Protein Engineering, Vol. 13, No. 10, 691-695,
October 2000
© 2000 Oxford University Press
Relationship between local structure and stability in hen egg white lysozyme mutant with alanine substituted for glycine
Graduate School of Pharmaceutical Sciences, Kyushu University, 62 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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Abstract |
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We prepared five mutant lysozymes in which glycines whose dihedral angles are located in the region of the left-handed helix, Gly49, Gly67, Gly71, Gly102 and Gly117, were mutated to an alanine residue. From analyses of their thermal stabilities using differential scanning calorimetry, most of them were more destabilized than the native lysozyme, except for the G102A mutant, which has a stability similar to that of the native lysozyme at pH 2.7. As for the destabilized mutant lysozymes, their X-ray crystallographic analyses showed that their global structures did not change but that the local structures changed slightly. By examining the dihedral angles at the mutation sites based on X-ray crystallographic results, it was found that the dihedral angles at these mutation sites tended to adopt favorable values in a Ramachandran plot and that the extent and direction of their shifts from the original value had similar tendencies. Therefore, the change in dihedral angles may be the cause of the slight local structural changes around the mutation site. On the other hand, regarding the mutation of G102A, the global structure was almost identical with that of the native structure but the local structure was drastically changed. Therefore, it was suggested that the drastic local conformational change might be effective in releasing the unfavorable interaction of the native state at the mutation site.
Keywords: differential scanning calorimetry/hen lysozyme/point mutation/protein stability/unfavorable interaction/X-ray crystallography
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Introduction |
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The conformation of a protein is often very unstable because the difference in free energy change between the native state and the denatured state is very small. When a protein is used under conditions such as extreme pH, high temperature and in the presence of a denaturant, it can easily lose its proper structure and activity. This instability may be required for activity or biological turnover. However, the instability of the protein is inconvenient for its practical use. Therefore, in order to stabilize a protein for pharmaceutical or industrial use, it is necessary to understand the mechanism of its stability.
Among all the amino acid residues, only a glycine residue does not have a ß-carbon, resulting in its having the largest conformational freedom. In a Ramachandran plot, the permissible regions at the glycine residue are known to be wider than those at other residues. The region of the dihedral angle around 
= 60° and 
= 60° corresponds to the region of a left-handed helix (Ramachandran et al., 1963). Most amino acid residues, which take a left-handed helix conformation in a protein, have similar dihedral angles. On the other hand, glycine residues are often present in the region with a dihedral angle around = 60° and = 60°, even though they are not in a left-handed helix. Therefore, glycine residues are unique and more attention should be given to the mutation of glycine to other residues in a protein. Matthews (1987) reported that the loss of entropy in the denatured state of a protein by sub-stituting alanine for glycine was estimated to be 10.0 J/mol·K, resulting in stabilization of a protein. On the other hand, substitution of alanine for glycine may induce an unfavorable interaction in the native state of a protein owing to steric hindrance, resulting in destabilization of the protein. Therefore, in order to improve the stability of a protein by such a mutation, we should investigate the conformational change accompanying the mutation in detail. So far, although there have been many examples of the mutation of glycine to alanine in proteins, there have been no systematic analyses of the mutation in a protein. Hen egg white lysozyme is the first enzyme whose tertiary structure has been elucidated (Blake et al., 1965
). Moreover, many data exist on thermal denaturation using differential scanning calorimetry (DSC). Therefore, we consider that it is a suitable protein with which to investigate the relationship between structure and stability in mutant lysozymes where glycines are mutated to alanines. There are 12 glycines in hen egg white lysozyme. The dihedral angles at eight of these 12 glycine residues are located in the region of the left-handed helix (Figure 1). Five of those glycines, Gly49, Gly67, Gly71, Gly102 and Gly117, are located at the surface of the lysozyme molecule (Blake et al., 1965). In this work, we prepared five one-point mutants of hen egg white lysozyme, whose glycines were mutated to alanines, and investigated the relationship between stability using DSC and structure using X-ray crystallography.
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Materials
Restriction enzymes, T4 polynucleotide kinase and DNA polymerase I (Klenow fragment) were purchased from Takara Shuzo (Kyoto, Japan) or New England Biolabs (Beverly, MA). DNA sequencing kits (Sequenase) were purchased from Amersham Japan (Tokyo, Japan). CM-Toyopearl 650M, a cation-exchange resin for the purification of secreted hen lysozyme, was obtained from Tosoh (Tokyo, Japan). All other chemicals were of analytical grade for biochemical use.
Purification and identification of mutant lysozymes secreted by yeast
Mutant lysozymes G49A, G67A, G71A, G102A and G117A were prepared as described (Hashimoto et al., 1996). Transformants of the yeast (Saccharomyces cerevisiae, AH22) were cultivated at 30°C for 125 h for expression and secretion of the lysozyme. Purification (ion-exchange chromatography) of the lysozymes was carried out as described (Inoue et al., 1992). Each mutation was confirmed by DNA sequencing.
Stabilities of mutant lysozymes determined using DSC measurements
DSC measurements and data analyses were carried out with a MicroCal VP-DSC system equipped with a Gateway personal computer. The scan rate was 1.0 K/min. The sample solutions were prepared by dissolution in 0.05 M glycine buffer (pH 2.4–3.2) and the protein concentrations were 0.7–1.5 mg/ml. Data analysis was performed using Origin software (MicroCal, Northampton, MA).
X-ray crystallography of mutant lysozymes
Mutant lysozymes were crystallized from 50 mM sodium acetate (pH 4.7) containing 0.6–0.9 M NaCl, the same conditions as those for crystallization of the native lysozyme (Blake et al., 1965). Mutant lysozymes were crystallized by vapor diffusion using the hanging-drop method. In shape, the crystals were isomorphous with that of native lysozyme; they belonged to the tetragonal system and the space group was P43212. The intensity data to below 2.0 Å were collected at room temperature on a Rigaku R-Axis IIC area detector using a Rigaku RU-300 rotating anode source operating at 40 kV and 120 mA. The intensity data were reduced using the Rigaku Process software package. Because the mutant lysozyme crystals were isomorphous with that of native lysozyme (P43212, cell dimensions a = b = 79.21 Å and c = 37.97 Å), the coordinates of native lysozyme were used as the starting model for the refinement. The structures of the mutant lysozymes were refined using XPLOR 3.1 (Brünger, 1991). The structures were fitted to sum-weighted difference-electron density maps using the program package TURBO-FRODO 5.1 (Roussel et al., 1990) on an SGI Indigo2.
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Results |
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DSC of mutant hen lysozymes
In order to determine the thermodynamic parameters for denaturation, DSC measurements of native and five mutant lysozymes were carried out at acidic pH between 2.4 and 3.2. In this pH region, the denaturation of the native lysozyme was reported to be reversible (Takano et al., 1997). We obtained DSC curves of the native lysozyme similar to those in a previous study (Ueda et al., 2000). In all mutant proteins, we also obtained a shape of the DSC curves similar to that of native lysozyme. The denaturation temperature (Td), the calorimetric enthalpies (
Hcal) and the van't Hoff enthalpies (HvH) for the denaturation of lysozymes were obtained directly from analyses of these curves (Table I).
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The Td values were sensitive to pH and increased linearly with increase in pH. The temperature dependences of
Hcal for the five Gly mutant lysozymes were linear. Each Cp value was obtained from each slope by least-squares fitting of the plots of Hcal against Td (Table I
). The thermodynamic parameters for denaturation of the mutant lysozymes at the denaturation temperature of the native protein, 65.6°C, and at pH 2.7 were calculated (Table I) using the following equations:
H(T) = H(Td) – Cp(Td – T) (1)
S(T) = H(Td)/Td – Cpln(Td/T) (2)
G(T) = H(T) – TS(T) (3)
and assuming that Cp does not depend on temperature (Privalov and Khechinashvili, 1974).
As shown in Table I, the effects of the addition of one methylene group by mutating glycine to alanine on stability of hen egg white lysozyme were different in each mutant protein. The difference in G between native and mutant lysozymes ranged from 0.1 to –7.9 kJ/mol.
X-ray structure of mutant hen lysozymes
The crystals of lysozymes were formed at pH 4.7, a condition commonly used for the crystallization of lysozymes. Data collection and refinement for the native and its five mutant lysozymes are summarized in Table II. The first refinements dropped the R-values of mutant lysozymes below 23.0%. Introductions of water molecules were then carried out. The final refinements of the structures including water molecules gave R-values <20.0%, Rfree <25.0%, R-merge <10% and completeness >85%. The root mean square deviations (r.m.s.d.s) between the native and all mutants are shown in Figure 2. The mutant lysozymes were found to have essentially the same folding pattern as the native lysozyme except around the mutated residues. The local structures around the mutation sites are shown by superimposition of the mutant structures on the native structures in Figure 3. The local structures did not change very much from that of native lysozyme except for the G102A mutant. In the mutation at position 49 where the glycine residue is located at the fourth residue in the I type turn structure among two ß-sheets and at position 117 where the glycine residue is located at the third residue in the turn structure, the mutation had little effect on the local structure of the lysozyme molecule (Figure 2A and E). Only the carbonyl-oxygen of the main chain of G49A moved slightly (Figure 3A). In the mutation at position 67 where the glycine residue is located in the loop structure and at position 71 where the glycine residue is located at the third residue in the turn structured, the main chain at the mutation site moved owing to the mutation (Figure 2B and C). There were large r.m.s.d.s around residue 70 of G49A and G67A because even in native lysozyme the main chains of the sites were very flexible and were not fixed. In G49A, G67A, G71A and G117A lysozymes, the distances between the ß-carbon at the mutation site and the preceding carbonyl oxygen were 2.93, 3.11, 3.07 and 3.10 Å, respectively, which may induce an unfavorable interaction in the native state. On the other hand, at position 102 where the glycine residue is located in the long loop region, the mutation induced considerable structural change at the mutation site, resulting in contact being avoided between the ß-carbon at the mutation site and the preceding carbonyl oxygen. Also, a new hydrogen bond between the carboxyl group at Asp101 and the nitrogen of the amide bond at Gly104 formed in G102A. However, because a hydrogen bond between the amide bond at Asp101 and a fixed water molecule disappeared in the native lysozyme, the enthalpic factor will be cancelled.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Based on the loss of entropy change in the denatured state in the mutation of glycine to alanine (Matthews, 1987
), the lysozyme mutants where glycine was mutated to alanine were expected to be more stable by 3.4 kJ/mol than the native lysozyme at 65.6°C, the denaturation temperature of native lysozyme at pH 2.7. However, four of the five mutant lysozymes employed here were less stable, although one (G102A) had a stability similar to that of native lysozyme. Therefore, in the present case, it was concluded that some unfavorable interactions occurred in the native state and overcame the extent of the destabilization energy in the denatured state. These results were consistent with the results of Kimura et al. (1992) in which the mutant ribonuclease HI was more stable by mutating Lys95 whose dihedral angle is located in the region of the left-handed helix to glycine.
Considering the local structural change, we divided the five mutant lysozymes into two groups. One group included G49A, G67A, G71A and G117A, whose local structural changes are minor. The other was G102A, whose local structural change is major. In Figure 4, the dihedral angles at the mutation sites in the former group are shown. The dihedral angles at the corresponding sites in native lysozyme are also shown. Interestingly, the dihedral angles at the mutation sites tend to adopt possibly favorable values in Ramachandran plots and the extent and direction of their shifts from the original values had similar tendencies. The change in dihedral angles may be the cause of the slight local structural changes in these mutations. These local conformational changes are suggested to occur in order to avoid the unfavorable interaction in the native state accompanying the mutation. Moreover, because the observation was common in these four mutant lysozymes, the finding may be applicable to the other cases.
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We found that there were differences in the free energy change in these four mutant lysozymes. Yutani et al. (1991) discussed the effect of the mutation on the stability of the
-subunit of tryptophan synthetase from the view point of the B-factor at the mutation site in the native structure and showed that there was a relationship between the effect of the mutation on the protein stability and the B-factor at the mutation site. Plots of the difference in free energy change (G) of denaturation between mutant and native lysozymes against the B-factor at the mutation site in native lysozyme are shown in Figure 5. A slight correlation was observed between them. Therefore, it is suggested that the differences in the extent of destabilization among the five mutant lysozymes were involved in the restriction of the movement of the main chain at the mutation site.
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For G102A, because the dihedral angles at position 102 are far from the other angles (Figure 1
), the stability of the mutant lysozyme where Gly102 is mutated to alanine is expected to be more unstable than the other mutant lysozymes. However, G102A was more stable than the other mutant lysozymes and had a stability similar to that of native lysozyme. This may be due to the local conformational change where the dihedral angle at the mutation site ( = 120° and = –30°) in native lysozyme is altered to that of a favorable one ( = –60° and = –30°) in the G102A lysozyme. The preceding carbonyl oxygen at the mutation site rotated about 180°, resulting in it locating at the opposite site of the ß-carbon of Ala102. The drastic local conformational change at the mutation site may be allowed by the situation where Gly102 is located in a region fully exposed to the solvent and in the flexible long loop structure. Such a phenomenon in which the drastic local structure change compensates for instability may be very unique as a releasing mechanism in the unfavorable interaction in the native state of the protein. Consequently, in this work, we investigated the relationship between the effect of the mutation of glycine to alanine on the stability of lysozyme and the local structural change accompanying the mutation. By using a series of the mutant lysozymes, we obtained information on the releasing mechanism of the unfavorable interaction in the native state of lysozyme accompanying the mutation. This finding is novel and will be significant in understanding the stability of the protein. Therefore, these results will be useful in 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 and Mr Kazuhide Tokuyama for helpful discussions.
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References |
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Received March 21, 2000; revised July 18, 2000; accepted July 26, 2000.
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