| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 CEA, Département d'Ingénierie et d'Etudes des Protéines–Bâtiment 152, C.E. Saclay, F-91191 Gif-sur-Yvette, France
2 Dipartimento di Chimica Organica e Biochimica, Università di Napoli "Federico II," 80126 Napoli, Italy
3 CEA Valrho, DSV/DIEP/SBTN, F-30207 Bagnols-sur-Cèze, France
Reprint requests to: Mireille Moutiez, CEA, Département d'Ingénierie et d'Etudes des Protéines–Bâtiment 152, C.E. Saclay, F-91191 Gif-sur-Yvette, France; e-mail: mireille.moutiez{at}cea.fr; fax: 33-1-6908-9071.
(RECEIVED December 11, 2001; FINAL REVISION March 17, 2002; ACCEPTED March 21, 2002)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4960102.
 |
Abstract |
|---|
 TOP Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Keywords: DsbA; catalytic cycle; peptide-enzyme complex; limited proteolysis; mass spectrometry
Abbreviations: Asp-N, endoproteinase Asp-N • DTT, dithiothreitol • ESIMS, electrospray ionization mass spectrometry • GdmCl, guanidinium chloride • Glu-C, endoproteinase Glu-C • IAM, iodoacetamide • MALDIMS, matrix assisted laser desorption ionization mass spectrometry • PDI, protein disulfide isomerase • PID, peptide derived from DsbB sequence • SDS, sodium dodecyl sulfate • TFA, trifluoroacetic acid • thioredoxin domain "lower" part, strands ß1, ß4, ß5, helix 
7, and the loop connecting ß5 and 7 • thioredoxin domain "upper" part, strands ß2 and ß3
|
Introduction |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
The very high oxidizing potential of DsbA is accompanied with two particular features. First, the pKa of the first cysteine of the active site, Cys30, is especially low (3–3.5) (Wunderlich and Glockshuber 1993; Nelson and Creighton 1994). At physiological pH, Cys30 is hence completely ionized, which makes it highly reactive for thiol-disulfide reactions. Second, although disulfides generally stabilize proteins, the reduced form of DsbA is much more stable than the oxidized one (Moutiez et al. 1999). Structural studies have pointed out the essential role of the electrostatic stabilization of the thiolate of Cys30 for the stability of the reduced form (Gane et al. 1995), but these same studies do not address the role of the dynamics of the protein in the activity, particularly the role of domain motions. Data still remain to be investigated to explain the different steps of the catalytic cycle: the specific binding of reduced, denatured substrates to the oxidized form; the oxidation and release of substrate; and the binding of the reduced protein to its membrane partner DsbB, followed by reoxidation and dissociation of the complex DsbA-DsbB (Bardwell et al. 1993; Bader et al. 1998).
The three-dimensional structure of DsbA can be divided into two domains: the family consensus domain that possesses a thioredoxin fold and an additional helical domain usually named B-domain. Although the structure of the oxidized enzyme has been solved since 1993 (Martin et al. 1993), the structure of the reduced form has been unavailable for a long time. In 1998, both a nuclear magnetic resonance (NMR) and an X-ray structure of reduced DsbA have been published (Guddat et al. 1998; Schirra et al. 1998). However, these structures do not bring all the expected explanations about the properties of the protein and are even contradictory on some points (e.g., the relation between domain motions and redox state).
The results presented in this paper aim at providing a description of the conformational variations associated to the different phases of the DsbA catalytic cycle. We focused on the two extreme stages of the enzyme cycle, the oxidized and the reduced forms, and on an intermediate stage, a stable complex between DsbA and a peptidic ligand. In a previous work, we have designed a stable ligand-enzyme complex in which the labile cystine bond that normally forms during the catalytic cycle has been replaced by a cysteine-homoalanine thioether bond (Couprie et al. 2000). This approach enables the direct use of the wild-type form of the protein, which is the most relevant one to obtain precise insights into the enzymatic mechanism. Moreover, the chemical stability of such a complex makes possible studies in physiological conditions of pH and temperature. As an investigative tool, we focused on a strategy that combines limited proteolysis with mass spectrometry methodologies. This procedure had been developed to probe the surface topography of proteins and to investigate interface regions in protein complexes (Zappacosta et al. 1996; Scaloni et al. 1998). Only the protein regions both exposed and flexible are accessible to proteolytic cleavages. Stereochemical barriers prevent the occurrence of cleavages within the highly structured core of the protein or at least slow down their kinetics.
The results reported here show that reduction essentially results in a loss of mobility in both domains of DsbA, which is probably a determining factor for the course of the catalytic cycle. The complex shows an intermediate flexibility between the oxidized and the reduced states, a tendency that can be connected with the results previously obtained by calorimetry (Couprie et al. 2000).
|
Results |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Topographical study of oxidized DsbA
The surface topography of oxidized DsbA was probed using the limited proteolysis/mass spectrometry approach based on the mass spectral analysis of the fragments released from DsbA by controlled proteolysis experiments. Limited proteolysis experiments were performed using trypsin, chymotrypsin, Asp-N, Glu-C, and subtilisin as proteolytic probes according to the strategy previously described (Zappacosta et al. 1996). Oxidized DsbA was incubated with each protease at pH 7.5 and 37°C with an appropriate enzyme-to-substrate ratio (see Materials and Methods). The extent of the enzymatic hydrolysis was monitored on a time-course basis by taking samples from the incubation mixture at different interval times. After quenching of the hydrolysis reaction by freezing, each aliquot was fractionated by reversed phase high-performance liquid chromatography (RP-HPLC) on a SMART system (Pharmacia). The mass values of proteolytic fragments were determined by mass spectrometric techniques with sufficient accuracy to enable unambiguous identification of the peptides, hence, the assignment of the cleavage sites in the amino acid sequence of DsbA. Only the low specificity of subtilisin made necessary further MS/MS analyses of the proteolytic fragments to confirm assignment of the peptide identity.
Special care was taken to define the appropriate conditions in terms of time of incubation and enzyme-to-substrate ratio. For the investigation of protein topography by limited proteolysis, a prerequisite is the conformational homogeneity of this protein. In the case of DsbA, the protein has been largely studied by many biophysical techniques. NMR studies (Couprie et al. 1998), chromatographic behavior, and calorimetric studies (Couprie et al. 2000) all converge on the existence of a unique monomeric conformation of DsbA in solution (within the experimental limits of detection related to each of these methods). Each protease used in this study was investigated with different ratios of protease-to-oxidized DsbA. The use of higher protease/DsbA ratios than those finally retained resulted in a sharp decrease in the selectivity of hydrolysis and a consequential increase of proteolytic fragments. Indeed, for specific proteases as well as for unspecific ones, the proportion of peptides originated from subdigestion of larger fragments greatly increased with the ratio of protease/DsbA, as already described in other studies (Zappacosta et al. 1996). On the basis of the preliminary experiments, the conditions of limited proteolysis were selected to maximize the selectivity of cleavages to preferred sites.
As an example, Figure 1
shows the HPLC chromatograms of the aliquots withdrawn at 15, 30, and 60 min of tryptic digestion. Mass spectral identification of the tryptic peptides observed within the proteolysis experiment is reported in Table 1. The occurrence of few fragments in the HPLC profiles and the presence of a large amount of undigested protein (peak 14) clearly indicated that under the experimental conditions used, the activity of the proteases was restricted to few specific sites. Three fragments rapidly appeared and were identified as peptides 184–189, 159–183, and 104–132 (peaks 1, 6, and 8, respectively), indicating the occurrence of early cleavage sites at Arg103, Lys132, Lys158, and Lys183. At longer hydrolysis time additional fragments were detected. Special attention was brought to the analysis of these new fragments to distinguish between peptides resulting from subdigestion of larger ones and fragments released from native DsbA with slower proteolysis kinetics. This analysis revealed the presence of four minor cleavage sites at Lys7, Lys48, Lys58, and Lys70, as inferred from the identification of the peptides 8–48 and 59–70 (peaks 12 and 5, respectively).
|
|
and Figures 2 and 3. DsbA proved to be highly resistant to the proteolysis regardless of the protease used as shown by the high enzyme/substrate ratio needed to observe proteolysis and by the low number of fragments released in the chromatograms. Preferential cleavage sites were classified as "major" or "minor" on qualitative evaluation according to their kinetics of appearance (at early or longer times of incubation) during the time-course experiments. Most of the major cleavages occurred in the thioredoxin domain, especially in the "lower" part (strands ß1, ß4, ß5, and helix 7). Minor cleavage sites accumulated in the "upper" part of the ß-sheet (strands ß2 and ß3) and in helix 2, a region described as the hydrophobic core of the protein (Hennecke et al. 1999). No region of the protein appeared to be really protected against proteolysis with the exception of helix 3 in which no cleavage sites could be detected.
|
|
|
Figure 4A and B show the HPLC profiles corresponding to the 60-min aliquots of the tryptic hydrolysis of the oxidized and the reduced form of DsbA, respectively. Peak identifications are reported in Table 1
. The comparison of both chromatograms indicates clearly that reduced DsbA is much more resistant to proteolysis than oxidized DsbA. The time-course analysis of the tryptic experiment on the reduced protein showed again the early occurrence of cleavages at the level of Arg103, Lys132, Lys158, and Lys183. The identification of peptides showed the increased accessibility of Lys7 as shown by the early appearance of the peptide 1–7. Conversely, all the other peptides corresponding to minor cleavage hydrolysis of the oxidized form have disappeared (peaks 12 and 5). A new cleavage site appeared at Arg148, as indicated by peak 9 (peptide 149–189), that increased at a rate comparable to peaks 1 or 8.
|
and Figure 5. Regardless of the protease used, the kinetics of appearance of proteolytic fragments were dramatically slowed down, indicating an increase in the overall compactness of the protein. In the case of the Glu-C endoproteinase hydrolysis, no fragments could even be detected with the enzyme-substrate ratio 1/20 previously used with oxidized DsbA. It was necessary to increase the ratio up to 1/10 to observe significant hydrolysis reactions during the usual experiment duration.
|
2) where a disappearance of the minor cleavage sites was observed in the reduced form. Of special interest is the disappearance of the hydrolysis site at the level of Phe129, a residue located at the hinge between helices 5 and 6, indicating a loss of flexibility in that region. It should also be noticed that proteolysis at Asp110 (helix 4) tends to be slower, whereas no cleavage was observed at the neighboring residue Phe112. Finally, all the chymotryptic cleavages occurring in strands ß4, ß5, and helix 7 appeared later in the time-course experiment, indicating a decreased accessibility of the "lower" part of the thioredoxin domain. Although most of the differences between oxidized and reduced DsbA concerned the disappearance of proteolytic sites, two new hydrolysis sites were identified in the reduced protein at Arg148, as mentioned previously, and at Glu94 where a cleavage by Glu-C was observed.
Topographical studies of the DsbA-PID complex
The experimental approach described previously was finally used to investigate the surface topography of the covalent complex between DsbA and the peptide PID (Fig. 6) as a mimic of the DsbA-substrate disulfide intermediate (Couprie et al. 2000).
|
shows the HPLC profile of the aliquot withdrawn after 60 min of tryptic hydrolysis of the DsbA-PID complex. The comparison of this profile with the chromatograms obtained for oxidized and reduced DsbA (Fig. 4A, B) indicated that the complex was resistant to proteases with a proteolytic behavior more resembling the reduced form of DsbA than the oxidized species. Analysis of the peptidic fragments indicated again the occurrence of major cleavages at residues 103, 132, 158, and 183. Moreover, as for reduced DsbA, all the peptides generated by minor cleavages occurring within the hydrophobic core of the protein were missing. Nevertheless, several features distinguished the conformation of the complex from the reduced protein: Lys7 was not recognized by proteases anymore and an increased rate of appearance of peptides resulting from cleavage after Arg148 (peptide 149–189—peak 9—and its subdigested fragment 149–183) was detected. Further information brought by this set of experiments concerned the sensitivity to proteolysis of the peptide PID itself within the protein complex. After 1-hr incubation with trypsin, most of the peptide lost the C-terminal residue, the major proteic species being henceforth the complex between full DsbA and PID 1`-13`.
The overall results of the limited proteolysis investigation of the DsbA-PID complex are reported in Table 2 and Figure 5. When these data are compared with those obtained on the oxidized and reduced forms of the protein, a number of considerations can be drawn. For all the proteases used, the HPLC profiles obtained for the proteolyzed complex are similar to those obtained for the reduced enzyme (data not shown). As for the reduced species, the minor proteolytic sites present in oxidized DsbA and mainly located in strands ß2, ß3, and helix 2 disappeared in the complex. The kinetics of appearance of most of the proteolytic fragments analyzed, as judged from the HPLC profiles, were intermediate between those observed for the oxidized and reduced form. In the case of Glu-C, as for reduced DsbA, it was necessary to increase the enzyme–substrate ratio up to 1/10 to obtain exploitable data. In addition to the differences already underlined for the tryptic experiments, other differences concerning residues Glu94, Thr168, Tyr178, and Phe129 could be inferred from the data of Table 2. Hydrolysis at the level of Glu94 by Glu-C (peptide 95–121) occurred very early in the time-course experiments, indicating that this residue is more sensitive to proteolysis than in the reduced protein. Contrary to the overall kinetic trend of the complex that seemed intermediate between those of the redox forms, hydrolysis at the level of Thr168 and Tyr178 by subtilisin was very slow. Finally, hydrolysis at the level of Phe129 that had completely disappeared in reduced DsbA occurred in the complex, although at very late stages of the experiments.
Analysis of the data obtained for the PID peptide within the complex showed a very high sensitivity of the peptide bond Arg13`-Phe14` that was easily cleaved by trypsin and in a very nonspecific manner by both chymotrypsin and Asp-N. Control experiments performed on the isolated peptide with these latter proteases did not show any cleavage at this specific position. As expected, Asp-N cleaved the isolated peptide at Asp9`, whereas chymotrypsin cleaved PID at Phe5` and Phe10`. The comparison of the proteolytic patterns obtained for isolated and complexed PID peptide indicated that Asp9` and Phe10` were protected against proteolysis in the complex. No cleavages at these sites were in fact observed when PID was complexed to DsbA, whereas hydrolysis at Phe5` remained unchanged.
|
Discussion |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
The most immediate observation for DsbA concerns the high stability of the protein. In agreement with thermodynamic measurements (Moutiez et al. 1999; Couprie et al. 2000), whatever its state, DsbA proved to be highly resistant to proteolysis. The protease to substrate ratios effective in causing the appearance of preferred predominant cleavage sites were particularly high. The kinetics of cleavage of the different forms of the enzyme globally follow the same tendency as 
G values (Couprie et al. 2000), namely the reduced form is much more stable against proteolysis than the oxidized protein, whereas the complex shows an intermediate stability.
In the conditions in which proteolysis occurs, the analysis of the preferred cleavage sites in the oxidized state shows that, although these sites are spread on most of the protein (with the exceptions previously mentioned), they are especially concentrated in the "lower" part of the thioredoxin domain (as defined in Results). They occur not only in loops but more surprisingly also in defined secondary structure elements such as helix 7 and ß-strands (ß1, ß4, and ß5). After prolonged incubation time, some cleavages also appear in the "upper" part of the ß-sheet (ß2 and ß3), a region poorly accessible to the solvent according to three-dimensional structures. In the ß-sheet, cleavage sites are found particularly at the extremities of ß-strands. The exact position of the secondary structural elements is usually somehow uncertain, and in the case of DsbA these elements have been noticed to differ sometimes by one or two residues between NMR and x-ray studies (Couprie et al. 1998). However, the finding of recurring cleavages in these regions may be more important than being merely a question of delimitation of secondary structure elements. The most likely interpretation of our data is that the whole ß-sheet region is mobile enough to enable interaction and reaction with the active site of proteases. Nevertheless, the fact that some of the observed cleavages could result from a modification of the topographical properties of the protein after a first proteolytic event cannot, of course, be ruled out. It is interesting to notice that the "lower" part of the thioredoxin domain contains most of the putative substrate-binding site (Guddat et al. 1997). The physiological role of DsbA in its oxidized state is to introduce disulfide bonds into secreted or exported proteins. The great mobility of this region in the oxidized form could facilitate the processes of recognition and binding of its large panel of proteic substrates that show various size, chemical properties, and perhaps degrees of folding.
When considering the reduced form, the kinetics of cleavage were found more or less reduced at nearly all positions. The minor cleavage sites located in ß-strands ß2 and ß3 even completely disappear. The related loss of mobility can reasonably be attributed to a tighter packing of the ß-sheet, especially in the "upper" part. We can assume that ß2 and ß3 probably have a more ordered structure resulting in stronger contacts between the strands. These observations corroborate well with the results obtained by NMR from the comparison of oxidized and reduced 1H, 15N, and 13C chemical shifts. This region is one of the four that undergoes large chemical shift variations (Couprie et al. 1998). Previous work of Frech et al. (1996) indicated that the folding of the substrate would decrease its affinity for DsbA and facilitate its dissociation from the enzyme. Our results offer new insights on the catalytic mechanism. The rigidity of the thioredoxin domain increases from the oxidized form to the reduced one, with an intermediate situation found in the complex. Although the great mobility of the oxidized form in this region probably enables accommodation of various substrates, the increasing rigidity from the complex to the reduced form could help the release of oxidized substrates. Moreover, the rigidity of the thioredoxin domain in the reduced state could be a major determinant for the specific recognition by DsbB, the DsbA oxidase, that presumably interact at least partially at the same binding site. The alternative null hypothesis cannot, however, be excluded. In that case, the flexibility would play no role in the intermolecular interactions of DsbA but would be strictly linked to the lower stability of the oxidized form in which some intramolecular interactions are lost or weakened to accommodate the active site disulfide bond.
The rigidifying of the thioredoxin domain after reduction is not an isolated phenomenon; the whole DsbA protein is more or less affected. In the helical domain, we also observe that helices 2, 4, and 6 probably have tighter interactions. The proteolytic sites of these regions either disappear or appear at longer incubation times. In particular, in the reduced enzyme no more cleavage occurs at Phe129, the residue that makes the junction between helices 6 and 7. It is noteworthy that this residue had already been pointed out by NMR experiments as undergoing significant chemical shift variation on reduction (Couprie et al. 1998). The loss of mobility that occurs on both domains of the protein may lead to a larger exposure to proteolysis of the last turns of helix 3 with the appearance of a cleavage site at Glu94 in both reduced and complexed forms.
At the junction between B-domain and thioredoxin domain, the turn formed by residues 63 to 66 constitutes a hinge around which domain motions occur. All structural studies agree today on the fact that domain movements do happen in DsbA (Guddat et al. 1997; Guddat et al. 1998; Schirra et al. 1998). The conflicting point is what causes these movements and when. The resolution of the reduced structure by NMR has indicated that these motions were related to the redox state (Schirra et al. 1998); however, this hypothesis was based on the comparison of their structure with the oxidized structure obtained from x-ray experiments in different experimental conditions (Martin et al. 1993; Guddat et al. 1997). The crystallographic data proposed from the comparison of a panel of structures obtained for both reduced and oxidized DsbA that domain motion is a consequence of substrate binding (Guddat et al. 1998). Our data are clearly insufficient to bring a definite answer to this question; nevertheless, several observations can be made. First, the hinge region is surrounded by helix 2 (B-domain) and strand ß3. Despite the apparent poor accessibility to the solvent of these elements, proteolysis does occur in these regions in the oxidized form. This area becomes clearly less sensitive to proteases in the other states with the cleavage sites disappearing in both the complex and the reduced forms. The second observation is the increased accessibility to proteolysis of Glu94, a residue of helix 3 (B-domain), close in space to the thioredoxin domain, once more in both the complexed and reduced forms. We can hypothesize that domain movements are an integral part of the overall mobility of the oxidized form and also contribute to the process of interaction with substrates. This comes close to the conclusion drawn from the x-ray studies and is supported by the large variation of domain orientations in the different oxidized structures (Guddat et al. 1998). In both the complexed and reduced states, the loss of mobility is sufficient to explain the absence of hydrolysis in this part of the molecule and no hypothesis can be drawn about the occurrence of domain movements or not.
Preliminary analyses performed on the DsbA-PID complex by NMR have indicated that the peptide was in an extended conformation and that preferential noncovalent interactions do exist between both partners. However, calorimetric experiments have shown that these interactions were relatively weak (Couprie et al. 2000). Our present data reinforce this conclusion: No zone of DsbA is significantly protected by its interaction with PID. The putative binding site is bounded by the active site helix 1/1` and the loop connecting strand ß5 and helix 7. A hydrophobic pocket is located inside this hydrophobic groove and contains residues of 1/1`, ß4, ß5, and 7 (Guddat et al. 1997). The disappearance/lower rate of hydrolysis at residues Lys7 and Thr168 (loop connecting ß5 and 7) is in favor of the actual presence of the peptide in this area. The other favorable argument is relative to the behavior of Arg148. Schirra et al (1998) have pointed out that the side chain of Arg148 was protruding over the entrance of the hydrophobic pocket in the reduced form and could shield the entrance of substrates into the groove in this redox state. The reduction of DsbA makes possible the hydrolysis at the level of Arg148 and, more interestingly, the cleavage at Arg148 is even easier in the complex. We can assume that the presence of the peptide in the groove can completely push the side chain of Arg148 toward the solvent.
As for the peptide itself, the great sensitivity of the bond Arg13`-Phe14` to hydrolysis, regardless of the protease used, indicates that its size exceeds the size of the binding site of the protein. The interaction with DsbA protects residues 9` and 10` against proteolysis. Because Phe5` and Phe10` are at the same distance in regard to the homoserine involved in the covalent linkage with DsbA, the occurrence of a proteolytic event only at Phe5` could be explained by a preferential orientation of the PID peptide in the binding site. However, this hypothesis requires more precise data to be confirmed; data that could not be obtained by the method used in this study. The resolution of the structure of the complex by NMR is currently in progress and will give a complete picture of both the peptide-binding site and the set of interactions between PID and the enzyme.
|
Materials and methods |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Formation of the DsbA–DsbB peptide complex
DsbA was purified as previously described (Wunderlich and Glockshuber 1993). DsbA-PID peptide complex was obtained as described (Couprie et al. 2000).
Protein reduction
Protein reduction was performed in 50 mM sodium phosphate buffer pH 7.5 using a 50 molar excess of DTT over the number of cysteine residues for 60 min at 37°C under argon atmosphere. DTT was removed by fast desalting on a Bio Gel P2 fine 45–90 µm column (Bio-Rad Laboratories) using an isocratic gradient with degassed 50 mM sodium phosphate buffer pH 7.5. Reduced protein was immediately used for limited proteolysis experiments performed under argon atmosphere. To show that the DsbA sample is reduced throughout the limited proteolysis experiments, the reduced protein was incubated in the conditions used for hydrolysis experiments for 1 hr and 3 hr at 37°C under argon atmosphere. It was then carboxyamidomethylated using a 10 molar excess of iodoacetamide (IAM) over the number of cysteine residues. The alkylation reaction was performed for 20 min at room temperature in the dark to minimize photolytic production of iodine that is a very potent oxidizing agent for thiols. Samples were desalted by reversed-phase chromatography by use of a µRPC C2/C18 SC 2.1/10 column (Pharmacia) on a SMART System (Pharmacia). The elution system consisted of 0.1% TFA (solvent A) and 0.07% TFA in 95% acetonitrile (solvent B) from 15% to 95% B at a flow rate of 200 µL/min. Eluted compounds were monitored at 220 nm and 280 nm and analyzed by ESIMS.
Proteolysis experiments
Limited proteolysis experiments were conducted by treating the isolated protein in the reduced, oxidized, or complexed form separately with an appropriate ratio of protease (trypsin, chymotrypsin, endoproteinase Glu-C, endoproteinase Asp-N, or subtilisin). Enzymatic digestions were all performed in 50 mM sodium phosphate buffer pH 7.5 at 37°C with a final protein concentration of 10 µM. The proteolysis experiments performed on the reduced DsbA were performed under argon atmosphere. For the limited proteolysis experiments performed with trypsin, an enzyme/substrate ratio of 1/70 was used; for those performed with chymotrypsin and Glu-C an enzyme/substrate ratio of 1 : 100 and 1 : 10 (or 1 : 20) were used, respectively. The hydrolysis with subtilisin and Asp-N were all performed with an enzyme/substrate ratio of 1 : 300 and 1 : 90, respectively. The extent of digestion was monitored on a time-course basis by sampling the reaction mixture at different reaction times ranging from 15 min to 3 hr. Proteolytically digested protein samples were analyzed and fractionated by reversed-phase chromatography with a µRPC C2/C18 S.C 2.1/10 column (Pharmacia) on a SMART System. Peptides were eluted by means of a linear gradient of 5% to 60% acetonitrile in TFA (0.1%) over 45 min; elution was monitored at 220 and 280 nm. Individual fractions were collected and identified by mass spectrometry (matrix assisted laser desorption ionization [MALDI] or ESIMS).
Mass spectrometry analyses
Matrix assisted laser desorption ionization mass spectrometry analyses (MALDIMS)
Peptides were analyzed with a reflectron MALDI-TOF mass spectrometer, Voyager DE (Perseptive Biosystem). The mass range was calibrated with bovine insulin (average molecular mass 5734.6 Da) and a matrix peak (379.1 Da) as internal standards. One µL of the peptide fractions collected after chromatographic separation was applied to a sample slide and allowed to air dry before applying 1 µL of -cyano-4-hydroxycinnamic acid (10 mg/mL) in ethanol/acetonitrile/0.1% TFA 1 : 1 : 1 (v:v:v). The matrix was allowed to air dry before collecting spectra. Mass spectra were generated from the sum of 50 laser shots and were performed by use of an accelerating voltage of 20 KV and a delay time of 100 ns. Raw data were analyzed by using Grams/386 software provided by the manufacturer.
Electrospray mass analyses
ESIMS and MS/MS analyses were performed by use of a QUATTRO II triple quadrupole mass spectrometer equipped with an electrospray ion source (Micromass). Protein and peptide samples derived from the chromatographic separation were injected directly into the ion source by Harward Apparatus with a flow rate of 6 µL/min. Data were acquired at 10 sec/scan and elaborated by MassLynx software provided by the manufacturer. Mass scale calibration was performed by means of multiply charged ions from a separate injection of horse heat myoglobin (average molecular mass 16951.5).
|
Acknowledgments |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
References |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Bardwell, J., Lee, J.-O., Jander, G., Martin, N., Belin, D., and Beckwith, J. 1993. A pathway for disulfide bond formation in vivo. Proc. Natl. Acad. Sci. 90: 1038–1042.
Couprie, J., Remerowski, M., Bailleul, A., Courçon, M., Gilles, N., Quéméneur, E., and Jamin, N. 1998. Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy. Protein Sci. 7: 2065–2080.[Abstract]
Couprie, J., Vinci, F., Dugave, C., Quéméneur, E., and Moutiez, M. 2000. Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex. Biochemistry 39: 6732–6742.[CrossRef][Medline]
Ferrari, D. and Söling, H.-D. 1999. The protein disulphide-isomerase family: Unravelling a string of folds. Biochem. J. 339: 1–10.
Foloppe, N., Sagemark, J., Nordstrand, K., Berndt, K.D., and Nilsson, L. 2001. Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: Comparison with functionally related proteins. J. Mol. Biol. 310: 449–470.[CrossRef][Medline]
Forman-Kay, J.D., Clore, G.M., Wingfield, P.T., and Gronenborn, A.M. 1991. High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution. Biochemistry 30: 2685–2698.[CrossRef][Medline]
Frech, C., Wunderlich, M., Glockshuber, R., and Schmid, F.X. 1996. Preferential binding of an unfolded protein to DsbA. Embo. J. 15: 392–398.[Medline]
Gane, P.J., Freedman, R.B., and Warwicker, J. 1995. A molecular model for the redox potential difference between thioredoxin and DsbA, based on electrostatics calculations. J. Mol. Biol. 249: 376–387.[CrossRef][Medline]
Grauschopf, U., Winther, J.R., Korber, P., Zander, T., Dallinger, P., and Bardwell, J.C. 1995. Why is DsbA such an oxidizing disulfide catalyst? Cell 83: 947–955.[CrossRef][Medline]
Guddat, L., Bardwell, J., and Martin, J. 1998. Crystal structures of reduced and oxidized DsbA: Investigation of domain motion and thiolate stabilization. Current Biol. 6: 757–767.
Guddat, L.W., Bardwell, J.C., Zander, T., and Martin, J.L. 1997. The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding. Protein Sci. 6: 1148–1156.[Abstract]
Hennecke, J., Sebbel, P., and Glockshuber, R. 1999. Random circular permutation of DsbA reveals segments that are essential for protein folding and stability. J. Mol. Biol. 286: 1197–1215.[CrossRef][Medline]
Holmgren, A., Soderberg, B.O., Eklund, H., and Branden, C.I. 1975. Three-dimensional structure of Escherichia coli thioredoxin-S2 to 2.8 A resolution. Proc. Natl. Acad. Sci. 72: 2305–2309.
Holst, B., Tachibana, C., and Winther, J. 1997. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum. J. Cell Biol. 138: 1229–1238.
Hu, S.H., Peek, J.A., Rattigan, E., Taylor, R.K., and Martin, J.L. 1997. Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae. J. Mol. Biol. 268: 137–146.
Huber-Wunderlich, M. and Glockshuber, R. 1998. A single dipeptide sequence modulates the redox properties of a whole enzyme family. Fold. Des. 3: 161–171.
Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M., and Creighton, T.E. 1996. Structure determination of the N-terminal thioredoxin-like domain of protein disulfide isomerase using multidimensional heteronuclear 13C/15N NMR spectroscopy. Biochemistry 35: 7684–7691.[CrossRef][Medline]
Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M., and Creighton, T.E. 1997. The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules. Curr. Biol. 7: 239–245.[CrossRef][Medline]
Kemmink, J., Dijkstra, K., Mariani, M., Scheek, R.M., Penka, E., Nilges, M., and Darby, N.J. 1999. The structure in solution of the b domain of protein disulfide isomerase. J. Biomol. NMR 13: 357–368.[CrossRef][Medline]
Kortemme, T., Darby, N.J., and Creighton, T.E. 1996. Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase. Biochemistry 35: 14503–14511.[CrossRef][Medline]
Martin, J.L., Bardwell, J.C., and Kuriyan, J. 1993. Crystal structure of the DsbA protein required for disulphide bond formation in vivo. Nature 365: 464–468.[CrossRef][Medline]
McCarthy, A., Haebel, P., Törrönene, A., Rybin, V., Baker, E., and Metcalf, P. 2000. Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli. Nat. Struct. Biol. 7: 196–199.[CrossRef][Medline]
Mössner, E., Huber-Wunderlich, M., and Glockshuber, R. 1998. Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases. Protein Sci. 7: 1233–1244.[Abstract]
Moutiez, M., Burova, T., Haertlé, T., and Quéméneur, E. 1999. On the non-respect of the thermodynamic cycle by DsbA variants. Protein Sci. 8: 106–112.[Abstract]
Nelson, J.W. and Creighton, T.E. 1994. Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo. Biochemistry 33: 5974–5983.[CrossRef][Medline]
Scaloni, A., Miraglia, N., Orrù, S., Amodeo, P., Motta, A., Marino, G., and Pucci, P. 1998. Topology of the calmodulin-melittin complex. J. Mol. Biol. 277: 945–958.[CrossRef][Medline]
Schirra, H., Renner, C., Czisch, M., Huber-Wunderlich, M., Holak, T., and Glockshuber, R. 1998. Structure of reduced DsbA from Escherichia coli in solution. Biochemistry 37: 6263–6276.[CrossRef][Medline]
Wunderlich, M. and Glockshuber, R. 1993. Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli. Protein Sci. 2: 717–726.[Abstract]
Zappacosta, F., Pessi, A., Bianchi, E., Venturini, S., Sollazzo, M., Tramontano, A., Marino, G., and Pucci, P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of minibody. Protein Sci. 5: 802–813.[Abstract]
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Kadokura, H. Tian, T. Zander, J. C. A. Bardwell, and J. Beckwith Snapshots of DsbA in Action: Detection of Proteins in the Process of Oxidative Folding Science, January 23, 2004; 303(5657): 534 - 537. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
