Graduate School of Pharmaceutical Sciences, Kyushu University,Fukuoka 812-8582, Japan
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Abstract |
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Keywords: cross-linking/differential scanning calorimetry/lysozyme/thermal stability
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Introduction |
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Introduction of intramolecular cross-links by point mutation or chemical modification effectively increases the stability of mesophilic proteins (Johnson et al., 1978; Lin et al., 1984
; Ueda et al., 1985
, Pantoliano et al., 1987
; Pace et al., 1988
; Matsumura et al., 1989a
,b
). Matsumura et al. (1989b) have demonstrated that disulfide bonds, each of which singly contributes to the stability of T4 lysozyme, can achieve substantial overall improvement in stability when used in combination. However, since the denaturation temperature of mutant T4 lysozymes, in which cysteinyl residues are introduced, were all lower than that of wild type T4 lysozyme under reduced conditions, the unfavorable interactions in the folded state of these mutants would have occurred by point mutations to cysteines. Therefore, while Matsumura et al. (1989a,b) successfully improved the stability of T4 lysozyme by the incorporation of multiple intramolecular cross-links, further stabilization of T4 lysozyme by multiple intramolecular cross-linking may be possible by avoiding unfavorable interactions in the folded state.
Hen egg-white lysozyme is a small protein, which has four disulfide bonds (Imoto et al., 1972). Trials to improve its stability by point mutations or chemical modification have been conducted extensively (Imoto, 1996
, 1997
). So far, the greatest increase in stability has been the result of an ester bond cross-link between Glu35 and Trp108 (35108 CL lysozyme), which stabilized the lysozyme molecule by 5.2 kcal/mol over that of wild type, at pH 2, in the presence of 1.94 M guanidine hydrochloride (Johnson et al., 1978
). On the other hand, a cross-link between Lys1 and His15 introduced after treatment with N,N'-bisbromoacetyltetramethylene-diamine (115 CL lysozyme) also stabilized the lysozyme molecule by 2.3 kcal/mol over that of wild type, at pH 5.5, in the presence of 3 M guanidine hydrochloride (Ueda et al., 1985
).
Chemical modification of the folded protein rarely affects the structure of the protein to any great extent. Indeed, few unfavorable interactions in the folded state of hen lysozyme were reported as a result of the introduction of cross-links between Glu35 and Trp108, and between Lys1 and His15 (Beddell et al., 1975; Ueda et al., 1985
). Therefore, in this paper, in order to accumulate information directed towards the problem of `how effectively can proteins from mesophiles, such as lysozyme, be stabilized by multiple intramolecular cross-linking?', we prepared a doubly intramolecularly cross-linked hen lysozyme by simultaneous introduction of cross-links Glu35Trp108 and Lys1His15, and examined its stability against thermal denaturation.
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Materials and methods |
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Five-times recrystallized wild-type hen egg-white lysozyme was donated by QP (Tokyo).
Preparation of cross-linked lysozymes
115 CL lysozyme (Ueda et al., 1985) and 35108 CL lysozyme (Imoto and Rupley, 1973
) were prepared as described previously. For preparation of doubly CL lysozyme, where cross-links exist between both Lys1 and His15 and between Glu35 and Trp108, 50 mg 115 CL lysozyme was dissolved in 3 ml 1 mM acetate buffer at pH 5.5. Ten microliters of KI-I2 solution (0.48 M KI containing 0.02 M I2) was added to the protein solution five times at room temperature over 2 h. After the addition of KI-I2, the reaction mixture was applied directly to an ion-exchange chromatography column (Bio Rex 70, Bio-Rad, Hercules, CA) equilibrated with 0.02 M borate buffer at pH 10. The column (2x70 cm) was eluted with a gradient of 1000 ml 0.02 M borate buffer at pH 10 and 1000 ml of the same buffer containing 0.1 M NaCl. The protein elution was monitored by absorbance at 280 nm. The desired protein fraction (120 ml) was collected and diluted to 300 ml with H2O. In order to concentrate the protein solution, the solution was adjusted to pH 4 by adding acetic acid and applied to the ion-exchange chromatography column (CM-Toyopearl 650 M, 1.5x20 cm; Tosoh, Tokyo) equilibrated with 0.1 M acetate buffer at pH 4. The desired protein was eluted with the same buffer containing 1 M NaCl, dialyzed against distilled water at 4°C and lyophilized. The yield of doubly CL lysozyme based on 115 CL lysozyme was 10%.
Characterization of cross-linked lysozyme
Ultraviolet absorption spectrum measurements of doubly CL lysozyme were taken using a Perkin Elmer Lambda 10 spectrophotometer. For lytic activities, 50 µl lysozyme solution (50100 µg/ml) was added to 3 ml 0.25 mg/ml Micrococcus luteus solution (Sigma, St. Louis, MO) and the decrease in turbidity at 450 nm was monitored.
Differential scanning calorimetry (DSC)
Before the DSC measurements were taken, a second chromatography step was performed on the 35108 CL and doubly CL lysozymes using ion-exchange HPLC (CM-Toyopearl 650 S, 4.6x300 mm; Tosoh, Tokyo) to remove any impurities that had formed during the storage of these cross-linked lysozymes. The column was eluted with 200 ml 0.02 M borate buffer at pH 10 and 200 ml of the same buffer containing 0.3 M NaCl at a flow rate of 1.0 ml/min. The protein elution was monitored by absorbance at 280 nm. The desired fraction was collected, followed by dialysis against 50 mM GlyHCl buffer, pH 2.03.0 at 4°C, exhaustively. Other samples for DSC measurements were dissolved in 50 mM GlyHCl buffer at pH 2.03.0 and were dialyzed against the respective buffers at 4°C, exhaustively. Differential scanning calorimetry measurements were carried out with a VP-DSC calorimeter (Microcal, Northampton, MA). The scan rate was 0.5 or 1.0 K/min. Protein concentrations (2545 µM) were checked using a amino acid analyzer after acid hydrolysis. Data analyses were done using the Origin Software (Microcal).
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Results and discussion |
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The obtained lysozyme had no activity against M.luteus. From its UV spectrum, the ratio of absorbance at 250 nm to that at 280 nm was 1.96, which is consistent with the formation of an ester bond between Glu35 and Trp108 (Imoto and Rupley, 1973). Moreover, amino acid analysis of the collected protein confirmed the formation of a cross-link between Lys1 and His15 in the presence of the alkyl reagent (data not shown). From these results, we concluded that the collected protein did indeed contain two cross-links, Lys1His15 and Glu35Trp108 (doubly CL lysozyme). Since the cross-link between Glu35 and Trp108 in 35108 CL lysozyme may be unstable due to the ester bond, a second chromatography step was performed on doubly CL lysozyme and 35108 CL lysozyme immediately before the DSC measurements to remove any impurities. In Figure 1
, a typical chromatograph of doubly CL lysozyme shows two distinct peaks. The derivative in the main peak was collected and dialyzed against 50 mM GlyHCl buffer, pH 2.03.0 at 4°C, exhaustively. The derivative in the minor fraction may be an oxindolealanine 108 lysozyme derivative, judging from its ultraviolet spectrum, which is a product of 35108 CL lysozyme hydrolysis (Imoto et al., 1973
).
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The thermodynamic parameters of the wild type and 115 CL lysozymes for reversible thermal denaturation at pH 2.7 were calculated using denaturation temperatures at different pHs (Table I). Also, the denaturation temperatures of doubly CL lysozyme and 35108 CL lysozyme are shown in Table I
. Consistent with previous reports (Johnson et al., 1978
; Ueda et al., 1985
), it was confirmed that both 35108 CL lysozyme and 115 CL lysozyme were more stable than wild type (Table I
). Doubly CL lysozyme was more stable by 20.8°C than wild type lysozyme (Table I
). In order to measure the strict contribution of the cross-link on the stabilization of the lysozyme molecule, it is necessary to evaluate
H and
Cp of doubly CL lysozyme and 35108 CL lysozyme for reversible thermal denaturation. However, since the thermal denaturations of doubly CL lysozyme and 35108 CL lysozyme, which both contain an ester bond between Glu35 and Trp108 in the lysozyme molecule, proceeded irreversibly to some extent, we could not evaluate the thermodynamic parameters of these cross-linked lysozymes for reversible thermal denaturation. Therefore, to understand better the contribution of each cross-link on the stabilization of the lysozyme molecule, each free energy change of denaturation of the modified lysozyme [
G (T)] was calculated according to the equations below.
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In this paper, we succeeded in the preparation of a modified lysozyme effectively stabilized against thermal denaturation by the introduction of two intramolecular cross-links. We found that the thermal denaturations of doubly CL lysozyme and 35108 CL lysozyme proceeded irreversibly to some extent, and as a result, the denaturation temperatures of doubly CL lysozyme and 35108 CL lysozyme obtained by DSC measurements were unfortunately underestimated. However, we consider that the irreversibility of the denaturation of doubly CL lysozyme and 35108 CL lysozyme only affect their thermal denaturations in a small way, because their thermal transitions were apparently single for the denaturation because their peak shapes were similar to that of the wild type lysozyme. Therefore, the present finding that the sum of the difference in the increasing denaturation temperature between the wild type lysozyme and each cross-linked lysozyme was nearly equal to that between the wild type lysozyme and doubly cross-linked lysozyme may be significant to show that the efficient stabilization of a protein molecule can be attainable by such modifications. This information is of help when designing de novo proteins, and the finding will contribute to protein engineering.
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Notes |
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References |
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Received April 15, 1999; revised December 8, 1999; accepted December 9, 1999.