Temperature-, SDS-, and pH-Induced Conformational Changes in

May 26, 2005 - Emilia Pedone,† Michele Saviano,† Simonetta Bartolucci,*,‡ Mose` Rossi,‡,§ Alessio Ausili,|. Andrea Scire`,| Enrico Bertoli,| ...
0 downloads 0 Views 451KB Size
Temperature-, SDS-, and pH-Induced Conformational Changes in Protein Disulfide Oxidoreductase from the Archaeon Pyrococcus furiosus: A Dynamic Simulation and Fourier Transform Infrared Spectroscopic Study Emilia Pedone,† Michele Saviano,† Simonetta Bartolucci,*,‡ Mose` Rossi,‡,§ Alessio Ausili,| Andrea Scire` ,| Enrico Bertoli,| and Fabio Tanfani| Istituto di Biostrutture e Bioimmagini, C.N.R.,Via Mezzocannone 16, 80134, Napoli, Italy, Dipartimento di Biologia Strutturale e Funzionale, Universita` degli Studi di Napoli Federico II, via Mezzocannone 16, 80134 Napoli, Italy, Istituto di Biochimica delle Proteine, C.N.R., via P. Castellino 111, 80131, Napoli, Italy, and Istituto di Biochimica, Universita` Politecnica delle Marche, via Ranieri, 60131 Ancona, Italy Received May 26, 2005

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of R-helices and β-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites. Keywords: protein structure • protein disulfide oxidoreductase • Pyrococcus furiosus • thermostability • FT-IR spectroscopy • MD simulations

1. Introduction Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyze dithiol-disulfide exchange reactions. These enzymes share a CXXC sequence motif at their active sites. The two cysteines can undergo reversible oxidation-reduction by shuttling between a dithiol and a disulfide form in the catalytic process. Protein disulfide oxidoreductases comprise the families of thioredoxin (Trx),1 glutaredoxin (Glx), protein disulfide isomerase (PDI), Dsb (Disulfide bond forming) and their homologues. While thioredoxin and glutaredoxin mainly catalyze the reduction of disulfides, PDI and Dsb catalyze the formation or rearrangement of disulfide bridges in the protein folding process. Protein disulfide oxidoreductases (PDO) were isolated from the hyperthermophilic archaea Sulfolobus solfataricus (SsPDO), Pyrococcus furiosus (PfPDO), and Pyrococcus horikoshii (PhPDO) showing an unusual molecular mass of about 26 kDa, compared to the molecular weight observed in Trxs and Glxs (12 kDa).1,2 The proteins have two potential active sites with the conserved CXXC sequence motif: a CPYC sequence, typical of the glutaredoxin family, is unusually located at the * To whom correspondence should be addressed. Tel: +39-81 679052. Fax: +39-81 679053. E-mail: [email protected]. † Istituto di Biostrutture e Bioimmagini, C.N.R. ‡ Dipartimento di Biologia Strutturale e Funzionale, Universita` degli Studi di Napoli Federico II. § Istituto di Biochimica delle Proteine, C.N.R. | Istituto di Biochimica, Universita` Politecnica delle Marche.

1972

Journal of Proteome Research 2005, 4, 1972-1980

Published on Web 10/22/2005

C-terminal half. In addition, a CQYC sequence, which has never been observed in any other protein disulfide oxidoreductase, is present at the N-terminal half of the protein. PDO displayed dithiothreitol-dependent insulin reduction activity and thioltrasferase activity by catalyzing the reduction of disulfide bonds in omocysteine.3,4 The PfPDO crystal structure provided some intriguing challenges to the understanding of the enzyme’s function.5-8 The protein consists of two homologous structural units with low sequence identity (18%). Each unit contains a thioredoxin fold with a distinct CXXC active site motif. The presence of two homologous units in the same protein resembles the structure of eukaryotic PDI; in fact, the PDI molecule possesses two thioredoxin-like domains with two active sites. The unusual structural features of PfPDO suggest that this enzyme represents a member of the protein disulfide oxidoreductase superfamily and a new type of isomerase referred to PDI and Dsb. Recently, functional studies confirmed that PfPDO represents an ancestor of the eukaryotic PDI.9 Sitedirected mutagenesis demonstrated that the C-terminal site CPYC is basic for oxidative and reductive activity, and that the two units function synergistically in isomerase activity. This protein represents the first example of an archaeal protein characterized with disulfide isomerase activity. To obtain new insights into structural-functional relationships in PfPDO, the structure and thermal stability of the protein were analyzed under different conditions by Fourier transform infrared spectroscopy and Molecular Dynamics simulations. The effects of SDS, pD, and temperature on the 10.1021/pr050152z CCC: $30.25

 2005 American Chemical Society

research articles

Conformational Changes in Protein Disulfide Oxidoreductase

protein structure were characterized. The data indicated that pD affects differently the thermostability of R-helices and β-sheets, and that 0.5% or higher SDS concentration have a significant repercussion on the structure. Moreover, the experiments allowed to detect a secondary structural reorganization at a definite temperature and pD which may correlate with previously observed high ATPase activity. Finally, the computational analysis revealed the different behavior of the N and C terminal segments, as well as of the two active sites.

2. Materials and Methods 2.1. Materials. Deuterium oxide (99.9% D2O) DCl and NaOD were purchased from Aldrich. All other chemicals were commercial samples of the purest quality. 2.2. Preparation and Purification of PfPDO. Pyrococcus furiosus protein disulfide oxidoreductase was prepared and purified as described.3 The protein was stored in lyophilized form. 2.3. Infrared Spectra. PfPDO was analyzed at three different pDs. The pD corresponds to the pH meter reading + 0.4.10 The buffers used were as follows: 50 mM piperazine/DCl pD 5.8 (buffer A); 50 mM Hepes/NaOD pD 7.0 (buffer B); 50 mM CAPS pD 10.0 (buffer C). In addition, the protein was analyzed at pD 7.0 (buffer B) in the presence of 0.1, or 0.2, or 0.5, or 0.7, or 4.0% SDS. Typically, 1.5 mg of lyophilized protein was dissolved in 200 µL of a suitable buffer and concentrated into a volume of approximately 35 µL using 10K Centricon microconcentrator (Amicon) at 3000 × g and at 4 °C. Then, further 200 µL of buffer were added, and the protein concentrated again. This procedure was repeated several times in order to fully hydrate the protein with the chosen buffer. Sample preparation took 24 h, which is the contact time of protein with D2O before spectra collection. Then, the concentrated protein solution was injected into a thermostated Graseby Specac 20500 cell (Graseby-Specac Ltd, Orpington, Kent, UK) fitted with CaF2 windows and a 25 µm spacer. For the experiments in the presence of detergent, the proper amount of SDS solution was added to the concentrated protein sample. The concentration of SDS in the sample was checked using a calibration curve obtained by monitoring the intensity of the symmetric methylene stretching vibration band (2854 cm-1) of SDS11 as a function of SDS concentration. FT-IR spectra were recorded by means of a Perkin-Elmer 1760-x Fourier transform infrared spectrometer using a deuterated triglycine sulfate detector and a normal Beer-Norton apodization function. At least 24 h before, and during data acquisition the spectrometer was continuously purged with dry air at a dew point of -40 °C. Spectra of buffers and samples were acquired at 2 cm-1 resolution under the same scanning and temperature conditions. FT-IR spectra were recorded at temperatures ranging from 20 °C to 99.5 °C, with typical 5 °C increments, using an external bath circulator (HAAKE F3). The actual temperature in the cell was controlled by a thermocouple placed directly onto the window. Spectra were processed using the SPECTRUM software from Perkin-Elmer. Correct subtraction of 2H2O was adjusted to the removal of the 2H2O bending absorption close to 1220 cm-1.12 Second derivative spectra were calculated over a 9-data point range (9 cm-1). The deconvoluted parameters for the amide I band were set with a gamma value of 2.5 and a smoothing length of 60. The estimation of the secondary structure composition was carried out by curve fitting of deconvoluted amide I′ band13

Figure 1. Absorbance (A), deconvoluted (B), and second derivative spectra (C) of PfPDO at 20 °C. Continuous and dashed lines refer to the spectra of the protein dissolve in buffer (B) prepared in D2O or H2O, pD or pH 7.0, respectively.

using the peak fitting module of the Origin software (OriginLab Corporation, Northampton, MA 01060 USA). 2.4. Molecular Dynamic Simulations. All calculations and graphical analyses were run on a Silicon Graphics Octene2 workstation. The INSIGHT/DISCOVER package (Accelrys, San Diego, CA) was used to perform energy minimization and molecular dynamic simulations in vacuo at two different temperatures (293, 373 K) and at three different pH values (5.8, 7.0, and 10.0), with the consistent valence force field (cvff).14,15,16 The starting structure used in structural analysis and simulations was that obtained from the Brookhaven Protein Data Bank (Accession No. 1a8l).6 Energy minimization to eliminate hot spots using conjugate gradient method was performed on the X-ray structure as an initial conformation.17 In all simulations, performed with a time step of 1.0 fs and a distance-dependent dielectric constant, the systems were equilibrated for 160 ps. After this first step, an additional 240 ps of simulations without rescaling were carried out, since energy conservation was observed and the average temperature remained essentially constant around the target values. The time span of the runs was optimized, checking that the prolongation of the simulations did not influence the results. Coordinates and velocities for the four simulations were dumped to a disk every 100 steps and were used for the statistical analysis. Computational conditions were chosen to avoid any boundary effects.18

3. Results 3.1. Secondary Structure of PfPDO. Figure 1 shows the absorbance, deconvoluted, and second derivative spectra of PfPDO in 50 mM Hepes/NaOD pD 7.0 (buffer B) prepared in H2O or D2O. The exchange of the medium from H2O to D2O induces changes in the infrared spectrum, as band downshift in wavenumber and changes in band intensity. In particular, the 1657.1 cm-1 band, that in H2O may be assigned to R-helix and unordered structures,19 shifts to 1655.1 cm-1 in the spectrum of the protein prepared in D2O. Indeed, the analysis of the spectrum in D2O allows to separate the absorption of Journal of Proteome Research • Vol. 4, No. 6, 2005 1973

research articles

Figure 2. Effect of SDS concentration on the secondary structure of PfPDO at 20 °C and pD 7.0. The figure shows deconvoluted spectra of the protein in the absence (continuous lines) and in the presence of SDS (dashed lines) at 0.1, 0.2, 0.5, 0.7, and 4.0%.

the R-helix from that of unordered structures that, in this medium, absorb at 1655.1 and 1647.2 cm-1, respectively.19 The 1632.7 and 1689.2 cm-1 bands displayed in the infrared spectrum obtained in D2O medium may be attributed to β-sheet, while the 1665 cm-1 shoulder is due to turns.19 The origin of the 1672.7 cm-1 band is less certain, because both turns and coupled high-frequency vibrations of β-segments can contribute in the spectral region between 1670 and 1690 cm-1.20 The peak close to 1550 cm-1 represents the residual amide II band, i.e., the amide II band (1600-1500 cm-1 range) after H/D exchange of the amide hydrogens of the polypeptide chain. The amide II band is particularly sensitive to the exchange of amide hydrogen with deuterium. In experiments performed in H2O medium, the intensity of the band is about 2/3 of that of amide I band, while in D2O medium it decreases significantly (see Figure 1).21,22 The bigger the intensity decrease, the bigger the H/D exchange. A big H/D exchange indicates that the protein structure is very accessible to the solvent (D2O). The fact that the infrared spectrum of PfPDO displays a residual amide II band indicates that at 20 °C the protein segments were not completely accessible to the solvent. The other peaks below 1620 cm-1 shown in Figure 1 are due to amino acid side-chain absorption.23,24 3.2. Effect of SDS on the Secondary Structure of PfPDO. Figure 2 shows the deconvoluted spectra of PfPDO in the absence (control) and in the presence of different concentrations of SDS. The secondary structure of the protein is not affected by 0.1 or 0.2% of SDS since the amide I′ band contour in the spectra of the protein in the absence and in the presence of the detergent is the same. However, in the presence of detergent the intensity of the residual amide II band is lower as compared to the control, indicating that SDS allows a deeper contact of the solvent with the protein segments. Since SDS does not change the secondary structure of the protein, the lower residual amide II band intensity is most likely due to a more relaxed tertiary structure.25 In the presence of 0.5%, the intensity of the R-helix band decreases markedly while the intensity of the β-sheet band is 1974

Journal of Proteome Research • Vol. 4, No. 6, 2005

Pedone et al.

Figure 3. Effect of pDs on the secondary structure of PfPDO at 20 °C. (A), continuous and dashed lines refer to the spectra of the protein at pD 7.0 and 5.8, respectively. (B), continuous and dashed lines refer to the spectra of the protein at pD 7.0 and 10.0, respectively.

slightly affected. In the presence of 0.7% SDS the β-sheet band intensity decreases further and in the presence of 4% SDS it results very low. At this SDS concentration a broad band centered at 1644 cm-1, due to unordered structures, characterizes the spectrum. These data indicate that SDS concentrations higher than 0.5% affect the secondary structure of the protein and that R-helices are more sensible to the detergent than β-sheets. Moreover, the data indicate that in the presence of 4% SDS the protein is largely unfolded (denatured). Figure 2 shows also that the residual amide II band intensity decreases with the increase of the concentration of the detergent. At SDS concentrations higher than 0.2% the H/D exchange is probably due to both the relaxation of the structure and to the unfolding of the secondary structures.25 At 4% SDS concentration the large H/D exchange, as indicated by the very low residual amide II band intensity, is most likely due to the large unfolding of the protein. 3.3. Effect of pD on the Secondary Structure of PfPDO. Figure 3 compares the spectra of PfPDO at different pDs. At pD 5.8 and 7.0 the secondary structure of the protein is very similar being the amide I′ band contour at the two pDs slightly different. In particular, the R-helix band intensity is slightly higher at pD 5.8 than at pD 7.0. At pD 10.0, the R-helix and unordered structures band intensities are markedly lower and higher, respectively than at pD 7.0 indicating that at pD 10.0 the protein undergoes partial unfolding. Thus, Figure 3 shows that R-helices are sensible to pD, whereas β-sheets are not, since the β-sheet band intensity and position remains the same at different pDs. These observations are confirmed by the curve-fitting calculations applied to the deconvoluted amide I′ band of the PfPDO spectrum obtained at different pDs. As an example Figure 4 shows the amide I′ band contour fitted with individual component bands. The results of the calculations are reported in Table 1 that shows a decrease in R-helix content at pD 10.0, whereas the β-sheet content remains unaltered at different pDs. Figure 3 shows also that the residual amide II band intensity is higher at pD 5.8 than at pD 7.0 suggesting that the acidic pD induces a more compact structure. On the other hand, at basic pD, the residual amide II band

Conformational Changes in Protein Disulfide Oxidoreductase

research articles

Figure 5. Effect of temperature on deconvoluted spectra of PfPDO at pD 5.8 and pD 7.0. Panel (A) and panel (B) show the spectra recorded at 20, 30, 40, 50, 55, 60, 65, and 70 °C and at pD 7.0 and 5.8, respectively. Panels (C) and (D) show the spectra recorded at 75, 80, 85, 90, 95, 98, 99, and 99.5 °C and at pD 7.0 and 5.8, respectively. The increase in temperature leads to a decrease in band intensity. The arrows indicate the temperaturedependent decrease in intensity of the corresponding bands. In panel D, there is a temperature-dependent increase in absorption value at 1624 cm-1 (arrow) due to protein aggregation (a). Panel C and panel D display a spectrum (c) obtained upon cooling to 20 °C the sample treated at 99.5 °C. The spectrum (c) reported in panel D shows an aggregation band (a).

Figure 4. Deconvoluted amide I′ band contour with the best fitted Gaussian/Lorentzian individual component bands for PfPDO at pD 7.0, 5.8, and 10.0. The deconvoluted parameters were set with a gamma value of 2.5 and a smoothing length of 60. Table 1. Calculated Positions (cm-1) and Fractional Areas (%) of the Amide I′ (1700-1600 cm-1) Component Bands for PfPDO at PD 7.0, 5.8, and 10.0a,b band position (cm-1) at pD 7.0

% at pD 7.0

band position (cm-1) at pD 5.8

% at pD 5.8

band position (cm-1) at pD 10.0

% at pD 10.0

1625.1 (β) 1632.7 (β) 1640.2 (β) 1646.6 (u) 1654.3 (R) 1661.6 (t) 1671.2 (β/t) 1677.1 (β/t) 1689.1 (β)

1.6 17.7 8.8 14.4 38.0 10.1 2.1 5.7 1.6

1625.9 (β) 1632.8 (β) 1640.1 (β) 1646.6 (u) 1654.4 (R) 1661.7 (t) 1671.1 (β/t) 1677.5 (β/t) 1689.3 (β)

2.3 16.8 8.5 14.0 38.5 9.9 2.0 6.3 1.5

1624.7 (β) 1632.4 (β) 1639.6 (β) 1645.3 (u) 1654.0 (R) 1661.0 (t) 1672.2 (β/t) 1679.9 (β/t) 1688.4 (β)

3.0 18.9 7.6 21.9 31.0 10.3 5.3 0.6 1.3

a The symbols (R), (β), (t), and (u) stand for R-helix, β-sheet, turn, and unordered structures, respectively. b The values of χ2 at pD 7.0, 5.8, and 10.0 were 6.03 × 10-6, 7.81 × 10-6, and 6.23 × 10-6, respectively

intensity is lower than at neutral pD. In this case, the low residual amide II band intensity is probably due to the partial unfolding of the R-helices. 3.4. Temperature-Dependent Spectral Changes at pD 5.8 and 7.0. Proteins from thermophilic and hyperthermophilic

organism are particularly resistant to high temperatures. In many instances, they denature about at 100 °C allowing to follow their thermal denaturation by spectroscopic approaches. In other cases, hyperthermophilic proteins have a Tm above 100 °C, and in order to follow the thermal denaturation, it is necessary to study the protein under high pressure or in the presence of destabilizing agents as detergents, organic solvents, or extreme pHs.22,26 Figure 5 shows the effect of temperature on deconvoluted spectra of PfPDO at pD 5.8 and pD 7.0. The R-helix, and β-sheet bands decrease in intensity with the increase in temperature, but the spectra recorded at 99.5 °C (Figure 5C and 5D) are still well structured and the R-helix and β-sheet band are still visible. Although the decrease in intensity of the above-mentioned bands may suggest some small losses in R-helix and β-sheet content, the result indicates that the secondary structural elements are quite resistant toward high temperatures. This is particularly true for the β-sheets of sample prepared at pD 7.0 since between 75 and 99.5 °C (Figure 5C) the β-sheets band is almost unchanged, while the R-helix band decreased constantly with the increase of the temperature. At pD 5.8, the β-sheets and the R-helix bands decrease in intensity constantly with the increase in temperature (Figure 5B,D) suggesting that low pD affects the stability of the β-sheets. Figure 5D (pD 5.8; T 75-99.5 °C) shows a significant increase in absorbance close to 1620 cm-1, indicating that the protein undergoes aggregation. In fact, this band concomitantly with another small band close to 1680 cm-1, is usually formed as a consequence of protein intermolecular interactions (aggregation) brought about by protein denaturation.22 Due to protein aggregation the thermal denaturation process is irreversible. Indeed, the spectrum of the protein obtained upon cooling the sample to 20 °C from 99.5 °C (spectrum c) resembles that obtained at 99.5 °C and not that of native protein at Journal of Proteome Research • Vol. 4, No. 6, 2005 1975

research articles

Pedone et al.

R-helix band decreases in intensity with respect to pD 7.0, whereas the β-sheet band intensity remains the same. From the combination of these results, it may be concluded that a population of R-helices is particularly pD-sensitive and that the remaining part of R-helices is more temperature-resistant with respect to β-sheets.

Figure 6. Effect of temperature on deconvoluted spectra of PfPDO at pD 10.0. Panel (A) shows the spectra recorded at 20, 30, 40, 50, 55, 60, 65, and 70 °C. Panel (B) shows the spectra recorded at 75, 80, 85, 90, 95, 98, 99, and 99.5 °C. The increase in temperature lead to changes in band intensity and/or in band position. The arrow V or v indicate a decrease or increase in intensity of the corresponding band. The arrow f indicate a shift to lower wavenumber of the corresponding band. The inset shows the temperature-induced shift of the β-sheet band and the temperature-dependent decrease in intensity of the β-sheet band.

20 °C (see panel 5B, top spectrum). The spectrum (c) also indicates that the cooled protein remained aggregated as shown by the aggregation band (a). The spectrum of the protein at pD 7.0 (Panel C) obtained upon cooling the sample to 20 °C (spectrum c) does not contain evident aggregation bands. However, since the spectrum does not resemble that of native protein at 20 °C (see panel 5A, top spectrum) and because it is similar to that obtained at 99.5 °C, the data indicate that the cooled protein did not assume the original conformation. Figure 5 shows also that the residual amide II band intensity decreases constantly with the increase in temperature in all cases. The phenomenon is probably due to the conformational changes of the protein and to the temperaturedependent protein dynamics. From all data, it may be concluded that high temperatures does not denature completely the protein but change slightly the content of secondary structural elements and that at pD 7.0, the R-helices are less thermostable than β-sheets (Figure 5C), whereas at pD 5.8, the thermostability of the two secondary structural elements is similar. 3.5. Temperature-Dependent Protein Structural Reorganization at pD 10.0. Figure 6 shows the effect of temperature on deconvoluted spectra of PfPDO at pD 10.0. In the 20-70 °C temperature range (Figure 6A), the β-sheet band intensity decreases with the increase in temperature, whereas the R-helix band intensity remains almost constant, suggesting that at pD 10, the R-helices are more thermostable than β-sheets. On the other hand, Figure 3 shows that at pD 10.0 and at 20 °C the 1976

Journal of Proteome Research • Vol. 4, No. 6, 2005

Figure 6B shows that the spectra collected in the 75-99.5 °C temperature interval undergo remarkable changes, and in particular the β-sheet band changes considerably. Indeed, the band decreases in intensity till 95 °C; it starts shifting to lower wavenumbers at 90 °C, and it starts increasing in intensity at 98 °C (see also inset). The first event may represent a partial loss of β-sheets that is different between 20 and 70 °C and 75-95 °C temperature ranges (inset) since between 75-95 °C the rate of decrease in intensity of the β-sheet band is higher than between 20 and 70 °C (see inset). The spectra show also an increased rate of H/D exchange between 90 and 98 °C as monitored by the decrease in intensity of the residual amide II band. This phenomenon may be associated to the downshift in wavenumber of the main β-sheet band that starts significantly at 90 °C (inset). Other minor temperature-dependent changes in band intensity/position are reported in the figure and indicated by the arrows. The onset of band shift is observed at 90 °C, and the shift continues till 99.5 °C. In some instances the phenomenon of band shift may be used as a tool to detect a molten globule-like state in proteins, as described in our previous study concerning the structural characterization of thioredoxin.25 In that paper, we described the existence of a molten globule-like state in a particular range of temperatures preceding (and close to) the temperature of protein denaturation (Tm). The downshift in wavenumber of a β-sheet band and an increase in H/D exchange without a remarkable loss of secondary structure reflected the relaxation of the tertiary structure, a typical example of the presence of a molten globule state.25 It was shown that the structure of PfPDO contains two thioredoxin fold units,6 and thus, the possibility of the presence of a molten globule like-state at a specific temperature exists. In this study, the situation seems to be more complex since the shift of the β-sheet band, observed between 90 and 99.5 °C is accompanied first by a decrease, and then by an increase in intensity of the band. The result suggests that with the increase in temperature the protein undergoes partially unfolding with a concomitant relaxation of the tertiary structure, followed by a reorganization of part of the structure in a new β-conformation. Hence, due to partial unfolding of the protein and to the reorganization of the structure at specific temperature ranges, the phenomenon seems not to fit with a typical molten globule-like state. To check the hypothesis of a temperature-dependent secondary structure reorganization at pD 10.0, we performed curve fitting calculations. The results (Table 2) shows that the R-helix content is slightly affected by the increase in temperature while, consistently with the qualitative observations, the amount of the β-sheets belonging to main β-sheet band (band close to 1632 cm-1) decreases at 95 °C and then increases at 99.5 °C to a value close to that observed at 70 °C. In addition, the calculation shows that the amount of β-sheet belonging to the band close to 1640 cm-1 also changed with the increase in temperature. It must be pointed out that the bands close to 1632 and 1640 cm-1 observed at 70 °C, shifted close to 1627 and 1636 cm-1, respectively at 99.5 °C. In conclusion, these results seem to support the hypothesis that at pD 10 and with the increase in temperature the β-sheets undergo reorganization.

Conformational Changes in Protein Disulfide Oxidoreductase Table 2. Calculated Positions (cm-1) and Fractional Areas (%) of the Amide I′ (1700-1600 cm-1) Component Bands for PfPDO at PD 10.0 and at 70.0, 95.0, and 99.5 °Ca,b band position (cm-1) at 70.0 °C

% at 70.0 °C

band position (cm-1) at 95.0 °C

% at 95.0 °C

1632.8 (β) 1640.5 (β) 1645.3 (u) 1653.7 (R) 1660.9 (t) 1671.6 (β/t) 1678.8 (β/t) 1687.8 (β)

18.2 9.4 24.6 26.4 15.0 4.3 1.4 0.7

1629.1 (β) 1636.2 (β) 1643.9 (u) 1653.1 (R) 1660.7 (t) 1670.4 (β/t) 1676.0 (β/t) 1690.0 (β)

9.8 15.5 24.5 24.9 13.3 5.5 4.1 2.4

band position (cm-1) at 99.5 °C

% at 99.5 °C

1627.4 (β) 1636.6 (β) 1643.5 (u) 1653.0 (R) 1662.8 (t) 1672.0 (β/t) 1681.4 (β/t)

17.9 11.0 25.5 24.1 12.4 6.3 2.8

a The symbols (R), (β), (t), and (u) stand for R-helix, β-sheet, turn, and unordered structures, respectively. b The values of χ2 at 70.0, 95.0, and 99.5 °C were 5.91 × 10-6, 4.18 × 10-6, and 2.30 × 10-5, respectively.

Figure 7. Effect of temperature on deconvoluted spectra of PfPDO in the presence of different amounts of SDS at pD 7.0. Panel (A) shows the spectra recorded at 20, 30, 40, 50, 55, 60, 65, and 70 °C. Panel (B) shows the spectra recorded at 75, 80, 85, 90, 95, 98, 99, and 99.5 °C. The increase in temperature lead to a decrease in band intensity. The arrows indicate the decrease in intensity of the corresponding bands.

3.6. Effect of SDS on the Thermal Stability of PfPDO. As shown in Figure 2, the secondary structure of the protein is affected by 0.5% or higher SDS concentrations. Furthermore, the data suggested that at 0.1% or 0.2% SDS, the structure of PfPDO is more relaxed than the control. Hence, we have to expect that SDS affects also the protein thermal stability. Indeed, Figure 7 shows that the destabilization of secondary structural elements induced by high temperatures is SDS concentration-dependent. In particular, the deconvoluted spectra collected between 20 an 70 °C (panel A) show that, in the range of 0.1-0.5% SDS, the β-sheet band is not remarkably affected by the detergent while the R-helix peak decreases in

research articles intensity with the increase in temperature. It is interesting to note that at 70 °C, the spectra are very similar to the spectrum of PfPDO at pD 7.0 and at 99.5 °C (Figure 5, panel C). This finding may suggest that low SDS concentrations reduce the thermal stability of the protein of about 30 °C. At 70 °C in the presence of 0.7% or 4% SDS concentration, the β-sheet band is present to a very low extent or absent, respectively indicating a strong thermal destabilization of the secondary structural elements induced by the detergent. Moreover, all spectra collected in the presence of 4% SDS show a main broad band centered at about 1651 cm-1 that is present also in all spectra collected above 70 °C (panel B). The band, that is typical for R-helices,19 has been found also in the spectra of other proteins in the presence of high SDS concentration and at high temperatures,22 and it was suggested that the band may be due to the combined absorption of unordered structures and R-helices. In the 75-99.5 °C temperature range (panel B), the β-sheet band decreases in intensity with the increase in temperature in all cases, indicating that even low SDS concentrations (i.e., 0.1-0.5%) destabilize the secondary structure at high temperatures, and that the extent of the destabilization is SDS concentration-dependent. At 0.7 and 4.0% SDS concentration and at 99.5 °C, the amide I′ band is broader, is devoid of typical secondary structural bands, but it is characterized by the 1651 cm-1 band, suggesting that the protein underwent denaturation with the probable and concomitant formation of some R-helices. It is interesting to note that the thermal denaturation of the protein in the presence of high SDS concentrations did not cause protein aggregation (intermolecular interactions), as indicated by the lack of characteristic aggregation bands close to 1680 and 1620 cm-1.22 3.7. Molecular Dynamic Analysis. Molecular dynamic techniques were used to analyze the dynamic behavior of the protein. In particular, molecular dynamic simulations were carried out in vacuo at three different pH values, increasing the temperature of the systems from 293 to 373 K to obtain information on the thermal and structural behavior of the protein at different pH values. In Figure 8, the maximum mean square displacement of each residue with respect to the minimized form as a function of the residue number at pH 5.8 (B), 7.0 (C), and 10.0 (D) for the simulations at 293 and 373 K is reported. The comparative analysis of the graphs demonstrates that the protein structure at pH 5.8 is less flexible and more globular than that found for the other two pHs. An accurate inspection of the simulations (Figure 8) reveals that the R-helix regions are more sensible to increase of pH and temperature and pH with respect to the β-sheets. This different stability at temperature is in agreement with dynamic studies on different proteins,27 suggesting that R-helical regions undergo the largest deviations and that, at higher temperatures, these may be the regions of the protein that first unfold with a consequent extension of β-sheets (Figure 9). A comparison of conformational behavior at different pHs and temperatures gives the following features that confirm the FTIR data: (1) The two active sites show a different behavior. At pH 5.8, pH not optimum for the activity, and at both temperature values, the R2 helix region, where the N-terminal active site is localized, is less flexible than at pH 7.0 and pH 10.0. On the contrary, the C terminal active site at 293 K shows a reduced flexibility at all pH values, while at 373 K the flexibility increases Journal of Proteome Research • Vol. 4, No. 6, 2005 1977

research articles

Pedone et al.

Figure 8. Maximum mean-square displacement (MSD) of each residue with respect to the minimized form for PfPDO as a function of the residue number. (A) Secondary structure of PfPDO. The MD simulations carried out in vacuo at pH (B) 5.8, (C) 7.0, and (D) 10.0 are reported as bold lines for T ) 293 K and as thin lines for T ) 373 K.

with the pH, with a high value at pH 7.0. This findings are in good agreement with the structural evidence that the Nterminal cysteine of the active site is normally exposed to the solvent and has an unusually low pKa value, whereas the C-terminal one is buried and has a higher pKa value; (2) Only at high temperature and pH 10.0, the β3-β4 region, the R6-β6 region, and the 310 helix (residues 178-180) show an high degree of flexibility. It is noteworthy that putative interactions site for ATP are located in these regions; showing that the optimum of ATPase activity is at high temperature and at pH 10.0; (3) At pH 10.0, the temperature increase induces a increase of flexibility of some β-strands (β4, β5, β7, β8) and the analysis of average conformation in these simulation conditions shows that this behavior is related to an extension of β-sheets. 1978

Journal of Proteome Research • Vol. 4, No. 6, 2005

Figure 9. Superimposition of minimized structure ofPfPDO (blue) and its average structure (red) as obtained by MD simulation in vacuo. The MD simulations carried out in vacuo at T ) 373 K at pH (A) 5.8, (B) 7.0, and (C) 10.0. The structures are represented as solid ribbons.

In addition, an accurate inspection of the simulations reveals two important structural features: (a) A high flexibility of R7 helix region, specially at pH 10, that confirms the different conformation properties of helix 7 in groove C with respect to the corresponding helix 3 in groove N; (b) a larger mobility of the N-terminal segment with respect to the C-terminal segment. This behavior is in agreement with X-ray data. In fact, the average B-factors for the heavy atoms

Conformational Changes in Protein Disulfide Oxidoreductase

research articles

for the first six residues 1-6 in the N-terminal segment and for the last six residues in the C-terminal segment are 52.9 Å2 and 32.0 Å2, respectively.

the negative charges of the adsorbed detergent could induce the formation of helical elements in which the negative charges would be exposed to the solvent on the surface of the helices. Moreover, the repulsion between the negative charges of adsorbed SDS is probably the cause of the lack of protein aggregation at high temperatures.

4. Discussion Insufficient information is available on protein disulfide oxidoreductases from archaea to define their physiological function(s) with any certainty and the inadequacy of our current knowledge leads us still to search for their function. Recent computational genomics study argue for the existence of intracellular bonds within several themophilic archaeal and bacterial intracellular proteins. The pattern of disulfide abundance in the tree of life asserts that these intracellular disulfide bonds play a role in thermostability,28 highlighting the importance of the protein disulfide oxidoreductase system in these microorganisms. Pyrococcus species inhabit environments with extremely high temperatures such as undersea hot vents. Optimal growth conditions include a pH level of about 7.0, a salt concentration around 2.5%, and a temperature around 98 °C. Studying Pyrococcus helps give insight to possible mechanisms used to endure extreme environmental conditions such as high temperatures and high pressure. In addition, the protein disulfide oxidoreductase from P. furiosus may give an important contribution toward understanding the function of protein disulfide oxidoreductases in hyperthermophilic archaea. Functional studies have showed that PfPDO is able to catalyze the oxidation of dithiols, as well as the reduction and the rearrangement of disulfides. Sitedirected mutagenesis experiments have demonstrated that the active site at C-terminal is basic for oxidative and reductive activities and that the two units do not seem to be functionally independent in the refolding the scrambled RNase.9 In addition, the resolution of the three-dimensional structure revealed that PfPDO resembles eukaryotic PDI as it has two thioredoxin-like motifs 6. In this paper, we described the effect of several environmental factor such a pH, temperature and SDS, on the structure and stability of PfPDO. Structural data obtained at different pDs highlighted the different thermostability of R-helices with respect to β-sheets. In particular, at pD 7.0 the R-helices are less thermostable than β-sheets, whereas at pD 5.8, the thermostability of the two secondary structural elements is similar. Loss of R-helices was observed at pD 10.0 and at 20 °C, whereas at high temperature and pD 10.0, the experiments revealed a peculiar behavior of β-sheets suggesting a structural reorganization of these secondary structural elements. Although the PfPDO thermostability was differently affected at different pDs, it was not possible to obtain a fully denatured state of the protein that instead was achieved in the presence of appropriate amount of SDS. The lower thermostability of R-helices than β-sheets was also observed in the presence of detergent. At 0.5% or higher SDS concentration and at 20 °C the infrared spectra revealed a significant decrease in intensity of the R-helix band, whereas the decrease in intensity of β-sheet band occurred significantly at 0.7% SDS, and at 4% SDS concentration, the protein lost markedly both R-helices and β-sheets. The formation of a broad band observed at high temperature and in the presence of 0.7% or 4% SDS has been described as the possible combination of absorptions due to unordered structures and R-helices.22 The latter structure might be formed as a consequence of the interaction of some unfolded protein stretches with SDS. The repulsion between

The property of PfPDO to bind and hydrolyze ATP supports its correlation to PDI.29 In fact, an ATP binding site and an ATPase activity related to its chaperone role have been reported in PDI.30 When assayed in the pH range 4.0-10.0, PfPDO catalyzed hydrolysis of ATP with a maximum around basic values (not far from its physiological pH value). Assays performed in the temperature range 30°-90 °C showed that at 90 °C PfPDO is still fully able to hydrolyze ATP. Interestingly, FT-IR experiments conducted at pD 10.0 showed a protein structural reorganization at that value of pD, with the shift of the β-sheet band, observed between 90 and 99.5 °C accompanied first by a decrease, and then by an increase in intensity of the band. The FTIR and computational results may suggest that the protein undergoes partially unfolding with a concomitant relaxation of the tertiary structure, followed by a reorganization of part of the structure in a new β-conformation. This reorganization could explain the maintenance of the ATPase activity for PfPDO. The ATPase activity does not seem to be linked to the isomerase or redox activities. This is in full agreement with a report stating that the site of phosphorylation, and thus probably the ATPase active site, lies somewhere within the central domain of the PDI,31 and that this site is far away from the redox active sites in the sequence. Furthermore, the measurements of the rates of PDI-catalyzed refolding of scrambled RNase A, in the absence or in the presence of ATP, show that ATP has little or no effect on this activity. In addition, the computational analysis underlines that the R-helix regions are more sensible to increase of pH and temperature and pH with respect to the β-sheets. These findings suggest that R-helical may be the regions of the protein that first unfold with extension of β-sheets. Moreover, simulation results are in agreement with crystallographic data where a larger mobility of the N-terminal segment is observed with respect to the C-terminal segment. Furthermore, there is also a good correlation between simulation and biochemical experiments because the C terminal active site, fundamental for the protein disulfide oxidoreductase activity, shows a reduced flexibility at 293 K and at all pH values examined, while at 373 K the flexibility increases with the pH, with a high value at pH 7.0, the optimal value for different activities of PfPDO. These results show that simulation procedures and FTIR techniques can be used all together to correlate biochemical data with structural information. Abbreviations. PfPDO, Pyrococcus furiosus protein disulfide oxidoreductase; FT-IR, Fourier transform infrared; Amide I′, amide I in D2O medium; SDS, sodium dodecil sulfate; MD, Molecular Dynamics.

Acknowledgment. This work was supported by a grant from Universita` Politecnica delle Marche (F. T., E. B.) and by grants from MIUR (PRIN 2002 and PRIN 2003). References (1) Guagliardi, A.; Nobile, V.; Bartolucci, S.; Rossi, M. A thioredoxin from the estreme thermophilic archaeon Sulfolobus solfataricus. Int. J. Biochem. 1994, 26, 375-380.

Journal of Proteome Research • Vol. 4, No. 6, 2005 1979

research articles (2) Kashima, Y.; Ishikawa, K. A hyperthermostable novel proteindisulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii. Arch. Biochem. Biophys. 2003, 418, 179-185. (3) Guagliardi, A.; de Pascale, D.; Cannio, R.; Nobile, V.; Bartolucci, S.; Rossi, M. The purification, cloning, and high level expression of a glutaredoxin-like protein fom the hyperthermophilic archaeon Pyrococcus furiosus. J. Biol. Chem. 1995, 270, 5748-5755. (4) Bartolucci, S.; De Pascale, D.; Rossi, M. Protein disulfide oxidoreductase from Pyrococcus furiosus: biochemical properties. Methods Enzymol. 2001, 334, 62-73. (5) Ren, B.; Tibbelin, G.; De Pascale, D.; Rossi, M.; Bartolucci, S.; Ladenstein, R. Crystallization and Preliminary X-ray Structure Analysis of a Hyperthermostable Thioltransferase from the Archaeon Pyrococcus furiosus J. Struct. Biol. 1997, 119, 1-5. (6) Ren, B.; Tibbelin, G.; De Pascale, D.; Rossi, M.; Bartolucci, S.; Ladenstein, R. A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units. Nat. Struct. Biol. 1998, 5, 602-611. (7) Ren, B.; Ladenstein, R. Protein disulfide oxidoreductase from Pyrococcus furiosus: structural properties. Methods Enzymol. 2001, 334, 74-88. (8) Freedman, R. B. Novel disulfide oxidoreductase in search of a function Nat. Struct. Biol. 1998, 5, 531-532. (9) Pedone, E.; Ren, B.;, Ladenstein, R.; Rossi, M.; Bartolucci, S. Functional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a member of a novel protein family related to protein disulfide-isomerase. Eur. J. Biochem. 2004, 271, 3437-3448. (10) Salomaa, P.; Schaleger, L. L.; Long, F. A. Solvent deuterium isotope effects on acid-base equilibria. J. Am. Chem. Soc. 1964, 86, 1-7. (11) Bellamy, L. J., Ed. The Infrared Spectra of Complex Molecules; Chapman and Hall: London, 1975. (12) Tanfani, F.; Galeazzi, T.; Curatola, G.; Bertoli, E.; Ferretti, G. Reduced beta-strand content in apoprotein B-100 in smaller and denser low-density lipoprotein subclasses as probed by Fourier transform infrared spectroscopy. Biochem. J. 1997, 322, 765-769. (13) Banuelos, S.; Arrondo, J. L. R.; Goni, F. M.; Pitaf, G. Surface-core relationships in human low-density lipoprotein as studied by infrared spectroscopy. J. Biol. Chem. 1995, 270, 9192-9196. (14) Hagler, A. T.; Dauber, P.; Lifson, S. Consistent force field studies of intermolecular forces in hydrogen bonded crystals. III. The CdO‚‚‚H-O hydrogen bond and the analysis of the energetics and packing of carboxylic acids. J. Am. Chem. Soc. 1979, 101, 5131-5141. (15) Hagler, A. T.; Lifson, S.; Dauber, P. Consistent force field studies of intermolecular forces in hydrogen bonded crystals. II. A benchmark for the objective comparison of alternative force fields. J. Am. Chem. Soc. 1979, 101, 5122-5130. (16) Hagler, A. T.; Stern, P. S.; Sharon, R.; Becker, J. M.; Naider, F. Computer simulation of the conformational properties of oligopeptides. Comparison of theoretical methods and analysis of experimental results. J. Am. Chem. Soc. 1979, 101, 68426852.

1980

Journal of Proteome Research • Vol. 4, No. 6, 2005

Pedone et al. (17) Brooks, C. L., III; Montgomery, Pettitt, B.; Karplus, M. in Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics; John Wiley and Sons: New York, 1988. (18) Saviano, M.; Aida, M.; Corongiu, G. Molecular dynamics simulation in vacuo and in solution of cyclolinopeptide A: a conformational study. Biopolymers 1991, 31, 1017-1024. (19) Arrondo, J. L. R.; Muga, A.; Castresana, J.; Gon ˜ i, F. M. Quantitative studies of the structure of proteins in solutions by Fourier transform infrared spectroscopy. Prog. Biophys. Mol. Biol. 1993, 59, 23-56. (20) Krimm, S.; Bandekar, J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv. Protein Chem. 1986, 38, 181-364. (21) Osborne, H. B.; Nabedryk-Viala, E. Infrared measurements of peptide hydrogen exchange in rhodopsin. Methods Enzymol. 1982, 88, 676-680. (22) D’Auria, S.; Barone, R.; Rossi, M.; Nucci, R.; Barone, G.; Fessas, D.; Bertoli, E.; Tanfani, F. Effects of temperature and SDS on the structure of β-glycosidase from the thermophilic archaeon Sulfolobus solfataricus. Biochem. J. 1997, 323, 833-840. (23) Chirgadze, Y. N.; Fedorow, O. W.; Trushina, N. P. Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water. Biopolymers 1975, 14, 679694. (24) Barth, A.; Zsherp, C. What vibrations tell us about proteins. Quart. Rev. Biophys. 2002, 35, 369-430. (25) Pedone, E.; Bartolucci, S.; Rossi, M.; Pierfederici, F. M.; Scire`, A.; Cacciamani, T.; Tanfani, F. Structural and thermal stability analysis of Escherichia coli and Alicyclobacillus acidocaldarius thioredoxin revealed a molten globule-like state in the thermal denaturation pathway of the proteins: An infrared spectroscopic study. Biochem. J. 2003, 373, 875-883. (26) Gogliettino, M. A.; Tanfani, F.; Scire`, A.; Ursby, T.; Adinolfi, B. S.; Bertoli, E.; De Vendittis, E. The role of Tyr41 and His155 in the functional properties of superoxide dismutase from the Archaeon Sulfolobus solfataricus. Biochemistry 2004, 43, 2199-2208. (27) Wei, G.; Tingting, W.; Jiang, Z.; Yunyu, S.; Haiyan, L.; Molecular dynamics simulation of the unfolding of the human prion protein domain under low pH and high-temperature conditions. Biophys. Chem. 2003, 104, 79-94. (28) Mallick, P.; Boutz, D. R.; Eisenberg, D.; Yeates, T. O. Genomic evidence that the intracellular proteins of archaeal microbes contain disulfide bonds. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9679-9684. (29) Quemeneur, E.; Guthapfel, R.; Gueguen, P. A major phosphoprotein of the endoplasmic reticulum is protein disulfide isomerase. J. Biol. Chem. 1994, 269, 5485-5488. (30) Guthapfel, R.; Gueguen, P.; Quemeneur, E. ATP binding and hydrolysis by the multifunctional protein disulfide isomerase. J. Biol. Chem. 1996, 271, 2663-2666. (31) Zapun, A.; Creighton, T. E.; Rowling, P. J.; Freedman, R. B. Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum. Proteins 1992, 14, 10-15.

PR050152Z