Oxidation of methionine residues in antithrombin. Effects on biological activity and heparin binding

Commercially available human plasma-derived preparations of the serine protease inhibitor antithrombin (AT) were shown to contain low levels of oxidation, and we sought to determine whether oxidation might be a means of regulating the protein's inhibitory activity. A recombinant form of AT, with similarly low levels of oxidation as purified, was treated with hydrogen peroxide in order to study the effect of oxidation, specifically methionine oxidation, on the biochemical properties of this protein. AT contains two adjacent methionine residues near the reactive site loop cleaved by thrombin (Met314 and Met315) and two exposed methionines that border on the heparin binding region of AT (Met17 and Met20). In forced oxidations with hydrogen peroxide, the methionines at 314 and 315 were found to be the most susceptible to oxidation, but their oxidation did not affect either thrombin-inhibitory activity or heparin binding. Methionines at positions 17 and 20 were significantly oxidized only at higher concentrations of peroxide, at which point heparin affinity was decreased. However at saturating heparin concentrations, activity was only marginally decreased for these highly oxidized samples of AT. Structural studies indicate that highly oxidized AT is less able to undergo the complete conformational change induced by heparin, most probably due to oxidation of Met17. Since this does not occur in less oxidized, and presumably more physiologically relevant, forms of AT such as those found in plasma preparations, oxidation does not appear to be a means of controlling AT activity.     

Relationship between protein structure and methionine oxidation in recombinant human alpha 1-antitrypsin

alpha 1-Antitrypsin is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors. Although it is known that methionine oxidation in the protein's active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of each of the protein's methionine residues. In this study, we have used peptide mapping to study the oxidation kinetics of each of alpha 1-antitrypsin's methionines in alpha 1-AT((C232S)) as well as M351L and M358V mutants. These kinetic studies establish that Met1, Met226, Met242, Met351, and Met358 are reactive with hydrogen peroxide at neutral pH and that each reactive methionine is oxidized in a bimolecular, rather than coupled, mechanism. Analysis of Met226, Met351, and Met358 oxidation provides insights regarding the structure of alpha 1-antitrypsin's active site that allow us to relate conformation to experimentally observed reactivity. The relationship between solution pH and methionine oxidation was also examined to evaluate methionine reactivity under conditions that perturb the native structure. Methionine oxidation data show that at pH 5, global conformational changes occur that alter the oxidation susceptibility of each of alpha 1-antitrypsin's 10 methionine residues. Between pH 6 and 9, however, more localized conformational changes occur that affect primarily the reactivity of Met242. In sum, this work provides a detailed analysis of methionine oxidation in alpha 1-antitrypsin and offers new insights into the protein's solution structure.     

Tertiary structural rearrangements upon oxidation of Methionine145 in calmodulin promotes targeted proteasomal degradation

The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.     

Redox modulation of cellular signaling and metabolism through reversible oxidation of methionine sensors in calcium regulatory proteins

Adaptive responses associated with environmental stressors are critical to cell survival. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met(144) or Met(145) results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylation-dependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in response to beta-adrenergic signaling in the heart. We suggest that under acute conditions, such as inflammation or ischemia, these types of mechanisms ensure minimal nonspecific cellular damage, allowing for rapid restoration of cellular function through repair of oxidized methionines by methionine sulfoxide reductases and degradation pathways after restoration of normal cellular redox conditions.     

Local flexibility facilitates oxidization of buried methionine residues

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.     

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.     

The oxidative inactivation of plasminogen activator inhibitor type 1 results from a conformational change in the molecule and does not require the involvement of the P1' methionine.

Plasminogen activator inhibitor 1 (PAI-1) is sensitive to oxidative inactivation, and it has been suggested that specific oxidation of a methionine residue, Met347, situated in the P1' position of the reactive center may be the cause of the inactivation. To test this hypothesis we have purified and biochemically characterized mutant proteins of PAI-1 in which Met347 and either of two other methionines, Met266 or Met354, has been replaced with oxidation-resistant valine residues. The mutant proteins were found to be equally sensitive to oxidation as wild-type PAI-1, suggesting that a specific oxidation of the P1' Met347 is not responsible for the inactivation. When PAI-1 was oxidized, circular dichroism analysis revealed a rapid conformational change that correlated to the loss of inhibitory activity. The oxidation sensitivity of PAI-1 was enhanced dramatically in the presence of 0.001% sodium dodecyl sulfate, and the circular dichroism spectrum was significantly different from that of untreated PAI-1, suggesting that the increased sensitivity to oxidation may be caused by a conformational change in the inhibitor molecule. Taken together, our data suggest that the oxidative inactivation of PAI-1 is not caused by the specific oxidation of the P1' methionine but results from a conformational change in the protein structure.     

3-Hydroxykynurenine oxidizes alpha-crystallin: potential role in cataractogenesis

The alpha-, beta-, and gamma-crystallins are the major structural proteins of mammalian lenses. The human lens also contains tryptophan-derived UV filters, which are known to spontaneously deaminate at physiological pH and covalently attach to lens proteins. 3-Hydroxykynurenine (3OHKyn) is the third most abundant of the kynurenine UV filters in the lens, and previous studies have shown this compound to be unstable and to be oxidized under physiological conditions, producing H2O2. In this study, we show that methionine and tryptophan amino acid residues are oxidized when bovine alpha-crystallin is incubated with 3-hydroxykynurenine. We observed almost complete oxidation of methionines 1 and 138 in alphaA-crystallin and a similar extent of oxidation of methionines 1 and 68 in alphaB-crystallin after 48 h. Tryptophans 9 and 60 in alphaB-crystallin were oxidized to a lesser extent. AlphaA-crystallin was also found to have 3OHKyn bound to its single cysteine residue. Examination of normal aged human lenses revealed no evidence of oxidation of alpha-crystallin; however, oxidation was detected at methionine 1 in both alphaA- and alphaB-crystallin from human cataractous lenses. Age-related nuclear cataract is associated with coloration and insolubilization of lens proteins and extensive oxidation of cysteine and methionine residues. Our findings demonstrate that 3-hydroxykynurenine can readily catalyze the oxidation of methionine residues in both alphaB- and alphaA-crystallin, and it has been reported that alpha-crystallin modified in this way is a poorer chaperone. Thus, 3-hydroxykynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens.     

Role of individual methionines in the fibrillation of methionine-oxidized alpha-synuclein

The aggregation of normally soluble alpha-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. We have previously established that oxidation of all four methionine residues in alpha-synuclein (to the sulfoxide, MetO) inhibits fibrillation of this protein in vitro and that the MetO protein also inhibits fibrillation of unmodified alpha-synuclein. Here we show that the degree of inhibition of fibrillation by MetO alpha-synuclein is proportional to the number of oxidized methionines. This was accomplished be selectively converting Met residues into Leu, prior to Met oxidation. The results showed that with one oxidized Met the kinetics of fibrillation were comparable to those for the control (nonoxidized), and with increasing numbers of methionine sulfoxides the kinetics of fibrillation became progressively slower. Electron microscope images showed that the fibril morphology was similar for all species examined, although fewer fibrils were observed with the oxidized forms. The presence of zinc was shown to overcome the Met oxidation-induced inhibition. Interestingly, substitution of Met by Leu led to increased propensity for aggregation (soluble oligomers) but slower formation of fibrils.     

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met[O]) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.Key words: immunoglobulin gamma antibody, methionine sulfoxide, oxidation, photo-oxidation, methionine sulfoxide reductase     

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met(O)) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.     

Site-specific methionine oxidation in calmodulin affects structural integrity and interaction with Ca2+/calmodulin-dependent protein kinase II

Methionine oxidation in the ubiquitous calcium signaling protein calmodulin (CaM) is known to disrupt downstream signaling and target CaM for proteasomal degradation. The susceptibility of CaM to oxidation in the different conformations that are sampled during calcium signaling is currently not well defined. Using an integrative mass spectrometry (MS) approach, applying both native MS and LC/MS/MS, we unravel molecular details of CaM methionine oxidation in the context of its interaction with the Ca(2+)/CaM-dependent protein kinase II (CaMKII). Sensitivity to methionine oxidation in CaM was found to vary according to the conformational state. Three methionine residues (Met71, 72, 145) show increased reactivity in calcium-saturated CaM (holo-CaM) compared to calcium-free CaM (apo-CaM), which has important consequences for oxidation-targeted proteasomal degradation. In addition, all four methionines in the C-terminal lobe (Met109, 124, 144 and 145) are found to be protected from oxidation in a peptide-based model of the CaMKII-bound conformation (cbp-CaM). We furthermore demonstrate that the oxidation of Met144 and 145 inhibits the interaction of CaM with CaMKII. cbp-CaM, in contrast to apo- and holo-CaM, maintains its ability to bind CaMKII under simulated conditions of oxidative stress and is also protected from oxidation-induced unfolding. Thus, we show that the susceptibility towards oxidation of specific residues in CaM is tightly linked to its signaling state and conformation, which has direct implications for calcium/CaM-CaMKII related signaling.     

Oxidation of Met144 and Met145 in calmodulin blocks calmodulin dependent activation of the plasma membrane Ca-ATPase

Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met(144) and Met(145) near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met(144) and Met(145). Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM. Likewise, oxidation of individual methionines has a minimal effect on the CaM concentration necessary for half-maximal activation of the PM-Ca-ATPase. These results are consistent with previous suggestions that no single methionine within CaM is essential for activation of the PM-Ca-ATPase. Oxidation of either Met(144) and Met(145) or all nine methionines in CaM results in an equivalent inhibition of the PM-Ca-ATPase, resulting in a 50-60% reduction in the level of enzyme activation. Oxidation of Met(144) is largely responsible for the decreased extent of enzyme activation, suggesting that this site is critical in modulating the sensitivity of CaM to oxidant-induced loss-of-function. These results are discussed in terms of a possible functional role for Met(144) and Met(145) in CaM as redox sensors that function to modulate calcium homeostasis and energy metabolism in response to conditions of oxidative stress.     

Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.     

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 +/- 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 +/- 4 microM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.     

Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.

The biological activity of some proteins is known to be sensitive to oxidative damage caused by a variety of oxidants. The model protein staphylococcal nuclease was used to explore the effect on protein structural stability of oxidizing methionine to the sulfoxide form. These effects were compared with the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Wild-type nuclease and various mutants were oxidized with hydrogen peroxide. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Oxidation destabilized the wild-type protein by over 4 kcal/mol. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the protein native state. Comparison of mutant protein's stability losses upon oxidation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32. While substitution of methionine 98 carried as great an energetic penalty as oxidation, substitution at position 65 was less disruptive than oxidation. Thus a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.     

Redox proteomics of protein-bound methionine oxidation

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.     

Thioredoxin-dependent redox regulation of cellular signaling and stress response through reversible oxidation of methionines

The sensitive oxidations of sulfur containing amino acids (i.e., cysteines and methionines) commonly control protein function, and act as important signaling mechanisms to modify metabolic responses to environmental stressors. Mechanisms associated with cysteine oxidation to form sulfenic acid and disulfides (i.e., cystine and glutathione adducts), and their reversibility through thioredoxin-dependent mechanisms, are broadly appreciated as important regulatory mechanisms that control the function of a range of different proteins. Less commonly understood are the cellular consequences of methionine oxidation to form methionine sulfoxide, as the structural requirements for their thioredoxin-dependent reduction by methionine sulfoxide reductases limit the reversibility of methionine oxidation to sequences within surface exposed and conformationally disordered regions of proteins. Surface exposed methionines are commonly involved in molecular recognition between transient protein signaling complexes, where their oxidation disrupts productive protein-protein interactions linked to a range of cellular responses. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress responses in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the physical basis for differences in the sensitivity of individual methionines within plant and animal calmodulin to reactive oxygen species (ROS), the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes. It is suggested that, in combination with high-throughput proteomic methods and current generation informatics tools, these mechanistic insights permit useful predictions of oxidatively sensitive signaling proteins that act as redox and stress sensors in response to methionine oxidation.     

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.