Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 727011021, USA
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Abstract |
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Keywords: methionine oxidation/methionine sulfoxide/mutagenesis/protein stability/staphylococcal nuclease
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Introduction |
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Methionine, cysteine, tryptophan, tyrosine and histidine residues are susceptible to oxidation. The oxidation of methionine to the sulfoxide form is of particular interest as it has been shown to occur in a wide variety of proteins and often reduces or eliminates biological activity (Brot and Weissbach, 1991; Vogt, 1995
). Natural biological oxidants (Glaser et al., 1992
; Fliss and Menard, 1994
; Fujii et al., 1994
), cigarette smoke (Gadek et al., 1979
; Boudier et al., 1983
) and other environmental oxidants (Banerjee and Mudd, 1992
; Blaurock et al., 1992
) have all been demonstrated to cause methionine sulfoxide formation in proteins and peptides. Methionine oxidation is of serious concern when proteins are used as pharmaceuticals (Fransson et al., 1996
; Gitlin et al., 1996
; Berti et al., 1997
; Konz et al., 1998
; Liu et al., 1998
; Kornfelt et al., 1999
) because of the possible effects on activity and the possibility of oxidation during processing or storage.
While it is not always understood how methionine oxidation changes the biological activity of a protein, oxidation is known in some cases to destabilize a protein's native structure (Volkin et al., 1997; Gao et al., 1998b
; Liu et al., 1998
; DalleDonne et al., 1999
) with obvious consequences for activity. It is well known that side chain substitution can have profound effects on protein stability and structure. In effect oxidation can be regarded as a chemical `mutagenesis' which substitutes the methionine side chain with methionine sulfoxide, a larger and more polar side chain. It should not be surprising that it can greatly disrupt protein structure and stability.
A potential strategy for protecting a protein against the effects of methionine oxidation is to replace those residues with some side chain which is resistant to oxidation. This has been attempted in a few cases, usually directed at preventing methionine oxidation that directly affects activity by altering an active or binding site. 1-Antitrypsin (Rosenberg et al., 1984
), subtilisin (Estell et al., 1985
; Bott et al., 1988
) and D-amino acid oxidase (Ju et al., 2000
) have all had the effect of methionine replacement on oxidative inactivation assessed. However, in only one case that we are aware of has the effect of methionine oxidation upon stability been measured combined with mutagenesis to desensitize the protein toward oxidation. Even that study (Lu et al., 1999
), on human granulocyte colony-stimulating factor, examined only the stability effects of oxidation itself and not the stability effects of mutagenesis.
Such a comparison is important since it is plausible that a site that is sensitive to the effects of oxidation might be equally sensitive to the effects of side chain substitution. The oxidation of methionine presumably affects protein structure and stability by reducing side chain hydrophobicity, increasing the capacity for hydrogen bonding and altering the size and shape of the methionine. The relative importance of these factors will depend on the particular environment of a given methionine. For example, somewhat counterintuitively it has been shown (Chao et al., 1997) that methionine oxidation can actually increase the hydrophobicity of a protein surface, presumably through conformational changes of some sort, but the energetic consequences of such rearrangement are not clear and neither are the effects of substitution. It is equally plausible to imagine that if the cause of destabilization upon oxidation is the increase in side chain polarity then the effects of substitution with a non-polar side chain may be minor.
At our present state of knowledge it is difficult to predict the degree of stability change upon oxidation of a given methionine residue, much less whether or not side chain substitution is a viable approach to moderate those effects. To further understanding of these issues, in this study we examined the effects of methionine oxidation upon the stability of staphylococcal nuclease, a well-characterized model protein. We determined the relative effects of oxidizing each methionine and we compared oxidation stability effects with those of mutating each methionine to alanine, glycine, isoleucine or leucine.
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Materials and methods |
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Procedure for methionine oxidation
Hydrogen peroxide, a well-known oxidizing agent fairly specific for methionine and cysteine (Shechter et al., 1975), was used. To 0.12 µmol (192.3 µl) of wild-type or mutant (M26I, M32I, M65L, M98L) staphylococcal nuclease at a concentration of 2.6 mg/ml (pH 7.0, 25 mM sodium phosphate, 100 mM NaCl) were added 11.2 µl of 31.6% (w/w) hydrogen peroxide (~950 equiv. for each of the four methionines in wild-type). This solution was stirred for 30 min at room temperature. The reaction mixture was then quenched with a large excess (25.2 µl, ~3000 equiv. based on four methionines) of ß-mercaptoethanol. The oxidized proteins were stored at 20°C until stabilities were determined.
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Results |
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Discussion |
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There are four methionines in wild-type staphylococcal nuclease. Their positions are shown in Figure 2. The stabilities of the isoleucine, leucine and alanine substitutions for each of the four methionines are summarized in Table I
. Data for the effects of glycine substitution are available and are also included for completeness, although such a mutation would usually be expected to be far too destabilizing for use in engineering oxidative resistance. In general, the effects of mutagenesis are relatively minor at positions 26 and 32, with much less than 1 kcal/mol of stability lost upon leucine or isoleucine substitution. Methionine 98 is clearly the position most sensitive to substitution, as expected given its lack of solvent exposure (Shortle et al., 1990
). Methionine 65 shows the most complicated behavior, with leucine substitution having relatively little effect, whereas isoleucine has a significantly greater effect. The alanine substitution at 65 is only slightly worse than the alanine substitution at 26 or 32, the least sensitive sites, while glycine substitution at 65 is severely destabilizing.
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More telling is the difference in stability between the oxidized and unoxidized proteins. This G value is nearly the same for wild-type, M32I and M26I, indicating that the removal of these residues does little to prevent the oxidative destabilization of staphylococcal nuclease. On the other hand, the removal of either methionine 98 or 65 reduced the value of
G by about 2.0 kcal/mol in each case, nearly half of the wild-type
G. This is consistent with these two residues accounting for most of the effects observed in wild-type.
The comparison of the effects of mutation to oxidation of each residue is informative. Not surprisingly, methionines 26 and 32 were not particularly sensitive to either mutation or oxidation. Indeed, substitution seemed, if anything, more disruptive than oxidation at these two sites. There would be little need to alter a methionine showing similar behavior in any protein unless such a residue interfered with activity because it was in an active or binding site. However, there would also be relatively little harm from a structural stability viewpoint in doing so if necessary.
The two residues that proved to cause most of the oxidative destabilization differed in their reaction to substitution. Oxidation of methionine 98 destabilized the protein by 2.0 kcal/mol, but the effects of substitutions were as bad or even worse. Hence although substitution of the methionine prevented oxidative damage in the case of positions such as methionine 98, it will also be necessary to make substitutions at other sites in the protein to find a stable structural solution. This is in contrast to the behavior of methionine 65. Oxidation of this side chain also destabilized the protein by 2.0 kcal/mol. Whereas three substitutions had effects that were about the same or worse than oxidation, one substitution, M65L, was significantly less destabilizing. The M65L substitution is not perfect, destabilizing the protein by 0.8 kcal/mol, but it is a noteworthy improvement over the effects of oxidation.
It has been proposed that methionine residues might act as intrinsic scavengers of various reactive oxygen species and thus protect other components of the cell against oxidative damage (Levine et al., 1999). This might lead to the supposition that removal of methionine would actually increase oxidative sensitivity. Our results show that this hypothesis is untrue in the specific case of staphylococcal nuclease, at least as far as damage to the protein itself is concerned. Of course, this does not mean that other sensitive components of a cell might be protected by sacrificial oxidation of methionine in a particular protein. However, the strategy suggested here is more likely to find application in situations where purified protein is being used and protection of other cellular components is moot.
In the future, as our ability to predict the effects of single and multiple mutations improves, it should be relatively easy to engineer a protein to resist oxidative destabilization. Unfortunately, our current state of knowledge is such that predicting the other changes needed to compensate completely for the effects of methionine substitution is not easy or reliable. Our results here show that simple substitution of some methionines helps to render a protein more structurally resistant to oxidation. Since every 1.34 kcal/mol represents a 10-fold increase in the amount of folded protein at 20°C, even modest prevention of stability loss can do much to retain activity, making this simple approach a viable strategy.
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Notes |
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2 To whom correspondence should be addressed. E-mail: wstites{at}uark.edu
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Acknowledgments |
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References |
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Received August 31, 2000; revised December 23, 2000; accepted February 22, 2001.