Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

Abstract

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.

Keywords: mass spectrometry (MS); methionine; oxidation-reduction; protein stability; proteomics.

Figure 1

Schematic representations of proposed model and methods.A, for buried methionines, protein unfolding precedes oxidation. Oxidation protection factors (PFs) of buried methionines are defined as the ratio of methionine oxidation rates from the unfolded state (k_intrinsic) and the observed oxidation rate (k_protein). According to a two-state folding model, PFs are related to folding stability (ΔG_folding) and the midpoint of denaturation (C_1/2) as indicated in the right side of the figure. B, the three proteomic methods employed in this study. Intrinsic oxidation rates (k_intrinsic) were measured using trypsin-digested proteins. Oxidation rates in intact proteins (k_protein) were measured using native protein lysates. Folding stabilities (C_1/2 values) were measured by SPROX. This method exposes native extracts to increasing concentrations chemical denaturants prior to oxidation. SPROX, stability of proteins from rates of oxidation.

Figure 2

k_intrinsicmeasurements and neighboring residue effects in the Escherichia coli proteome.A, experimental method. Trypsin-digested peptide samples were exposed to varying concentrations of H₂O_2. Fractional oxidation levels were measured with LC-MS/MS and used to calculate pseudo-first order oxidation rate constants. In the schematic, closed ovals indicate methionines in the native state, open squares indicate exposed methionines in peptides and red squares indicate oxidized methionines. B, relative distribution of k_intrinsic measurements in the E. coli proteome. The dotted line indicates the distribution median. C and D, effect of neighboring residues on k_intrinsic of methionines. E, effect of position relative to the N-terminus on k_intrinsic of methionines. For C–E, boxes indicate the interquartile range and whiskers show the entire range of values excluding outliers (>2 SD). ∗∗ indicates a p-value < 0.001 using a Mann-Whitney U test, corrected for family-wise error rate with a Holm–Šidák method (significance level = 0.001).

Figure 3

k_proteinmeasurements within native proteins and correlations with solvent accessibility (SA).A, experimental method. Native lysates were treated with H₂O₂ using a range of oxidation times. Fractional oxidation levels were measured with LC-MS/MS and used to calculate pseudo-first order oxidation rate constants. In the schematic, ovals indicate methionines in the native state, squares indicate exposed methionines in peptides, black indicates unoxidized methionines, and red indicates oxidized methionines. B, the relative log₁₀ distribution of k_protein measurements. The dotted line indicates the distribution median. Gray and white bars indicate protected and unprotected methionines as defined in the text. C, the relative distribution of PFs in the E. coli proteome. D, the relationship between PF and SA measurements. Methionines were categorized into three groups (buried, partially buried, and exposed) based on their SA values. Boxes indicate the interquartile range and whiskers show the entire range of values excluding outliers (>2 SD). The total pairwise comparison between PFs and SAs had a significant level of correlation (Spearman rank test ρ <0.001). PFs, protection factors.

Figure 4

C_1/2measurements within native proteins and correlations with PFs.A, experimental method (SPROX). Native lysates were treated with increasing concentrations of denaturant (GdmCl) prior to oxidation with H₂O₂ and subsequent digestion with trypsin. Fractional oxidation levels were measured with LC-MS/MS and used to measure C_1/2 values. Symbols in the schematic are described in Figure 3. B, the relative distribution of C_1/2 values in the Escherichia coli proteome. C and D, the relationship between PF and C_1/2 values for all methionines (C) and highly protected methionines (D). Boxes indicate the interquartile range and whiskers show the entire range of values excluding outliers (>2 SD). The Spearman rank correlation values indicate the correlation for total pairwise comparisons. PFs, protection factors; SPROX, stability of proteins from rates of oxidation.

Figure 5

Comparison of C_1/2and PF measurements between Escherichia coli and Thermus thermophilus proteomes.A, comparison of the distribution of C_1/2 values for E. coli and T. thermophilus at 25 °C. B, comparison of the distributions of PF values for buried methionines within E. coli and T. thermophilus at varying temperatures. C, comparison of the distributions of SAs for methionines analyzed in (B). Boxes indicate the interquartile range and whiskers show the entire range of values excluding outliers (>2 SD). ∗ and ∗∗ indicate p-values greater than 0.05 and 0.001, respectively, using a Mann-Whitney U test. PF, protection factor; SA, solvent accessibility.