Protein Engineering, Vol. 14, No. 11, 829-833,
November 2001
© 2001 Oxford University Press
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Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
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Abstract |
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We prepared two dissected fragments of hen lysozyme and examined whether or not these two fragments associated to form a native-like structure. One (Fragment I) is the peptide fragment Asn59–homoserine-105 containing Cys64–Cys80 and Cys76–Cys94. The other (Fragment II) is the peptide fragment Lys1–homoserine-58 connected by two disulfide bridges, Cys6–Cys127 and Cys30–Cys115, to the peptide fragment Asn106–Leu129. It was found that the Fragment I immobilized in the cuvette formed an equimolar complex with Fragment II (Kd = 3.3x10–4 M at pH 8 and 25°C) by means of surface plasmon resonance. Moreover, from analyses by circular dichroism spectroscopy and ion-exchange chromatography of the mixture of Fragments I and II at pH 8 under non-reducing conditions, it was suggested that these fragments associated to give the native-like structure. However, the mutant Fragment I in which Cys64–Cys80 and Cys76–Cys94 are lacking owing to the mutation of Cys to Ala, or the mutant fragment in which Trp62 is mutated to Gly, did not form the native-like species with Fragment II, because the mutant Fragment I derived from mutant lysozymes had no local conformation due to mutations. Considering our previous results where the preferential oxidation of two inside disulfide bonds, Cys64–Cys80 and Cys76–Cys94, occurred in the refolding of the fully reduced Fragment I, we suggest that the peptide region corresponding to Fragment I is an initiation site for hen lysozyme folding.
Keywords: folding/lysozyme/peptide fragment/point mutation/surface plasmon resonance
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Understanding protein folding is one of the most important themes in the field of life science in the post-genome period. So far, advances in experiment and theory have led to considerable progress in our understanding of protein folding. However, the underlying factors that control the products of the protein folding still remain unknown. Elucidation of the factors involved in protein folding would help in understanding the mechanism of protein organization and contribute to the de novo design of proteins. Since the folding reaction of protein in vivo starts with its denatured and reduced polypeptide chain, in vitro analysis of protein folding with disulfide bond(s) should be carried out from its reduced form. In vitro analysis of protein folding is also meaningful, since the oxidant concentration in living cells is similar to that in the folding of the protein from its reduced form in vitro (Hwang et al ., 1992). One of the powerful approaches used to elucidate the factors involved in protein folding is to identify the folding intermediate (Kim and Baldwin, 1982
). It has been well shown by the pulse-labeling technique by means of NMR spectroscopy that many proteins fold through intermediates with local conformation (Roder et al ., 1988; Udgaonkar and Baldwin 1988; Miranker et al . 1991). However, there is little information about the intermediate in the case of the folding of protein with disulfide bond(s) from the reduced state.
Hen egg-white lysozyme is one protein whose folding process from the reduced form has been investigated. About 25 years ago, Anderson and Wetlaufer (1976) suggested that the two disulfide bonds involving Cys64, Cys76, Cys80 and Cys94 formed faster than those involving Cys6, Cys30, Cys115 and Cys127 in the folding of reduced hen lysozyme. We also showed that the preferential oxidation of two inside disulfide bonds in hen lysozyme, Cys64–Cys80 and Cys76–Cys94, occurred in the refolding of the fully reduced peptide fragment between Asn59 and homoserine-105 in hen lysozyme (dark-colored region in Figure 1) (Ueda et al ., 1996). These findings indicated that Cys64–Cys80 and Cys76–Cys94 disulfide bonds form in the early stage of the folding of reduced lysozyme. On the other hand, Dobson's group suggested recently that the disulfide equilibration step occurred before the formation of the folding intermediate with three disulfide bonds, since they did not detect any particular folding intermediate with one or two disulfide bonds by analysis of the folding process of reduced hen lysozyme using reversed-phase high-performance liquid chromatography (RP-HPLC) (van den Berg et al ., 1999a,b). Therefore, the region that forms in the early stage of the folding of reduced hen lysozyme is now controversial.
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Sancho and Fersht (1992) have shown the contributions of the N-terminal fragment containing an
-helix and the C-terminal fragment containing a ß-sheet to the organization of the whole barnase molecule by binding each individual dissected fragments. However, since we did not refer to how the formation of the peptide region was related to the organization of the whole hen lysozyme molecule in our previous paper (Ueda et al ., 1996), further investigation of the involvement of the peptide region on the organization of its tertiary structure seemed necessary. Therefore, in this work, in order to elucidate the involvement of the peptide region 59–105 in hen lysozyme (Fragment I) on the organization of its tertiary structure, we evaluated whether or not the association of Fragment I or its mutant fragments with the residual region of hen lysozyme (Fragment II) took place. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Materials
Five-times recrystallized hen egg-white lysozyme was donated by QP (Tokyo). Bovine pancreatic ribonuclease A was a product of Sigma. Columns of Asahipak ES-502C (7.6x100 mm) for ion-exchange HPLC was obtained from Asahi Chemical Industry (Tokyo). Fragment I, a peptide fragment Asn59–homoserine-105 derived from I58M lysozyme, and Fragment II, a peptide fragment where a peptide Lys1–homoserine-58 is connected to a peptide fragment Asn106–Leu129 through the disulfide bonds derived from I58M lysozyme, were obtained by cleavage of I58M lysozyme using BrCN according to the method in our previous work (Ueda et al ., 1996). All other chemicals were of the highest quality commercially available.
Site-directed mutagenesis and expression and purification of lysozymes
Site-directed mutagenesis and expression of W62G/I58M lysozyme, a mutant lysozyme where Trp62 in I58M lysozyme is mutated to Gly (Hashimoto et al ., 1996) or those of des 4Cys/I58M lysozyme, a mutant lysozyme where Cys64, Cys76, Cys80 and Cys94 are simultaneously mutated to Ala and Ile58 is mutated to Met (Mine et al ., 1997), was performed by the method in our previous work. These mutations in the lysozyme gene were confirmed using DNA sequence analysis.
Preparation of peptide fragments of lysozymes or peptide fragment of ribonuclease A
W62G Fragment I, a peptide fragment Asn59–homoserine-105 where Trp62 is mutated to Gly, was obtained by cleavage of W62G/I58M lysozyme using BrCN according to the method used in our previous work (Ueda et al ., 1996). Des 4Cys Fragment I, a peptide fragment Asn59–homoserine-105 where Cys64, Cys76, Cys80 and Cys94 are simultaneously mutated to Ala, was obtained by dissecting the modified des4Cys/ I58M lysozyme, which had been reduced and alkylated with N,N,N-trimethylammonio-N',N'-dimethylaminopropyl bromide bromide, with BrCN. In addition, a peptide fragment 1–31 of ribonuclease A (KETAAAKFERQHMDSSTSAASSSNYCNQMMK) was obtained by digestion of reduced and carboxymethylated ribonuclease A with lysylendopeptidase. These fragments were purifed using RP-HPLC. The confirmation of these peptides was carried out by amino acid analysis with a Hitachi L-8500 amino acid analyzer.
Preparation of Fragment I modified at Asp101
Modification of I58M lysozyme at Asp101 with 2-(2-aminoethyl)pyridyl disulfide was carried out according to the method in our previous work (Ueda et al ., 1991). Fragmentation of Asp101-modified I58M lysozyme by BrCN was performed according to the method in our previous work (Ueda et al ., 1996). The yield was ~30%.
Immobilization of Fragment I modified at Asp101 on the cuvette
Biospecific interaction analysis was carried out using a biosensor (IAsys, Fisons) based on the principle of surface plasmon resonance (Gorgani et al ., 1997; Krebs et al ., 1998). Asp101 in hen lysozyme is located in Fragment I and is considerably exposed to the solution in the folded structure (Imoto et al ., 1972). Since the carboxyl group at Asp101 was selectively modified by an alkyl chain with an amino group (Yamada et al ., 1981), the residue may be a candidate for the site to fix to the cuvette. First, 0.05 M phosphate buffer of pH 7.2 containing 0.15 M NaCl and 0.05% Tween (PBS/T buffer) was added to the cuvette placed in the instrument and then the solution was kept at 20°C for 10 min. After removing the solution, aminosilane, which is linked to the detecting surface of the cuvette in the instrument, was activated by the addition of 15 mM N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) dissolved in 10 mM phosphate buffer of pH 6.5 containing 30% ethanol. The cuvette was exhaustively washed with 10 mM phosphate buffer of pH 6.5 and then 100 mM DTT dissolved in the same buffer was added in order to liberate sulfopyridyl group, resulting in the production of a thiol group. After washing the cuvette with 10 mM phosphate buffer of pH 6.5, Fragment I modified at Asp101 dissolved in the same buffer (50 µM) was added to the cuvette in order to fix it by a disulfide bridge with the produced thiol group on the cuvette. After 6 h, the cuvette was washed with PBS/T buffer and the cuvette was refilled with 10 mM N-ethylmaleimide solution at pH 6 for 30 min. Finally, the N-ethylmaleimide solution was removed and the cuvette was filled with PBS/T buffer again before the kinetic experiments.
Kinetic measurement of the interaction between the immobilized Fragment I and Fragment II or the peptide fragment 1–31 of ribonuclease A
Both the association rate constant (kass) and dissociation rate constant (kdiss) were obtained in 0.1 M Tris–HCl buffer of pH 8 containing 0.1 M urea according to our previous paper (Ueda et al ., 1998). Briefly, the cuvette was filled with 200 µl of 0.1 M Tris–HCl buffer of pH 8.6 containing 0.1 M urea (reaction solution) and thermostated at 25°C for 15 min. An appropriate volume of the reaction solution was removed from the cuvette and then the same volume of a pre-warmed Fragment II solution (0.2–6.3 µM) at 25°C or a pre-warmed solution of the peptide fragment 1–31 of ribonuclease A (1.5–9.8 µM) at 25°C was added to the cuvette (association reaction). Each association reaction was monitored for 7 min. Each solution was then removed with a sucker and 200 µl of the reaction solution that had been pre-incubated for 25°C were immediately added to the cuvette (dissociation reaction). Each dissociation reaction was monitored for 3 min. After the association and dissociation reactions, the immobilized Fragment I was regenerated by two-step regeneration; the detecting surface of the cuvette in the biosensor was washed with 6 M guanidine hydrochloride (Gdn·HCl) solution (0.1 M Tris–HCl of pH 8.6 containing 5.37 mM EDTA and 6 M Gdn·HCl) and followed by replacement with 10 mM HCl. Each washing operation was repeated three times. After regeneration, the cuvette was refilled with 0.1 M Tris–HCl buffer of pH 8.6 as soon as possible.
The interaction between immobilized Fragment I and Fragment II or the peptide fragment 1–31 of ribonuclease A was monitored at 25°C as the change in R, that is, the respective amount of bound Fragment II or the peptide fragment 1–31 of ribonuclease A to the immobilized Fragment I measured as the surface plasmon resonance (SPR) response (resonance units) at time t. A plot of the slope of dR/dt versus R against the concentration of the peptide fragment that was added to the cuvette (the immobilized Fragment I) would give a good straight line with a distinct slope if the association of the peptide fragment with the immobilized Fragment II occurs stoichiometrically. The slope and the intercept of the straight line give kass and kdiss, respectively. The dissociation constant (Kd) is then calculated as kdiss/kass. The evaluation of Kd using Scatchard plots was carried out according to the standard protocol of IAsys.
Association of Fragment I or its derivative with Fragment II in solution
The lyophilized Fragment I, des 4Cys Fragment I, the peptide fragment Asn59–homoserine-105 where Cys64, Cys76, Cys80 and Cys94 are simultaneously mutated to Ala or W62G Fragment I, the peptide fragment Asn59–homoserine-105 where Trp62 is mutated to Gly and Fragment II were dissolved in 0.1 M Tris–HCl buffer (pH 8.0) containing 6 M Gdn·HCl and incubated at 40°C for 30 min under non-reducing conditions. Equimolar mixtures (at peptide concentrations of 12.5–200 µg/ml) of Fragment I solution, des 4Cys Fragment I solution or W62G Fragment I solution and Fragment II solution or individual Fragment I solution (16 µg/ml) and Fragment II solution (34 µg/ml) were gently and exhaustively dialyzed against 0.1 M Tris–HCl buffer (pH 8.0) in the presence or absence of 1 M urea at 4°C.
Analytical methods
For analysis of the interaction between Fragment I or its mutants and Fragment II, 500 µl of each dialyzate were applied to the ion-exchange column (Asahipak ES-502C, 7.6x100 mm) for HPLC. The column was eluted with a gradient of 40 ml of 0.1 M sodium acetate buffer at pH 5.0 and 40 ml of the same buffer containing 1.0 M NaCl at a flow-rate of 1.0 ml/min. The eluate was monitored by measuring the absorbance at 280 nm. Fluorescence spectra of Fragment I or its mutants and N-acetyltryptophan ethyl ester at a concentration of 0.2–1 µM in the presence of various urea concentrations were measured in 20 mM phosphate buffer at pH 8 and 20°C using a Hitachi F-2000 spectrofluorimeter. Circular dichroism (CD) spectra of intact lysozyme and the mixture of Fragment I and Fragment II in 50 mM Tris–HCl buffer at pH 8 and 20°C were measured with a JASCO-J 720 spectropolarimeter.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Evaluation of kinetic parameters of the interaction of immobilized Fragment I and Fragment II or the peptide fragment 1–31 of ribonulcease A by use of surface plasmon resonance
On analysis of the response curves obtained under the conditions where various amounts of peptide fragments were added to the cuvette using the FASTFIT program, we found that all association or dissociation phases were attributable to monophasic reactions. Plots of the slope (dR/dt versus R) against the concentration of Fragment II that was added to the cuvette are shown in Figure 2A. The plot shows a good straight line, indicating that the immobilized Fragment I associated with Fragment II. From the slope and the y-intercept, kass and kdiss were determined as 4.3x103 s–1 and 1.4 s–1 M, respectively. Based on the values of kass and kdiss, Kd was calculated as 3.3x10–4 M. The Kd value evaluated using Scatchard plots was 1x10–5 M. Clearly, these Kd values were not equal. Since the deviation of Kd obtained by using the Scatchard plots is larger, we employed the Kd value obtained from kass and kdiss. For comparison, the peptide fragment 1–31 of ribonuclease A was added to the cuvette under the same conditions. Plots of the slope (dR/dt versus R) against the concentration of the peptide fragment gave a line parallel to the x-axis (Figure 2B), indicating that there was no specific interaction between the immobilized Fragment I and the peptide fragment 1–31 of ribonuclease A. These data supported the result that the immobilized Fragments I and II formed an equimolar complex.
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Formation of the native-like molecule by the association of Fragment I with Fragment II
As was shown above, the association between the immobilized Fragments I and II was confirmed. Here, we investigated whether or not the association between Fragments I and II formed a native-like molecule. The solution containing denatured peptide fragments was slowly and exhaustively dialyzed against 0.1 M Tris–HCl buffer (pH 8.0) containing 1 M urea at 4°C, then the dialyzate was applied to an ion-exchange HPLC column. Only one peak was eluted at the same position as the native lysozyme. From comparison of the area of the resulting peak with that of a known amount of the native lysozyme, the yield of the native-like molecule was determined as 23% under the conditions employed. On the other hand, neither Fragment I nor Fragment II was adsorbed on the column under the above conditions. Because the net charge in the complex of Fragments I and II was almost identical with that in the native lysozyme under the HPLC conditions (pH 7), the association of Fragment I with Fragment II was found to result in the formation of a native-like molecule. In order to confirm this, we measured the CD spectrum of the dialyzate (Figure 3A). For comparison, the CD spectrum of intact lysozyme was also measured (Figure 3A). The dialyzate containing the mixture of Fragments I and II showed a pattern similar to that of intact lysozyme but slightly different owing to the presence of uncomplexed fragments. From the above results, we concluded that the association between Fragments I and II gave a native-like molecule.
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To find the optimum conditions, we examined the yield of the native-like molecule at various concentrations of an equimolar mixture of Fragment I and II solutions in 0.1 M Tris–HCl buffer (pH 8) in the presence (open circles in Figure 3B
) or absence (closed circles) of 1 M urea. Under both conditions, there were optimum concentrations of peptide fragments in the yield of the native-like molecule (50 µg/ml in the presence of 1 M urea or 25 µg/ml in the absence of urea). The yield of the native-like molecule at a concentration of 25 µg/ml was higher in the absence than in the presence of urea. This result may be derived from the favorable association between Fragments I and II in the absence of urea. On the other hand, the yield of the native-like molecule at concentrations over 50 µg/ml was lower in the absence than in the presence of 1 M urea. Because urea has a tendency to depress the aggregation, the limb at higher peptide concentration may depend on aggregation due to the non-specific interactions between the peptide fragments. Therefore, it is considered that the optimum yield of the native-like molecule from the association between Fragments I and II results from the association phase and the aggregation phase between these peptide fragments. We measured the dependence of the tryptophyl fluorescence intensity at 354 nm of Fragment I on urea concentration. With an increase in urea concentration, the tryptophyl fluorescence intensity at 354 nm of Fragment I showed a broad transition with increase in urea concentration, whereas the tryptophyl fluorescence intensity at 354 nm of N-acetyltryptophan ethyl ester increased monotonically. There was a distinct difference in the tryptophyl fluorescence intensity at 354 nm between Fragment I and N-acetyltryptophan ethyl ester at a lower concentration of urea whereas the intensities converged at higher concentrations of urea. Moreover, we discounted the possibility that the dependence of the tryptophyl fluorescence intensity at 354 nm in Fragment I resulted from the non-specific intermolecular interactions by performing the experiments with a more dilute solution of Fragment I (at a concentration of 0.2 µM). From the above results, we concluded that Fragment I had a local conformation and a propensity to associate with Fragment II to form a native-like molecule.
Requirement for local conformation in Fragment I in the organization of the native-like structure
In order to elucidate whether or not the peptide region 59–105 in hen lysozyme was an initiation site for lysozyme folding from the reduced form, association experiments were performed between Fragment I derived from mutant lysozymes and Fragment II. One was des 4Cys Fragment I and the other W62G Fragment I. Trp62 is essential for the correct formation of disulfide bonds (Cys64–Cys80 and Cys76–Cys94) in the peptide fragment Asn59–homoserine-105 (Ueda et al ., 1996). These mutant Fragment I had no local conformation at lower concentrations of urea, because the dependence of the tryptophyl fluorescence intensity at 354 nm of des 4Cys Fragment I or W62G Fragment I overlapped that of N-acetyltryptophan ethyl ester. Moreover, it was confirmed by performing the experiments with more dilute mutant Fragment I solution that disruption of the local conformation in mutant Fragment I was not due to non-specific intermolecular interactions.
After mixing des 4Cys Fragment I or W62G Fragment I with Fragment II, dialysis was carried out at pH 8 in the presence of 1 M urea at 4°C. During the mixing of des 4Cys Fragment I or W62G Fragment I with Fragment II, precipitations were also observed. Part of the dialyzate was subjected to ion-exchange HPLC. No peaks were detected on ion-exchange HPLC under the conditions employed above. The net charge in the native-like molecule derived from the mixture between des 4Cys or W62G Fragment I and Fragment II should be almost identical with that of the native lysozyme. Therefore, it was evidenced that an equimolar mixture between des 4Cys or W62G Fragment I and Fragment II did not give a native-like molecule under the conditions employed. Moreover, these dialyzates did not show CD spectra similar to that of intact lysozyme due to the uncomplexed fragments or the precipitations. These results may be explained by the idea that non-specific interactions occur between peptide fragments because mutant peptide fragments do not possess local conformation. Thus, since local conformation in Fragment I was required for the organization of the native-like structure of lysozyme, we confirmed that the peptide region 59–105 in hen lysozyme was an initiation site for lysozyme folding from the reduced form. Evidence for the initiation site would be significant for understanding the organization of tertiary structure of lysozyme, but also for de novo design of functional proteins since it gives information on structural units, resulting in a contribution to protein engineering.
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Notes |
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1 To whom correspondence should be addressed. E-mail: ueda{at}phar.kyushu-u.ac.jp
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Acknowledgments |
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We thank Dr Yoshio Hashimoto of our laboratory for his advice on gene engineering and protein expression and Mr Charles Pilkey for assistance with English. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a grant from the Rice Genome Project PR-2101, MAFF, Japan.
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Received January 24, 2001; revised July 12, 2001; accepted July 16, 2001.
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