Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
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Abstract |
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Keywords: hen egg white lysozyme/isoleucine/`safe' substitution/selenomethionine/X-ray crystallography
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Introduction |
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From a comparison of many closely related protein structures, the `safe' residue substitutions that do not disturb the protein structure were shown (Bordo and Argos, 1991) (Figure 1
). Leucine was shown to be the only candidate for `safe' residue substitution of methionine and has a similar hydrophobicity to methionine [difference ~0.5 kcal/mol (Finney et al., 1980
; Guy, 1985
)]. Experimental data presented in a previous report (Gassner et al., 1996
) showed that the loss of stability in the mutant T4 lysozyme where leucine was mutated to methionine was from 0.4 to 0.8 kcal/mol (for only one mutation was it 1.9 kcal/mol). However, all leucine residues cannot always be substituted for methionine residues and they may not be suitably located where a selenomethionine residue should be introduced. Therefore, another substituent of methionine would be required. As an isoleucine residue has a similar volume to a leucine residue, it should be the next candidate for the choice of a substituent for methionine. However, isoleucine was not considered to be a `safe' residue for substitutions of methionine (Bordo and Argos, 1991
). The change in hydrophobicity from isoleucine to methionine was larger than that from leucine to methionine by about 0.5 kcal/mol (Finney et al., 1980
; Guy, 1985
). The instabilities of two mutants of T4 lysozyme where isoleucine at the hydrophobic core was mutated to methionine were 1.5 and 1.6 kcal/mol (Gassner et al., 1996
). Hence it is desirable to examine the possibility of substitution of methionine for isoleucine. The secure substitution of methionine for isoleucine in a protein molecule means being able to introduce selenomethionine into the position where isoleucine is located. Therefore, it is useful to examine whether isoleucine in protein structures is replaced with methionine for the development of a method for the determination of protein structure using X-ray crystallography. So far, substitutions of methionine for isoleucine have been reported in several proteins (Leahy et al., 1994
; Skinner et al., 1994
; Gassner and Matthews, 1999
). However, these substitutions of methionine for isoleucine were conducted only in hydrophobic cores or in completely buried positions, and not systematically.
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Materials and methods |
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Restriction enzymes, T4 polynucleotide kinase and DNA polymerase I (Klenow fragment) were purchased from either Takara Shuzo (Kyoto, Japan) or New England Biolabs (Beverly, MA). Micrococcus luteus and DNA sequencing kits (Sequenase) were purchased from Sigma and Amersham-Pharmacia Japan (Tokyo, Japan), respectively. CM-Toyopearl 650M, a cation-exchange resin for the purification of secreted hen lysozymes, was obtained from Tosoh (Tokyo, Japan).
Mutant lysozymes
Mutation of the lysozyme gene was carried out by site-directed mutagenesis using the method of Kunkel et al. (Kunkel et al., 1987), as described previously (Hashimoto et al., 1996
). The DNA oligonucleotides used as the 5'- and 3'-mutagenic primers (about 20 bases long) were designed such that the codon for Ile58, -78 or -98 was changed from ATC to ATG for Met. For the construction of the single mutant proteins at three isoleucine residues (58, 78 and 98), the wild-type lysozyme (WT) gene in the M13 phage pKP1700 was used as a template. The mutant lysozymes where Ile58, -78 and -98 were mutated to Met are abbreviated to I58M, I78M and I98M, respectively.
Purification and identification of lysozyme secreted by yeast
Each transformant of Saccharomyces cerevisiae AH22 was cultivated at 30°C for 125 h for expression and secretion of the respective mutant lysozyme (Hashimoto et al., 1996). Purification (ion-exchange chromatography) and identification (peptide mapping, amino acid sequencing and amino acid composition) of the lysozymes were carried out as reported previously (Ueda et al., 1993
).
Unfolding equilibrium
The unfolding equilibrium of lysozymes by guanidine hydrochloride (Gdn.HCl) was determined at pH 5.5 and 35°C by measuring the fluorescence at 360 nm (excitation at 280 nm). The protein concentration was 0.9x107 M. The buffer used was 0.1 M sodium acetate adjusted to pH 5.5 with HCl. It has been found experimentally that the free energy of unfolding of proteins in the presence of Gdn.HCl is linearly related to the concentration of denaturant (Pace, 1986). Details of the analysis were as described (Inoue et al., 1992
) and the average m value of WT and isoleucine to methionine (IM) mutants was employed.
X-ray analysis
Crystallization was carried out using a hanging drop vapor diffusion technique at pH 4.7. Intensity data collection for WT and IM mutants was carried out with an R-AXIS IIC automated oscillation camera system (Rigaku), equipped with an imaging plate detector, on a Cu rotating-anode generator operated at 40 keV and 120 mA at room temperature. Refinement of the structure was carried out using the programs TURBO-FRODO (Roussel et al., 1990) and X-PLOR (Brunger, 1992
) installed on an SGI Indigo2. The crystallographic data for WT and IM mutants are shown in Table I
. Each sample was measured at a resolution in the range 1.631.91 Å, R-merge was <9% and data completeness was >84%. The refinements of the mutant lysozymes were carried out as described previously (Ohmura et al., 1997
). Coordinates for WT have been deposited in the Protein Data Bank (Brookhaven National Laboratory) (accession number 1RFP) and coordinates for all IM mutants (I58M, I78M and I98M) have also been deposited in the Protein Data Bank.
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Results |
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We designed three mutants in which Ile is mutated to Met in lysozyme (IM mutant). Position 58 is located in an anti-parallel ß-sheet and buried in the interior of the lysozyme molecule. Position 78 is located on the surface in lysozyme molecule but partially covered by the nearby residues. Position 98 is located in an -helix and buried in the interior of the lysozyme molecule. Stabilities of all the IM mutants were measured by a Gdn.HCl denaturation experiment. I58M was less stable than WT by about 1.2 kcal/mol (Table II
). Both I78M and I98M were less stable than WT by 0.9 kcal/mol (Table II
).
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After the refinement of the crystal structures, R-factors of all mutants fell to ~17%. The overall and local structures of all IM mutants were similar to those of WT (Figures 3 and 4). The side chains of all substituted methionine residues were placed mimicking the C
1C
1 chain of isoleucine residue in WT (Figure 4
). The dihedral angle and average B-factor of the side chain of the mutated residue are shown in Table III
. There were no unfavorable contacts in these structures (Figure 5
). The solvent-accessible surface areas at the mutated residue in IM mutants (I58M, 3; I78M, 36; and I98M, 10 Å2) were almost the same as those of WT (I58, 1; I78, 34; and I98, 8 Å2).
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Discussion |
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The side chain atoms (Cß, C and S
) in methionine of the respective mutant lysozymes were located at overlapped positions with the respective side chain atoms (Cß, C
1 and C
1) of isoleucine in WT (Figure 4
). As a result, there were few changes in local structures and in the solvent-accessible surface areas in mutant lysozymes. The crystal structure of gene V protein where Ile47 is mutated to Met has been solved at 1.8 Å resolution (Skinner et al., 1994
). The conformation of methionine in this mutant protein was also located at an overlapped position in side chain atoms of isoleucine in the intact protein. This result was consistent with the present results. Therefore, these coincidences of the side chain conformation in a protein where isoleucine residues are mutated to methionine residues indicated that the side chain conformation of methionine in a mutant protein may mimic that of isoleucine in an intact protein.
The dihedral angles of the side chain of the mutated residues did not variy except for 1 of I78M (Table III
). The change of conformation energy calculated for
1 of residue 78 was small. Therefore, it would hardly influence the protein stability. The average B-factors of the side chains of the mutated residues were slightly larger than that of WT (Table III
). The increased B-factor of the side chain may reflect the increased entropy and/or the decreased enthalpy of the mutated residue. Funahashi et al. reported on the protein stability of IM mutant at positions 56 and 59 of human lysozyme (Funahashi et al., 1999
). The I59M in human lysozyme corresponds to I58M in hen lysozyme. Decreased enthalpies were observed in IM mutants of human lysozyme. The same explanation would be applicable to IM mutant of hen lysozyme, although the result for human lysozyme could not be applied simply to that for hen lysozyme. X-ray crystallographic data show that the errors in the data for mutant lysozymes were slightly larger than that in WT (Table I
). The largest B-factor was seen in mutated residue 78. Residue 78 was buried 81% and the B-factor seen here was comparable to those of the residues having similar accessibility. As a result, it could be considered that the observed increase in B-factors in the mutant residues deserves no special attention. Hence we can conclude that the substitution of methionine for isoleucine did not cause substantial structural changes.
Other workers also reported the security of the substitution of methionine for isoleucine (Leahy et al., 1994; Skinner et al., 1994
; Gasnner and Matthews, 1999,). Thus, in general, the substitution of methionine for isoleucine would be considered as a `safe' residue substitution in a protein. Although Gasnner and Matthews mentioned that the second-best substitution is Phe
Met rather than Ile
Met, we thought that the second-best substitution would be Ile
Met in terms of hydrophobicity, side chain volume and Dayhoff mutation probability.
In conclusion, it has been shown that the substitution of methionine for isoleucine in a protein could be performed without any significant change of the overall and local structure. Thus, isoleucine would be the second candidate after leucine as the substituent of methionine. Hence this substituent could be considered as a `safe' residue substitution but the reverse substitution remains unknown. Our present findings provide valuable information on point mutations of amino acid residues in a protein and may contribute to the determination of novel protein structures with the mutation from isoleucine to selenomethionine by using the multiple-wavelength anomalous dispersion method.
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Notes |
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References |
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Received October 12, 2000; revised February 21, 2001; accepted March 5, 2001.