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Pressure Effects on the Structure and Stability of the Hyperthermophilic Trehalose/ Maltose-Binding Protein from Thermococcus litoralis Stephane Marchal,† Maria Staiano,‡ Anna Marabotti,§ Annalisa Vitale,‡ Antonio Varriale,‡ Reinhard Lange,† and Sabato D’Auria*,‡ INSERM, U710, Montpellier, F-34095, France. UniVersity Montpellier 2, Montpellier, F-34095, France, EPHE, Paris, F-75007, France, Laboratory for Molecular Sensing, IBP-CNR, Naples, Italy, Laboratory of Bioinformatics, ISA-CNR, AVellino, Italy ReceiVed: May 27, 2009; ReVised Manuscript ReceiVed: July 21, 2009
In this work, we investigated the effect of pressure on the structure and stability of the recombinant D-trehalose/ D-maltose-binding protein isolated from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). The spectroscopic results obtained both in the absence and in the presence of maltose or trehalose revealed that the TMBP-Mal complex exhibits a larger structural stability under high pressure values than TMBP-Tre complex. In addition, the results also pointed out that pressure induces reversible denaturation transitions of the protein structure. By combining the fluorescence results obtained with 8-anilino-1-naphtalene sulfonate as extrinsic probe and the intrinsic indolic fluorescence of TMBP, it is evident that the protein structural changes above 400 MPa that involve the exposure to the solvent of a large portion of the hydrophobic protein domains are preceded by a partially unfolded protein structural state. The spectroscopic results have been interpreted and discussed by taking into account the X-ray structure of the protein and, in particular, the interactions of maltose and trehalose within the three-dimensional structure of TMBP. Introduction The D-trehalose/D-maltose-binding protein (TMBP) is one component of the trehalose (Tre) and maltose (Mal) uptake system that is mediated by a protein-dependent ATP-binding cassette (ABC) transporter in the hyperthermophilic archaeon Thermococcus litoralis.1,2 TMBP from T. litoralis is a monomeric 48 kDa macromolecule formed by two globular domains connected by a hinge region made of three short polypeptide segments. The two domains are made up of noncontiguous polypeptide stretches and exhibit a similar tertiary structure. The sugar-binding site is located in the deep cleft between the two domains, and the binding of sugar is accompanied by a movement of the two lobes as well as by conformational changes in the protein hinge region.3 These structural motifs are common to a number of other sugar-binding proteins.4-10 The recombinant TMBP from T. litoralis is highly thermostable and binds both trehalose and maltose with a Kd of 0.160 µM for both sugars.2 Ligand-binding proteins from protein-dependent ABC transport systems are good candidates for the design of highly specific fluorescence biosensors for small analytes.11 In particular, a sensor using TMBP as biological recognition element would have excellent stability and shelf life, and the presence of 12 tryptophan residues in this protein would allow their use as an intrinsic fluorescence probe. In fact, due to the high sensitivity of indole emission to local interactions and variations in microenvironment, structural fluctuations perturbing Trp fluorescence characteristics can be related to local events near or involving the fluorescence probe and its immediate environment * Corresponding author. Address: Laboratory for Molecular Sensing, IBPCNR, Via P. Castellino, 111, 80131 Napoli, Italy. Phone: +39-0816132250. Fax: +39-0816132277. E-mail:
[email protected]. † INSERM, University Montpellier 2, EPHE. ‡ Laboratory for Molecular Sensing. § Laboratory of Bioinformatics.
or to slower processes involving the entire protein structure.12 Moreover, since both maltose and trehalose are composed of two glucose subunits, it is possible to hypothesize that targeted modifications of this protein will help in the design of a new fluorescence biosensor for diabetic patients. When planning biotechnological applications involving modifications or labeling of proteins, a detailed knowledge of their structural properties, such as structural stability, and dynamic conformations in solution is of high interest. In fact, investigations of the resistance of proteins to physicochemical stresses such as high temperature and pressure are needed to design and realize stable biosensors able to work for a long time at the operative required conditions. This is especially true when developing implantable biosensors for the real-time monitoring of biological fluids, since implantable devices need to be sterilized before being inserted into the human body. The application of nonthermal processes as an alternative or together with conventional preservation methods can enhance their global antimicrobial effect.13 As a consequence, it is of interest to know if the biologic element of the sensor is able to resist these extreme treatments, such as the exposure at high pressure values. Hydrostatic pressure is known to modulate various biochemical processes, including ligand-protein interactions, viability of viral particles, velocity of enzyme reactions, and protein structure and dynamics.14-17 Some details on how high pressure can impact protein stability have been highlighted in the past.18,19 In a previous work,20 some of us studied how temperature can affect structural features of TMBP in the absence and in the presence of trehalose or maltose. In this work, we extended the characterization of the structural features of TMBP upon application of hydrostatic pressure. In particular, we investigated the effects of pressure on the stability of TMBP in the absence and in the presence of trehalose or maltose by steady-state fluorescence spectroscopy experiments. The obtained results were discussed by taking into account a
10.1021/jp904973y CCC: $40.75 2009 American Chemical Society Published on Web 08/27/2009
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TABLE 1: Distances between the Centroids of Sugars and of Aromatic Residues in TMBP trehalose ring residue TMBP distance (Å) % relative accessibility Glc 1
Glc 2
W73 Y177 W257 W295 Y121 Y259 W331
9.15 8.31 4.13 5.94 7.12 6.31 8.58
4.2 13.5 10.0 10.0 6.2 7.6 0.5
Glc 1
Glc 2
detailed inspection of the interactions of the substrates within the three-dimensional structure of the protein. Materials and Methods Materials. D-Maltose and D-trehalose were purchased from Sigma, deuterium oxide (99.9% 2H2O), 2HCl and NaO2H were purchased from Aldrich. All other chemicals were commercial samples of the best available quality. Purification of TMBP and Protein Concentration Determination. The Escherichia coli strain ready for the expression of the recombinant TMBP was produced as described.3 Twenty grams cell wet weight of the E. coli cell pellet containing the expressed TMBP was resuspended in 50 mM Tris-HCl, pH 7.5 and 500 mM NaCl (100 mL), ruptured by a French pressure cell at 16 000 psi, and centrifuged for 15 min at 19000g. The supernatant was heated to 90 °C for 20 min and centrifuged for 15 min at 19000g. The supernatant was collected and dialyzed for 12 h against 10 mM Tris-HCl, pH 7.5, at 4 °C. The solution was loaded onto a DEAE column previously equilibrated in 10 mM Tris-HCl, pH 7.5. After washing the column with 10 mM Tris-HCl, pH 7.5, TMBP was eluted by a NaCl gradient (0.0 M-1.0 M NaCl). Centricon 30 concentrator (Amicon) was used to concentrate and to dialyze TMBP against 10 mM Tris-HCl, pH 7.5. The purified protein was treated with a streptomycin sulfate step, and thereafter, the protein solution was passed through a Blue A affinity column (Amersham) to obtain DNAfree TMBP. The purity of TMBP was verified by SDS-PAGE and absorption spectra. The protein concentration was determined by the method of Bradford21 with bovine serum albumin as standard on a double-beam Cary 1E spectrophotometer (Varian, Mulgrade, Victoria, Australia). Pressure Experiments. TMBP was rather resistant to pressure between neutral pH and pH 9.5, where an increase in the pressure to 600 MPa led to only partial unfolding. A complete unfolding could be observed only at pH 10. Therefore, all experiments were carried out in 0.1 M glycine buffer at pH 10. Control experiments showed that folding/unfolding was rapid and complete within a few minutes after each change in pressure. Accordingly, samples were incubated for 5 min at each pressure prior to spectral recording. The fluorescence experiments were carried out at 25 °C using an SLM series 2 luminescence spectrometer (Aminco Bowman) modified to accommodate a high pressure cell. The excitation wavelength was set to 295 nm (4 nm slit). Emission spectra (8 nm slit) between 300 and 400 nm were recorded as the mean of 3 accumulations. The spectral changes were quantified by determining the center of spectral mass (csm) 〈ν〉:
〈Vp〉 )
∑ (Vi × Fi)/ ∑ Fi
maltose ring residue TMBP distance (Å) % relative accessibility
(1)
where Fi is the intensity of fluorescence emitted at wavenumber νi. This parameter reflects the mean exposure of tryptophan residues to water.22,23 8-Anilino-1-naphtalene sulfonate (ANS)
W73 Y177 W257 W295 Y121 Y259 W331
8.37 7.90 4.73 5.50 7.30 5.50 8.89
2.4 7.8 0 0 2.9 0.7 0.6
binding was measured upon excitation at 350 nm (4 nm slit), and emission spectra (8 nm slit) were recorded from 400 to 600 nm. The concentrations were 0.1 mg/mL protein, 200 µM ANS, and 2.6 mM substrate. The thermodynamic parameters of pressure-induced spectral changes were determined by fitting csm values to two- or threestate transitions, as previously described.24 Results and Discussion Analysis of 3D TMBP Structure. The analysis of the interactions between TMBP and the two sugars trehalose (Tre) and maltose (Mal) was made, respectively, on the structure of the complex TMBP/Tre obtained by X-ray crystallography3 and freely available in the Protein Data Bank25 (PDB code: 1EU8) and on the structure of the complex TMBP/Mal obtained with a docking procedure as described in our previous work.20 The tools of the program Insight II (version 2000.1, 2000; Accelrys) were used to evaluate the interactions between the sugar and the residues belonging to the active site of the protein. Residues with at least one atom within 3.50 Å distance from each oxygen atom of the sugars were taken into account to evaluate the contacts between amino acids and sugars. The TMBP sugar-binding site is located in the cleft between the two domains of the protein.3 The cavity is formed by both hydrophobic and polar residues to allow the accommodation of the apolar moiety of the sugar and also to provide the formation of H-bonds between the oxygen atoms of the sugar and polar amino acids. W295 and W257 form interesting stacking interactions with the Glc1 ring of both sugars, whereas the rings of W73 and of Y177 are farther distant from Tre than from Mal. The phenolic rings of Y259 and Y121 lie in proximity of Glc2 in both sugars, whereas the indole moiety of W331 is closer to Tre (Table 1). Other apolar residues forming the walls of the binding site are L292, G293, G294 and P258. We also made an evaluation of the solvent accessibility of aromatic residues near the binding site, in both complexes, using NACCESS.26 The results reported in Table 1 show that, in general, these residues are more exposed to solvent in the TMBP/Tre complex than in the TMBP/Mal complex. The main variation is visible for W257 and W295, the two principal amino acid residues forming stacking interactions with the sugar rings. They are completely shielded from solvent in TMBP/Mal but partially exposed in TMBP/Tre. The pattern of polar interactions between the protein and Mal appears also to be different from that of Tre. This is due both to the variation of the conformation of the amino acid residues when maltose is present in the binding site of TMBP and to the variation of the position of the rings. In fact, the Glc2 moiety of this sugar is in a similar position as the corresponding ring in Tre, whereas the Glc1 moiety is differently located in the protein binding site (Figure 1). From the analysis of polar contacts, it can be deduced that, with a few exceptions, the amino acid residues able to form
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Figure 1. Close-up of the binding pocket of TMBP with trehalose (A) and maltose (B). The Tre is cyan, the Mal is pink. Residues interacting with the substrates are in color atom code: carbon, green; oxygen, red; nitrogen, blue; sulfur, yellow; and labeled.
polar contacts are the same in both complexes. In addition, some amino acid residues, such as G294, R49, W257, and T46, interact only within the TMBP/Tre complex, and some other residues, such as C182, E178, and G179, interact only within the TMBP/Mal complex. In particular, for TMBP/Tre, R49 interacts with the O3′ atom in Tre. This atom corresponds to the O3 atom of Mal, but the different position of the ring in this last sugar impairs the formation of this contact. W257 is able to form an H-bond with O5 of Tre, and this interaction is missed in Mal, where the corresponding atom is a carbon; however, this residue is still able to interact with this sugar by means of hydrophobic interactions, as described above. In the TMBP/Mal complex, C182 interacts with atom O1′ of the Mal in the Glc1 moiety. This atom corresponds to atom O4 in Tre, which cannot interact with the same residue because the position of the ring is different. E178 interacts with oxygen O2′ of the Mal by means of its backbone oxygen; the same interaction with trehalose is not possible, since in the corresponding position, there is a carbon atom. The analysis of the binding of Tre and Mal to TMBP shows that, despite the similarity of the two sugars, their interactions within the two complexes are rather different and could also affect the exposure of Trp residues to solvent. Although it is hard to find a direct relationship between these observations and the following fluorescence results that reflect the structural state of the entire protein, it is clear that the different interactions of the two sugars with TMBP modulate the responsiveness of this protein to physicochemical stress. Pressure-Induced TMBP Unfolding. In a previous work, some of us showed that at pH 7.0, the TMBP structure was not affected by temperature increases up to 100 °C. In fact, to obtain information on the temperature-induced denaturation of the structure of TMBP, the experiments were performed with TMBP at pH values between 9.0 and 10.0.27 TMBP is still correctly folded in this range of pH values and is still able to bind the sugars with the same affinity.27 In the present study, we experienced the same high stability of TMBP at pH 7.0 toward high pressure values exposure. In fact, no structural changes of TMBP were monitored up to 400 MPa (data not shown). To obtain structural data upon exposure of TMBP at high pressures, the experiments performed in this
Figure 2. Effects of pressure on conformational stability of TMBP at 25 °C and at pH 10. (A) Fluorescence spectra under compression; (B) pressure-induced decrease of fluorescence maxima with increasing (solid circles) or decreasing (open circles) pressure; (C) pressure-induced changes in polarity of tryptophan environment as evidenced by plots of csm values versus pressure under compression (solid circles) and decompression (open circles), respectively; (D) binding of ANS, compression (solid circles), decompression (open circles).
work were performed at pH 10.0. As shown in Figure 2A, hydrostatic pressure induced tryptophan fluorescence quenching. Concomitantly, a pressure-dependent red shift is observed above 400 MPa. The combination of these two effects makes it difficult to interpret the plot of fluorescence intensity as a function of pressure (Figure 2B). This difficulty is further increased by the well-known intrinsic pressure dependence of tryptophan fluorescence efficiency, which has been found to depend on the physicochemical parameters of the solution, such as osmolarity and polarity of solutes and the pH of the solvent.28 An alternative, more suitable method of analysis is based on the quantification of the fluorescence spectral change by calculating the center of spectral mass (csm), which is related to the mean exposure of protein tryptophan residues to water (see the Materials and Methods section). Using this approach, the pressure-induced unfolding can be described as a multistep
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TABLE 2: Thermodynamic Parameters Calculated from Pressure Unfolding and Refolding Curves of TMBP at 25°C and at pH 10a A: under compression
TMBP TMBP/Tre TMBP/Mal TMBP/Glc (1) TMBP/Glc (2) a
∆V1, ml · mol-1
∆Gu1, kJ · mol-1
-33.7 ( 2.1 -15.3 ( 0.4
7.6 ( 0.6 4.4 ( 0.2
-21.4 ( 2.4 -55.5 ( 8.0
6.7 ( 0.4 113 ( 1.8
P1/2, MPa
B: under decompression ∆V2, ml · mol-1
Unfolding 230 -55.4 ( 2.1 287 -187.4 ( 3.2 -90.3 ( 5.1 313 -81.7 ( 6.6 204 -60.8 ( 2.1
∆Gu2, kJ · mol-1
P1/2, MPa
28.2 ( 0.9 95.4 ( 3.2 48.6 ( 2.6 38.3 ( 3.0 28.3 ( 0.9
509 509 536 468 465
TMBP TMBP/Tre TMBP/Mal TMBP/Glc (1) TMBP/Glc (2)
∆V, ml · mol-1
∆GF, kJ · mol-1
P1/2, MPa
Refolding -32.2 ( 2.3 -147.2 ( 8.2 -31.7 ( 1.0 -53.2 ( 5.2 -60.0 ( 5.4
9.7 ( 0.7 86.8 ( 6.9 10.7 ( 0.3 12.6 ( 1.4 15.4 ( 1.5
301 318 337 237 256
Glucose (Glc) was used at concentrations of 0.5 mM (1) and 10 mM (2).
transition (Figure 2C). Data were accurately fitted to three- and two-state models for compression and decompression, respectively. The thermodynamic parameters of the unfolding and refolding reactions are listed in Table 2. From the csm analysis (see Figure 2C), a reversible pressure-induced protein unfolding process with a hysteresis behavior under decompression was expected. However, the initial and final fluorescence profiles were not superimposable, and upon decompression, the initial fluorescence intensity at the maximum wavelength (336 nm) was not recovered (Figure 2B). This indicates a partial loss of soluble structured protein as a consequence of the pressure treatment. However, successive compression-decompression cycles gave rise to superimposable csm profiles (data not shown). This indicates that the remaining fluorescence protein stayed intact and recovered its native structure after decompression. For understanding the nature of these structural changes, we analyzed the pressure-induced unfolding of TMBP by using the extrinsic fluorescence probe ANS, an environmentally sensitive fluorophore that binds to water-exposed large structured hydrophobic domains of proteins. As shown in Figure 2D, TMBP bound ANS molecules only at pressures higher than 400 MPa; that is, in the same pressure range in which we observed TMBP unfolding by monitoring the protein intrinsic tryptophan fluorescence (Figure 2B). Again, hysteresis behavior was observed, indicating incomplete equilibrium during refolding on the time scale of experiments due to slow isomerization processes.29-31 Effects of Substrate Binding on Protein Stability under Pressure. As already published,32 different conformations of TMBP were detected whether trehalose or maltose is bound to the protein. Here, we investigated the effect of these sugars on the stability of TMBP under hydrostatic pressure. Experiments were performed at 25 °C and pH 10.0 in the presence of 2.6 mM substrate. As already observed, binary protein-substrate complexes showed different fluorescence properties. A spectral blue shift and a quenching of fluorescence were observed in the presence of Tre, whereas binding of Mal to TMBP induced mainly a 20% increase in fluorescence intensity and a 1.0 nm red shift of the maximum emission wavelength (Figure 3A). The different changes in fluorescence with maltose (increasing) and trehalose (decreasing) indicate that maltose is bound to TMBP such that the nonreducing glucose moiety (Glc2) is located remotely from Trp295. This placement can explain both the quenching of the fluorescence in TMBP by the van der Waals contact of Glc2 of trehalose with Trp295 and the absence of quenching by maltose.3 The pressure-induced unfolding of protein/substrate complexes was reversible, but a hysteresis phenomenon was also observed for decompression (data not shown). As shown in Figure 3B, the csm profiles of the transitions of protein and ANS fluorescence, obtained in the presence of Tre, were
Figure 3. Pressure-induced unfolding of TMBP in the absence and in the presence of substrate. (A) Fluorescence spectra of TMBP in the absence of substrate (s), TMBP in the presence of 2.5 mM Tre ( · · · ), TMBP in the presence of 2.5 mM Mal (- -) at 10 MPa. The maximum emission wavelength is reported. (B) The center of the spectral mass of tryptophan fluorescence (solid symbols) and ANS binding (open symbols) were used to study the effects of Tre (squares) and mal (triangles) on the conformational stability of TMBP as a function of pressure. For comparison, experimental data obtained for TMBP in the absence of substrate were included in the figure (circles).
qualitatively similar to that of the protein in the absence of substrate. The absolute differences in csm values are readily explained by the blue-shifted emission maximum of the Tre/ TMBP complex at atmospheric pressure values. However, the half-transition pressure values, p1/2, determined from ∆G0/∆V were similar to those exhibited by TMBP in the absence of substrate (Table 2). In contrast to Tre/TMBP complex, the Mal/ TMBP complex underwent a transition that could be ascribed as a two-state model with the unfolding process occurring only at high pressure values. In addition, Tre/TMBP complex also exhibited a relatively larger stability under pressure, as evaluated
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Marchal et al. phobic stabilization of the core of the protein, with an ultimate positive effect in terms of global entropy. Acknowledgment. The authors thank Dr. Fabienne Chevance, University of Utah, Salt Lake City, UT; and Prof. Winfried Boos, University of Konstanz, Germany, for supplying the E. coli strain ready for the expression of the recombinant TMBP. This work was realized in the frame of the CNR Commessa “Progettazione e Sviluppo di Biochip per la Sicurezza Alimentare e Salute Umana” (S.D., M.S.). This work has been also made in the frame of the CNR-Bioinformatics project. References and Notes
Figure 4. Concentration-dependent effects of monosaccharides on the structural stability of TMBP under pressure. The csm deviation as a function of pressure is represented by circles for TMBP alone (circles) and triangles, up and down, for TMBP in the presence of 0.5 mM and 10 mM glucose, respectively.
by the p1/2 value (536 MPa), as compared to that showed by TMBP in the absence of substrate (at 509 MPa). Recent studies on TMBP pointed out that at room temperature, TMBP is able to bind glucose.30 In this work, we extended our investigation to the effect of glucose on TMBP structure at different pressure values. In particular, we tested the protein stability under high pressure values in the presence of glucose. As shown in Figure 4 and Table 2, significant differences of the unfolding transitions of TMBP were observed. In particular, at lower pressure values, the unfolding profiles showed a decrease in p1/2 as a function of glucose concentration, indicating a marked sensitivity of the first unfolding step to substrateinduced structural changes (Table 2). At higher pressure values (the second unfolding step), this effect was less evident. In conclusion, the intrinsic tryptophan fluorescence of TMBP in the absence and in the presence of substrate revealed that high pressure values induce reversible structural transitions of TMBP to an unfolded state. These transitions, occurring at high pressure values (509 MPa for TMBP in the absence of substrate), reflect not only changes in the local environments of the tryptophan residues of TMBP but also the exposure of TMBP large hydrophobic domains. It is noteworthy that an additional pressure-induced TMBP conformational change of small spectral amplitude that is indicative of the presence of unfolded intermediate species was detected at lower pressure values, between 200 and 300 MPa. However, this TMBP structural variation disappeared when the protein was bound to Mal. Even if the specific effect of the interaction between Mal and TMBP is still unclear, it is possible to hypothesize that the observed differences in the behavior exhibited by TMBP are due to the different interactions between Mal or Tre in the binding site of TMBP. In fact, from a detailed inspection of the TMBP/Tre and TMBP/Mal structures, it appears that there are several aromatic residues of TMBP that form hydrophobic and stacking interactions with the rings of Mal: In the TMBP/Mal complex, these interactions are more shielded from the solvent than in the TMBP/Tre complex. This could result in a highly hydro-
(1) Xavier, K. B.; Martins, L. O.; Peist, R.; Kossmann, M.; Boos, W.; Santos, H. J. Bacteriol. 1996, 178, 4773. (2) Horlacher, R.; Xavier, K. B.; Santos, H.; DiRuggiero, J.; Kossmann, M.; Boos, W. J. Bacteriol. 1998, 180, 680. (3) Diez, J.; Diederichs, K.; Greller, G.; Horlacher, R.; Boos, W.; Welte, W. J. Mol. Biol. 2001, 305, 905. (4) Mowbray, S. L.; Smith, R. D.; Cole, L. B. Receptor 1990-1991, 1, 41. (5) Zou, J. Y.; Flocco, M. M.; Mowbray, S. L. J. Mol. Biol. 1993, 233, 739. (6) Quiocho, F. A.; Spurlino, J. C.; Rodseth, L. E. Structure 1997, 5, 997. (7) D’Auria, S.; Nucci, R.; Rossi, M.; Gryczynsky, I.; Gryczynsky, Z.; Lakowicz, J. R. Biophys. Chem. 1999, 81, 23. (8) Evdokimov, A. G.; Anderson, D. E.; Routzahn, K. M.; Waugh, D. S. J. Mol. Biol. 2001, 305, 891. (9) Marabotti, A.; D’Auria, S.; Rossi, M.; Facchiano, A. M. Biochem. J. 2004, 380, 677. (10) D’Auria, S.; Alfieri, F.; Staiano, M.; Pelella, F.; Rossi, M.; Scire´, A.; Tanfani, F.; Bertoli, E.; Grycznyski, Z.; Lakowicz, J. R. Biotechnol. Prog. 2004, 20, 330. (11) D’Auria, S.; Lakowicz, J. R. Curr. Opin. Biotechnol. 2001, 12, 99. (12) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed; Kluwer Academic/Plenum Publishers: New York, 1999; p 698. (13) Ross, A. I.; Griffiths, M. W.; Mittal, G. S.; Deeth, H. C. Int. J. Food Microbiol. 2003, 89, 125. (14) Silva, J. L.; Luan, P.; Glaser, M.; Voss, E. W.; Weber, G. J. Virol. 1992, 66, 2111. (15) Jaenicke, R. Annu. ReV. Biophys. Bioeng. 1981, 10, 1. (16) Gross, M.; Jaenicke, R. Eur. J. Biochem. 1994, 221, 617. (17) Mozhaev, V. V.; Heremans, K.; Frank, J.; Masson, P.; Balny, C. Proteins 1996, 24, 81. (18) Mombelli, E.; Shehi, E.; Fusi, P.; Tortora, P. Biochim. Biophys. Acta 2002, 1595, 392. (19) Scharnagl, C.; Reif, M.; Friedrich, J. Biochim. Biophys. Acta 2005, 1749, 187–213. (20) Herman, P.; Staiano, M.; Marabotti, A.; Varriale, A.; Scire`, A.; Tanfani, F.; Vecer, J.; Rossi, M.; D’Auria, S. Proteins 2006, 63, 754. (21) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (22) Silva, J. L.; Miles, E. W.; Weber, G. Biochemistry 1986, 25, 5780– 5786. (23) Ruan, K.; Weber, G. Biochemistry 1989, 28, 2144–2153. (24) Torrent, J.; Connelly, J. P.; Coll, M. G.; Ribo, M.; Lange, R.; Vilanova, M. Biochemistry 1999, 38, 15952–15961. (25) Berman, H.; Henrick, K.; Nakamura, H.; Markley, J. L. Nucleic Acids Res. 2007, 35(Database issue), D301. (26) Hubbard, S. J.; Campbell, S. F.; Thornton, J. M. J. Mol. Biol. 1991, 220, 507. (27) Fessas, D.; Staiano, M.; Barbiroli, A.; Marabotti, A.; Schiraldi, A.; Varriale, A.; Rossi, M.; D’Auria, S. Proteins 2007, 67, 1002–1009. (28) Ruan, K.; Tian, S.; Lange, R.; Balny, C. BBRC 2000, 269, 681– 686. (29) Sinclair, J. F.; Ziegler, M. M.; Baldwin, T. O. Nat. Struct. Biol. 1994, 1, 320. (30) Fuchs, A.; Seiderer, C.; Seckler, R. Biochemistry 1991, 30, 6598. (31) Schuler, B.; Rachel, R.; Seckler, R. J. Biol. Chem. 1999, 274, 18589. (32) Herman, P.; Barvik, I., Jr.; Staiano, M.; Vitale, A.; Vecer, J.; Rossi, M.; D’Auria, S. Biochim. Biophys. Acta 2007, 1774, 540.
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