Loss of Conformational Stability in Calmodulin upon Methionine Oxidation

Abstract

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM.

Selective Degradation of Oxidized Calmodulin by the 20 S Proteasome

“…Calcium-dependent Changes in Secondary Structure of CaM Correlate with Degradation Rate-To distinguish whether the increased degradation rate of CaM ox by the proteasome reflects a preferential recognition of methionine sulfoxide or, rather, to the resulting loss of CaM secondary structure, we took advantage of the fact that the ␣-helical content of extensively oxidized CaM increases dramatically upon calcium binding, so that calcium-saturated CaM ox assumes a native-like structure (22,32). Accordingly, we have compared the calcium dependence of the degradation rate of extensively oxidized CaM containing 8.2 Ϯ 0.5 methionine sulfoxides with changes in the secondary structure, as measured by the molar ellipticity at 222 nm.…”

“…Mass Spectrometric Analysis-Electrospray ionization mass spectrometry (ESI-MS) was used to 1) quantify the distribution of CaM oxiforms and determine the average methionine sulfoxide concentration for each sample and 2) identify masses of CaM peptides resulting from proteolytic cleavage, essentially as described previously (17)(18)(19)32). Identification of released peptides was assisted by the software GPMAW (Lighthouse Data, Aalokken 14, DK-5250; Odense SV, Denmark), which permitted a unique identification for approximately onehalf of the released peptides.…”

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

“…Susceptible methionines are typically located on the surface of the protein and exposed to the solvent. [7][8][9][10] Met oxidation can have adverse effects on proteins, including decreased stability, 5,[11][12][13][14][15][16] and decreased biological activity. 5,8,12,[17][18][19][20] The immunoglobulin gamma (IgG) monoclonal antibody (mAb) has emerged as one of the most promising drug class in the biopharmaceutical industry.…”

Functional and Structural Aspects of Poplar Cytosolic and Plastidial Type A Methionine Sulfoxide Reductases

“…Enzymes named methionine sulfoxide reductases were found to catalyze the reduction of MetSO back to methionine residues (4,5). The consequences of this side-chain modification are variable and can be partial to protein unfolding (6,7) and modification of biological functions (8 -10). Sometimes surface methionine residues can undergo oxidation without much impact on the protein properties, and this modification can be seen as a mechanism to scavenge oxidative species in a detoxification process based on methionine sulfoxide reductase activity (11).…”