak and no intense bands were observed (Fig. 2C), probably due to the low content of aromatic residues. The spectrum showed a small band at 292 nm and a shoulder at 285 nm corresponding to the vibronic components of the 1 L b transition in tryptophan residues [38]. Because of lack of intense distinctive features, we did not use further the near-UV spectrum to map any temperature, pH or chemical denaturant changes. FTIR spectroscopy. FTIR is a powerful method for investigation of protein secondary structure. The main advantage in comparison with CD is that FTIR is more sensitive to the presence of b structure or random-coil. Measurements were only carried out at selected pH values from pH 2.5–7.5, because of the large amounts of protein Table 1. Quenching constants for HPr in acrylamide and KI. Errors are data fit errors to Eqn (1) with (acrylamide) or without (KI) the exponential factor. The K sv and v were obtained by fitting of fluores- cence intensity at 335 nm vs. concentration of quenching agent (similar values were obtained by fitting the intensities at 336, 337 and 338 nm, data not shown). Experiments were carried out at 25 °C. The value with a Ô–’ could not be measured due to HPr precipitation. Conditions Acrylamide KI K sv ( M )1 ) v ( M )1 ) K sv ( M )1 ) pH 3.3 4.5 ± 0.3 3.0 ± 0.1 – pH 7.5 3.4 ± 0.3 2.0 ± 0.2 0.82 ± 0.05 6 M urea 4.2 ± 0.5 7.9 ± 0.2 4.5 ± 0.1 Ó FEBS 2003 Stability of Streptomyces coelicolor HPr (Eur. J. Biochem. 270) 2259 used in the FTIR experiments and the impossibility of sample recovery. As the pH increased the maxima of the amide I bands moved from 1651.0 to 1645.6 cm )1 (Fig. 3B). The titration, as concluded from the position of the amide I band, followed the same sigmoidal behaviour than that observed by CD and fluorescence, but a reliable value for the pK a midpoint could not be determined. Deconvolution of the bands at the extreme explored pH values (pH 2.5 and 7.5) indicates that the helical structure remained basically unaltered (50%); conversely, antiparallel b structures (the 1628 and the 1666 cm )1 bands) were increased at lower pH values (it changed from 20% at pH 7.5–27% at low pH). Although the exact reasons of the increase in the b sheet bands are unknown, it could be due to formation of soluble oligomers at low pH values. The presence of those oligomer species would explain also the apparent thermal titration observed at low pH values in the FTIR experiments. Gel-filtration chromatography. Gel filtration provides a measurement of the compactness (hydrodynamic volumes) of the polypeptide chain [39]. HPr eluted at neutral pH at Fig. 2. CD of HPr under different conditions. (A) Far-UV CD spectra at different conditions (filled circles, pH 3; open circles, neutral pH; squares, 6 M urea). The conditions were: 20 l M of protein at 25 (± 0.1) °C; buffer concentration was 10 m M inallcases;spectrawereacquiredin0.1-cm pathlength cells. (B) pH-induced unfolding of HPr following the ellipticity at 222 nm in the far-UV CD. The line is the fitting to Eqn (2). (C) Near- UV of HPr. Continous line (circles) is the near-UV spectra at pH 7.5, 10 m M phosphate buffer. Dotted line (squares) is the near-UV at 6 M urea, pH 7.5, 10 m M phosphate buffer. Conditions were: 60 l M protein, 25 °C, spectra were acquired in 0.5-cm pathlength cells. (D) CD spectra at different concentrations at pH 3.0, after normalization by the smaller concentration used (10 l M ). Open circles (black lines) are spectra acquired at 10 l M ; open squares (red lines) are at 20 l M and filled circles (blue lines) at 40 l M protein concentration. The ellipticity units on the y-axis are the normalized raw ellipticity. Fig. 3. FTIR of HPr at different pH values. (A) Green (pH 2.5), blue (pH 3), red (pH 5) and black lines (pH 7). Vertical bars indicate absorbance units. (B) Position (cm )1 )ofthe amide I band at different pH values. Protein concentration was 6 mgÆmL )1 ; all other con- ditions as described under the Experimental procedures section. 2260 G. Ferna ´ ndez-Ballester et al.(Eur. J. Biochem. 270) Ó FEBS 2003 the volume expected for a globular folded protein of its size, 13.92 mL. The R s determined from Eqn (6) is 16.35 A ˚ , within the range expected for a globular protein of its size (Fig. 4A). At low pH values, the protein eluted close to the void volume of the column as a single peak (Fig. 4B), 8.55 mL at pH 3.5. From pH 4–4.5, HPr eluted in two different peaks: (a) the first peak eluted at the same volume as the pH-unfolded protein and (b) the second peak was very broad. These data indicate that the interconversion between the unfolded and native forms of the protein is slow as compared to the column retention time [39]. Similar behaviour has been observed in other partially folded species [40]. The fact that the elution volume of HPr at low pH values is close to the void volume suggests that either HPr is an oligomer or it has lost most of this globular shape. We can rule out the first explanation as: (a) glutaraldehyde cross- linking at pH 3.0 did not show the presence of oligomers at the concentrations explored, 20–100 l M (data not shown); (b) far-UV experiments at different concentrations, ranging from 14 to 295 l M , at pH 3.0 did not show any change either in the shape or the raw ellipticity after being normalized by the smallest concentration used (Fig. 2D); (c) if there was a large population of aggregated forms, two intense bands at 1620 cm )1 and 1685 cm )1 [30,31] should appear in the FTIR spectrum; at pH 2.5, even at the large amounts of protein used in the FTIR experiments, these bands were not found at 25 °C (Fig. 6B); however, the presence of an small amount of aggregated forms could explain the increase (7%) in the b sheet structure observed at this pH; (d) the gel filtration experiments carried out at different protein concentrations (20–60 l M )atthese low pH values did not show any difference in the elution volume and (e) the <k> is very sensitive to changes in oligomerization processes [23], and the value observed at thesepHvaluesisclose(2.84lm )1 ) (Fig. 1A) to that observed for urea-unfolded HPr (where aggregated forms are not present); furthermore, only below pH 2 a large increase in the <k> was observed (from pH 0.22–0.87, <k> shifted from 2.90 to 2.86 lm )1 , respectively); also, the maxima wavelength decreased as the pH was reduced (from pH 0.22–0.87, it shifted from 340 to 348 nm, respectively) (Fig. 1C); these findings indicate the presence of aggregated forms at pH values below 2. Then, all these probes suggest that at pH 3.0, HPr is monomeric, and the small elution volume indicates that HPr has lost most of its globular form. Below this pH small populations of aggregated species cannot be ruled out, but their contribution to the spectral properties is insignificant as suggested by the FTIR spectra. Thermal-denaturation experiments at different pH values In order to obtain the thermodynamic parameters charac- terizing the unfolding transition of HPr, we carried out thermal-denaturation experiments followed by CD, FTIR and DSC. Measurements trying to obtain a complete set of thermodynamic parameters by using fluorescence failed due to the large temperature dependence of the intrinsic Fig. 4. pH-induced unfolding of HPr followed by gel-filtration chromatography. (A) Determination of the Stokes radius by gel filtration chroma- tography on a HR Superdex G75 (Amersham Biosciences). The elution volume of HPr is indicated by an arrow and a square. The numeration corresponds, respectively, to the elution volumes of ribonuclease A (1), chymotrypsinogen A (2), ovalbumin (3), and albumin (4). (B) Elution volume vs. pH, the filled circles observed from pH 4–4.5 indicate one of the two peaks observed during protein elution. The open circles and squares indicate the elution volumes of two different measurements. (C) Chromatograms at selected pH values: continuous black lines and open circles, pH 3; dotted blue lines and open squares pH 4.3; dotted-and-dashed red lines and filled circles pH 7. Conditions were: 20–60 l M of protein at 25 °C, in 10 m M of the corresponding buffer and 150 m M NaCl. Ó FEBS 2003 Stability of Streptomyces coelicolor HPr (Eur. J. Biochem. 270) 2261 fluorescence of both the native and unfolded states of the protein (data not shown). Far-UV CD. We explored the thermal-denaturation beha- viour of HPr from pH 2.0–11.0 (Fig. 5), by following the ellipticity at 222 nm. We found four different pH regions, according to the reversibility and sigmoidal behaviour. (a) Between pH 2.0 and 3.0 the heating transition did not have a sigmoidal behaviour, but it was reversible. (b) Between pH 3.5 and pH 4.5 the heating transition was noncooper- ative and irreversible. (c) Between pH 5.0 and pH 5.5 the heating transition showed a sigmoidal behaviour, but was not reversible. (d) Between pH 6 and pH 9 the heating transition had a sigmoidal behaviour and it was reversible. The reversibility at those pH values was approximately 90–100%, as measured from the relative ellipticity recovery in the reheating experiments. At higher pH values, the thermal transitions were not reversible, probably due to deamidation processes [16,41]. A reliable determination of the DC p and DH cal between pH 6.0–9.0 could not be carried out, because of the absence of baseline in the unfolded state at some pH values. The thermal unfolding of HPr was pH-independent from pH 7.0–9.0, as concluded from the identical T m values (64.6 ± 0.6 °C), suggesting that protein stability was similar in that pH range. It is interesting to note here that the transition at pH 3.5 (Fig. 5A) showed a low degree of cooperativity at low temperatures, although not a proper and complete sig- moidal behaviour was observed. This could suggest the presence of a molten-globule species, as has been seen in a-lactalbumin and other proteins [42]. FTIR spectroscopy. Upon heating, the shape of the amide I band of HPr changed dramatically at pH 2.5 and 7.5, as shown by: (a) a substantial loss in the integrated intensity of bands arising from ahelix and (b) the appearance of a strong and weak bands at 1620 and 1680 cm )1 , respectively, which correspond to interactions between extended chains, and have been related to aggre- gation of thermally unfolded proteins [30] (Fig. 6). The measurement of the whole band width at half-height upon temperature allowed the characterization of the melting curve at pH 7.5, which resembled that found by CD experiments (Figs 5 and 6). Similar sigmoidal transitions were also obtained by following the change in intensity in the bands at 1652 cm )1 (where the a helix appears) and at 1630 cm )1 (where the b sheet is absorbing) (data not shown). At pH 7.5, the transitions were irreversible, precluding the determination of T m . The lack of reversibi- lity, when compared to CD and DSC results, was probably due to the high protein concentrations used. At pH 2.5 a thermal transition was also observed (Fig. 6A), in contrast to the experimental findings obtained by CD. The presence of this transition at pH 2.5 is not understood, but it could be due to aggregation processes occurring at the high protein concentrations and tempera- tures used in the FTIR experiments. DSC experiments. We studied the heat-induced denatur- ation of the protein by DSC at pH 7.5. The protein (1 mgÆmL )1 ,87.5l M ) was heated at a constant scanning rate (60 °CÆh )1 )upto95°C (scan), cooled down, and reheated under identical conditions (re-scan). The scan and re-scan experiments are equally well fitted by the two-state model [43] with van’t Hoff to calorimetric enthalpy ratios, DH VH /DH cal , of 1.02 and 1.01, respectively. The DSC results indicate that the heat-induced unfolding of HPr was characterized, under these experimental conditions, by a melting temperature, T m , of 65.4 ± 0.5 °C, a calorimetric enthalpy change upon unfolding, DH cal ¼ 60.3 ± 1.5 kcalÆmol )1 and an entropy change upon unfolding: DS(T m ) ¼ DH(T m )/T m ¼ 177.8 calÆK )1 Æmol )1 (Fig. 7). Chemical denaturation experiments As the stability of HPr does not change significantly around neutral pH, as concluded from thermal denatur- ation experiments, we decided to follow the chemical denaturation at pH 7.5 to compare with the stability Fig. 5. Thermal denaturation profiles of HPr followed by far-UV CD at 222 nm. Continuous line (circles) is the heating experiment and the dotted line (squares) is the reheating scan at (A) pH 3.5; (B) pH 5; and (C) pH 7.5. The ellipticity units on the y-axis are arbitrary. The con- ditions were: 20 l M of protein; buffer concentration was 10 m M ; spectra were acquired in 0.1-cm pathlength cells. The scan rate was 60 °CÆh )1 in all cases. 2262 G. Ferna ´ ndez-Ballester et al.(Eur. J. Biochem. 270) Ó FEBS 2003 results obtained in other HPr family members. Figure 8 shows the chemical denaturation curves of HPr at pH 7.5, 10 m M phosphate, followed by fluorescence, far-UV and gel filtration chromatography at 25 °C. The agreement between the three probes suggests that the chemical- denaturation can be described as a two-state model. Thermodynamic parameters from gel-filtration and CD measurements had a large error. We do not know the reasons for those large errors, but they could be due to the spread observed in the baselines of the transitions. Similar large slopes in chemical denaturation experiments followed by NMR have also been observed by following the GdmCl chemical-denaturation in the E. coli HPr [16], but they do not yield large errors in the thermodynamic parameters. Chemical denaturation followed by ANS binding did not show any sigmoidal titration (data not shown), suggesting that no intermediate with close solvent- exposed hydrophobic patches accumulated during the denaturation. The free energy, determined from the fluorescence measurements using the linear extrapolation method approach, yielded a value of 4.0 ± 0.2 kcalÆmol )1 , indicating that HPr is not a highly stable protein. The denaturation experiments were reversible in all cases. Discussion Equilibrium-unfolding of HPr at neutral and high pH values follows a two-state mechanism The chemical-denaturation folding of S. coelicolor follows a two-state mechanism at neutral and high pH values, as happens in other small proteins [44]. The denaturation of HPr can be described as a two-state reaction at neutral pH from the following evidence. (a) All the unfolding data can be fitted to a single transition curve using Eqn (3). (b) The denaturant transitions appear to be independent of the biophysical probe (fluorescence, far-UV CD and gel-filtra- tion chromatography) used (Fig. 8), and reversible for either folding and unfolding (data not shown). (c) The ratio of the van’t Hoff enthalpy of denaturation and the calorimetric enthalpy obtained from DSC is close to unity for the heating and the reheating scans (Fig. 7). The conformational stability of S. coelicolor is the smallest among that of the other family members reported so far (Table 2). This low stability is also confirmed by hydrogen exchange measurements at pH 7.5 using FTIR and NMR experiments, where most of the protons exchanged within 20 min (data not shown). Comparison of the sequence among 31 HPr homologues in the protein data bank indicates that the HPr of S. coelicolor forms a completely different cluster of sequence equally distant to the rest of the members of the family [9] (Fig. 9). The differences in stability, as the structure of HPr of S. coeli- color (unpublished results) is nearly identical to those of E. coli and B. subtilis, must rely on differences in the packing of their different side-chains. Fig. 7. Excess heat capacity function of HPr at pH 7.5 in 10 m M phosphate buffer. The continuous lines represent the fitting of the experimental data to a two-state reversible model. Fig. 6. Thermal denaturation profiles of HPr followed by FTIR. (A) Thermal denaturation profiles of HPr at pH 2.5 (filled squares) and pH 7.5 (filled circles). The lines at both pH values are drawn to guide the eye. (B) Thermal denaturation profiles of HPr at pH 7.5 (left side) and pH 2.5 (right side) at selected temperatures. Protein concentration was 6 mgÆmL )1 ; all other conditions as described under the Experi- mental procedures section. Ó FEBS 2003 Stability of Streptomyces coelicolor HPr (Eur. J. Biochem. 270) 2263 [...]... reflecting the presence of other acidic groups, whose titration causes the chain to acquire a native-like compactness We favour, however, the latter explanation as the thermal behaviour of HPr between pH 3.5 and 4.5 is different to that observed at lower pH values Although it is difficult to rationalize a native-like tertiary structure around the tryptophan without the acquisition of a native-like compactness,... titration towards the native state, as shown by ANS binding (Fig 1) Although the protein has attained native-like compactness, secondary, and tertiary structure, the ANS-binding experiments suggest that not all the hydrophobic patches are buried This titration could be associated either with: (a) the own ANS titration or (b) the presence of aggregated forms of the protein at those pH values Control experiments... that the sole tryptophan did not map correctly the acquisition of tertiary structure, and further structural rearrangements were required to attain a native-like tertiary compactness Preliminary NMR analysis at pH 4.5 indicate that the protein has native-like chemical shifts for the tryptophan moiety, but also non-native NOEs are observed (unpublished results) There is, still, a third titration towards... 0.2 a Data are from Nicholson and Scholtz [45] at 10 mM phosphate buffer, pH 7.0, 25 °C There is another complete set of data for the E coli HPr, obtained from Dobson and coworkers [16], which are similar to those described here, but obtained in the presence of GdmCl The value of the DG obtained from that set of data is 4.9 ± 0.4 kcalÆmol)1 b Data are from Scholtz [24] at 10 mM phosphate buffer, pH 7.0,... experiments in other proteins, followed by ANS binding, have also shown titration midpoints associated to the ionization of acid groups of the macromolecule [48] It seems, then, that the transition mapped by ANS binding is indicating another partially folded conformation, associated with either an acidic residue or, the active-site His, whose pKa measured in other HPrs, is small (pKa ¼ 5.6) [49] However,... nearly the same as that in the native state (approximately 50% of the whole percentage of structure) The difference between both techniques must rely either on: (a) the FTIR deconvolution procedures or (b) possible rearrangements of the aromatic residues, which also absorb at 222 nm in the CD spectra Then, the probes indicate that at low pH values the tertiary native-like structure is lost, but there... different pattern to that observed at lower and higher pH values These findings suggest that there are regions of the protein which are not well-fixed and upon heating become more disordered Then, the different biophysical probes suggest that at low pH values, HPr acquires a partially folded conformation, devoid of native-like tertiary structure, compactness and with solvent-exposed hydrophobic patches,... environment around the indole moiety, as concluded by the native-like maxima of fluorescence spectra and the native ellipticity (Figs 1 and 2) However, the compactness is not native-like, as judged by the gel-filtration experiments In a narrow pH range (i.e from pH 4–4.5) the protein attains its native compactness We do not know whether the acquisition of native-like compactness of the polypeptide chain is a consequence... in different proteins at low pH values have shown common characteristics, such as: (a) the presence of a pronounced amount of secondary structure; (b) the absence of most of the native tertiary structure, as a result of lack of the tight packing of side chains; (c) loosely packed hydrophobic core that increases the hydrophobic surface accessible to solvent and (d) almost a native-like compactness This... well -folded protein state) binds ANS weakly and thus fluoresces weakly, compared to the low- pH unfolded state Then, the apparent difference in the midpoint of the pH transitions (Fig 1) could be likely explained in this manner However, both objections can be ruled out considering that the thermal denaturations of HPr in this pH range, despite the sigmoidal behaviour during the heating scan, follow a different . heating, the shape of the amide I band of HPr changed dramatically at pH 2.5 and 7.5, as shown by: (a) a substantial loss in the integrated intensity of bands arising from ahelix and (b) the appearance. as a two-state process without the accumulation of intermediates at neutral and moderately basic conditions (pH 7–9) at 25 °C. The stability of the protein, at pH 7.5 and 25 °C, as obtained by chemical. cases. Discussion Equilibrium-unfolding of HPr at neutral and high pH values follows a two-state mechanism The chemical-denaturation folding of S. coelicolor follows a two-state mechanism at neutral and high pH values, as happens