Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G

Oxidation of methionine (Met) residues is one of the most common protein degradation pathways. Two Met residues, Met256 and Met432, of a recombinant fully human monoclonal IgG1 antibody have been shown to be susceptible to oxidation. Met256 and Met432 are located in the antibody CH2-CH3 interface and in close proximity to protein A and protein G binding sites. The effect of oxidation of these susceptible Met residues on the binding to protein A and protein G was investigated in the current study. Incubation of the antibody with 5% tert-butyl hydroperoxide (tBHP) resulted in a nearly complete oxidation of Met256 and Met432, while incubation with 1% tBHP resulted in mixed populations of the antibody with different degrees of Met oxidation. Oxidation of Met256 and Met432 resulted in earlier elution of the antibody from protein A and protein G columns when eluted with a gradient of decreasing pH. Analysis by ELISA and surface plasmon resonance (SPR) revealed decreased binding affinity of the oxidized antibody to protein A and protein G. It is therefore concluded that oxidation of the Met256 and Met432 residues of the recombinant monoclonal antibody altered its interaction with protein A and protein G resulting in a decrease in binding affinity.     

Chemical modifications in therapeutic protein aggregates generated under different stress conditions

In this study, we characterized the chemical modifications in the monoclonal antibody (IgG(2)) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met(246), His(304), and His(427) in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxide-treated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.     

Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

Methionine (Met) oxidation is a major degradation pathway of protein therapeutics. Met oxidation of a fully human recombinant monoclonal antibody was investigated under both chemically stressed conditions using tert-butylhydroperoxide (tBHP) and thermal stability conditions where the sample was incubated in formulation buffer at 25 degrees C for 12 months. This antibody has one Met residue on each of the light chains and four Met residues on each of the heavy chains. In the thermal stability sample, only Met residues 256 and 432 in the Fc region were oxidized to form methionine sulfoxide, while Met residues in the Fab region were relatively stable. The susceptibility of Met residues 256 and 432 was further confirmed by incubating samples with tBHP, which has been shown to induce Met oxidation. Further analysis revealed that the susceptible Met residues of each heavy chain were randomly oxidized in samples incubated with tBHP, while in the thermal stability sample, the susceptible Met residues of one heavy chain were preferentially oxidized.     

Free-energy simulations of the oxidation of c-terminal methionines in calmodulin

In the course of aging or under conditions of oxidative stress, methionine residues of calmodulin undergo oxidation, leading to loss of biological activity of the protein. We have performed free-energy simulations of the effects of C-terminal methionine side-chain oxidation on the properties of calmodulin. The simulation results indicate that oxidation should have a destabilizing effect on all three protein functional states: calcium free, calcium loaded, and with both calcium and target peptide bound. Because the different states are destabilized by different amounts, this leads to a more complex pattern in the observable effects on protein thermal stability, calcium affinity, and binding of a target peptide. The influence of oxidation on the free energy of CaM unfolding is estimated by comparing the free-energy cost of oxidizing a Met residue in a Gly-Met-Gly peptide and in the protein. The protein thermal stability of the oxidized forms is lowered by a moderate amount 1-3 kcal/mol, in qualitative agreement with experimental results of 0.3 kcal/mol. The calculated changes in affinity for calcium and for the target peptide show opposing trends. Oxidation at position 144 is predicted to enhance peptide binding and weaken calcium binding, whereas oxidation at 145 weakens peptide binding and enhances affinity for calcium. The lower affinity of Met 145-oxidized calmodulin toward the target peptide correlates with experimentally observed lowering of calmodulin-activated Ca-ATPase activity when oxidized calmodulin from aged rat brains is used. Thus, our simulations suggest that Met 145 is the oxidation site in the C-terminal fragment of calmodulin. The microscopic mechanism behind the calculated free energy changes appears to be a greater affinity for water of the oxidized Met side-chain relative to normal Met. Structures with Met exposed to solvent had consistently lower free energies than those with buried Met sidechains.     

Constitutive activation of met kinase in non-small-cell lung carcinomas correlates with anchorage-independent cell survival

Lung cancer is currently the most frequent cause of cancer death in North America. Hepatocyte growth factor (HGF) and its receptor Met are frequently over-expressed in non-small-cell lung carcinomas (NSCLC), but their potential role in tumor progression is not clearly known. To assess the role of HGF/Met signaling in lung carcinomas, we have examined the expression, activation status, and function of Met in NSCLC cell lines (n = 7), established from primary tumors or pleural fluids of cancer patients. We observed Met expression in three NSCLC cell lines, two of which exhibited constitutive tyrosine-phosphorylation of Met, and Met kinase activity. In addition, the observed constitutive activation of Met was sustained under anchorage-independent conditions, and correlated with phosphatidyl inositol 3-kinase-dependent cell survival. Immunoreactive HGF-like protein was secreted by two Met-positive and two Met-negative NSCLC cell lines. However HGF activity, as determined by the ability to induce cell scattering and tyrosine-phosphorylation of Met in reporter cell lines, was detected in conditioned medium from only one Met-negative NSCLC cell line: none of the conditioned media from Met-expressing NSCLC cell lines showed detectable HGF activity. Thus, constitutive activation of Met in NSCLC cell lines may occur at least in part through intracrine, or HGF-independent mechanisms. Interestingly, additional paracrine stimulation with exogenous recombinant HGF was required for DNA synthesis and correlated with increased activation of ERK1/2 in all Met-positive NSCLC cell lines, regardless of the basal activation status of Met. These findings indicate that a medium level of constitutive activation of Met occurs in some NSCLC cell lines, and correlates with survival of detached carcinoma cells; whereas additional paracrine stimulation by recombinant HGF is required for DNA synthesis. Thus constitutive and paracrine activation of Met may provide complementary signals that promote survival and proliferation, respectively, during tumor progression of NSCLC.     

Taurine chloramine-induced inactivation of cofilin protein through methionine oxidation

Cofilin regulates reorganization of actin filaments (F-actin) in eukaryotes. A recent finding has demonstrated that oxidation of cofilin by taurine chloramine (TnCl), a physiological oxidant derived from neutrophils, causes cofilin to translocate to the mitochondria inducing apoptosis (F. Klamt et al. Nat. Cell Biol. 11:1241–1246; 2009). Here we investigated the effect of TnCl on biological activities of cofilin in vitro. Our data show that TnCl-induced oxidation of recombinant human cofilin-1 inhibits its F-actin-binding and depolymerization activities. Native cofilin contains four free Cys and three Met residues. Incubation of oxidized cofilin with DTT does not lead to its reactivation. A double Cys to Ala mutation on the two C-terminal Cys shows similar biological activities as the wild type, but does not prevent the TnCl-induced inactivation. In contrast, incubation of oxidized cofilin with methionine sulfoxide reductases results in its reactivation. Phosphorylation is known to inhibit cofilin activities. We found that Met oxidation also prevents phosphorylation of cofilin, which is reversed by incubating oxidized cofilin with methionine sulfoxide reductases. Interestingly, intact protein mass spectrometry of the oxidized mutant indicated one major oxidation product with an additional mass of 16 Da, consistent with oxidation of one specific Met residue. This residue was identified as Met-115 by peptide mapping and tandem mass spectrometry. It is adjacent to Lys-114, a known residue on globular-actin-binding site, implying that oxidation of Met-115 disrupts the globular-actin-binding site of cofilin, which causes TnCl-induced inactivation. The findings identify Met-115 as a redox switch on cofilin that regulates its biological activity.     

Diastereoselective protein methionine oxidation by reactive oxygen species and diastereoselective repair by methionine sulfoxide reductase

Recent studies have shown that the "calcium-sensor" protein calmodulin (CaM) suffers an age-dependent oxidation of methionine (Met) to methionine sulfoxide (MetSO) in vivo. However, MetSO did not accumulate on the Met residues that show the highest solvent-exposure. Hence, the pattern of Met oxidation in vivo may give hints as to which reactive oxygen species and oxidation mechanisms participate in the oxidation of this important protein. Here, we have exposed CaM under a series of different reaction conditions (pH, [Ca(2+)], [KCl]) to various biologically relevant reactive oxygen species and oxidizing systems (peroxides, HOCl, peroxynitrite, singlet oxygen, metal-catalyzed oxidation, and peroxidase-catalyzed oxidation) to investigate whether one of these systems would lead to an oxidation pattern of CaM similar to that observed in vivo. However, generally, these oxidizing conditions led to a preferred or exclusive oxidation of the C-terminal Met residues, in contrast to the oxidation pattern of CaM observed in vivo. Hence, none of the employed oxidizing conditions was able to mimic the age-dependent oxidation of CaM in vivo, indicating that other, yet unidentified oxidation mechanisms may be important in vivo. Some oxidizing species showed a quite-remarkable diastereoselectivity for the formation of either L-Met-D-SO or L-Met-L-SO. Diastereoselectivity was dependent on the nature of the oxidizing species but was less a function of the location of the target Met residue in the protein. In contrast, diastereoselective reduction of L-Met-D-SO by protein methionine sulfoxide reductase (pMSR) was efficient regardless of the position of the L-Met-D-SO residue in the protein and the presence or absence of calcium. With only the L-Met-D-SO diastereomer being a substrate for pMSR, any preferred formation of L-Met-L-SO in vivo may cause the accumulation of MetSO unless the oxidized protein is substrate for (accelerated) protein turnover.     

Further characterization studies of the alpha-amylase protein inhibitor of gel electrophoretic mobility 0.19 from the wheat kernel.

A highly purified amylase protein inhibitor from the kernels of hexaplois wheat, designated 0.19 according to its gel electrophoretic mobility, has been characterized according to its circular dichroism spectra determined at different pH values and in the presence or absence of dissociating and reducing agents. The 0.19 albumin has also been characterized according to the specificity with which it inhibits 21 alpha-amylases from different origins and according to its sensitivity to a number of chemical and enzymatic treatments of its inhibitory action on human saliva and Tenebrio molitor L. larval midgut alpha-amylases. Inhibitory activity of 0.19 toward human saliva amylase significantly increased when the inhibitor was incubated with the enzyme before the addition of starch, but it was not affected by the preincubation of 0.19 with starch. Maltose reversed the inhibition of human saliva by 0.19 and showed some inhibitory activity toward the enzyme. However, maltose concentrations that only slightly affected amylase activity were very effective in restoring the amylase activity inhibited by 0.19. The inhibitory action of 0.19 on human saliva and T. molitor L. amylases were equally resistant to trypsin and thermal treatments, but 0.19 was readily inactivated by incubation with pepsin or by reduction of disulfide bonds. The inhibition of the mammalian amylase by 0.19 was adversely affected by a treatment with CNBr (1:100 ratio of methionine residues to CNBr) whereas the inhibition of the insect amylase was not. As shown by circular dichroism measurements in the far ultraviolet, 0.19 is a protein with about 50% of ordered structure. Significant and largely reversible changes have been observed in the aromatic CD spectrum of 0.19 at alkaline pH values or in the presence of sodium dodecyl sulfate. These changes, which were associated with a partial loss of inhibitory activity, indicate that ionizable tyrosine groups contribute significantly to the ellipticity bands of 0.19 in the near ultraviolet.     

Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin

Antioxidant properties of human serum albumin (HSA) may explain part of its beneficial role in various diseases related to free radical attack. In the present study, the antioxidant role of Cys and Met was studied by copper-mediated oxidation of human low density lipoproteins and by free radical-induced blood hemolysis which essentially assessed metal-chelating and free radical scavenging activities, respectively. Mild conditions were set up to specifically modify Cys and Met residues by N-ethylmaleimide (NEM) and chloramine T treatments, respectively. We found that Met and Cys accounted for 40–80% of total antioxidant activity of HSA. Copper binding to HSA was decreased by about 50% with chloramine T treatment of Met whereas no change was observed after NEM treatment of Cys. Although other amino acid residues are likely to be involved in anti-/prooxidant properties of HSA, from our data, we propose that Cys chiefly works as a free radical scavenger whereas Met mainly acts as a metal chelator.     

Inactivation of thrombomodulin by ionizing radiation in a cell-free system: possible implications for radiation responses in vascular endothelium

Normal tissue radiation injury is associated with loss of vascular thromboresistance, notably because of deficient levels of endothelial thrombomodulin (TM). TM is located on the luminal surface of most endothelial cells and has critical anticoagulant and anti-inflammatory functions. Chemical oxidation of a specific methionine residue (Met388) at the thrombin-binding site in TM reduces its main functional activity, i.e., the ability to activate protein C. We examined whether exposure to ionizing radiation affects TM in a similar manner. Full-length recombinant human TM, a construct of epidermal growth factor-like domains 4-6, which are involved in protein C activation, and a synthetic peptide containing the methionine of interest were exposed to gamma radiation in a cell-free system, i.e., a system not confounded by TM turnover or ectodomain shedding. The influence of radiation on functional activity was assessed with the protein C activation assay; formation of a TM-thrombin complex was assessed with surface plasmon resonance (Biacore), and oxidation of Met388 was assessed by HPLC and confirmed by mass spectroscopy. Exposure to radiation caused a dose-dependent reduction in protein C activation, impaired TM-thrombin complex formation, and oxidation of Met388. These results demonstrate that ionizing radiation adversely affects the TM molecule. Our findings may have relevance to normal tissue toxicity in clinical radiation therapy as well as to the development of radiation syndromes in the non-therapeutic radiation exposure setting.     

Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin

Antioxidant properties of human serum albumin (HSA) may explain part of its beneficial role in various diseases related to free radical attack. In the present study, the antioxidant role of Cys and Met was studied by copper-mediated oxidation of human low density lipoproteins and by free radical-induced blood hemolysis which essentially assessed metal-chelating and free radical scavenging activities, respectively. Mild conditions were set up to specifically modify Cys and Met residues by N-ethylmaleimide (NEM) and chloramine T treatments, respectively. We found that Met and Cys accounted for 40-80% of total antioxidant activity of HSA. Copper binding to HSA was decreased by about 50% with chloramine T treatment of Met whereas no change was observed after NEM treatment of Cys. Although other amino acid residues are likely to be involved in anti-/prooxidant properties of HSA, from our data, we propose that Cys chiefly works as a free radical scavenger whereas Met mainly acts as a metal chelator.     

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

NMR spectroscopy was used to study the effect of guanidinium chloride on the unfolding of horse heart and yeast iso-1 cytochrome c under mild alkaline conditions. The structural changes on the horse heart protein were detected through NOESY (Nuclear Overhauser Effect SpectroscopY) experiments whereas (15)N-(1)H heteronuclear NMR was used to monitor the behavior of the yeast protein. The latter represents the first characterization through (15)N-(1)H heteronuclear NMR spectroscopy of the guanidinium chloride induced unfolding of mitochondrial cytochrome c. The presence of denaturants decreases the temperature at which the native Met80 axial ligand is displaced from the iron center under the present mild alkaline conditions. The process can be described in terms of protein fragments behaving as unfolding units of different stability. The comparison between the two proteins indicates that the loop+helix connecting the proximal and distal sites, as well as the long Met80-containing loop immediately after a short helix, are structural characteristics of mitochondrial cytochrome c that appear to be responsible for the Met80-iron(III) bond fragility.     

Oxidation of methionine residues in coagulation factor VIIa

The oxidation of the activated form of recombinant coagulation factor VII (FVIIa) by hydrogen peroxide has been studied. The three predominant oxidation products observed at pH 7.5 have been characterized as methionine sulfoxide derivatives of the parent protein involving two of the four methionine residues of the protein, Met298 and Met306. We conclude that oxidation of FVIIa with hydrogen peroxide only affects methionine residues and selectively oxidizes those which are readily accessible to the solvent. The oxidation process has been studied in the pH range 3.5-9.5. The total rate of oxidation of FVIIa as well as the formation of the three oxidation products is consistent over the pH interval 7.5-9.5. However, under acidic conditions, significant variations have been observed indicating a conformational change of FVIIa. Oxidized FVIIa had the same amidolytic activity as the native protein. The binding to soluble tissue factor (TF) was weaker after oxidation as manifested by a threefold increase in dissociation constant and the amidolytic activity in complex with soluble TF was 80% compared to that of native FVIIa. In complex with lipid surface TF, the rate of factor X activation catalyzed by oxidized FVIIa was also reduced by approximately 20% compared to that of native FVIIa. However, native and oxidized FVIIa appeared to bind lipidated TF with indistinguishable affinities.     

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.     

Evidence for the involvement of histidine A(12) in the aggregation and precipitation of human relaxin induced by metal-catalyzed oxidation

The metal-catalyzed oxidation (ascorbate/cupric chloride/oxygen) of recombinant human relaxin (rhRlx, type II) was shown by Li et al. [Li, S., Nguyen, T. H., Sch?neich, C., and Borchardt, R. T. (1995) Biochemistry 34, 5762-5772] to result in the chemical modification of His A(12), Met B(4), and Met B(25). Considering the fact that His A(12) exists in an extended loop that joins two alpha-helices in this protein, we hypothesized that oxidation of this specific amino acid leads to alterations in the secondary and tertiary structures of the protein, resulting in the pH-dependent aggregation/precipitation phenomena observed in our earlier studies (i.e., at pH >6.0 most of the degradants of rhRlx are insoluble). Evidence obtained in the current study that supports this hypothesis includes the following: (i) oxidation of rhRlx with hydrogen peroxide (H(2)O(2)), which leads only to modification of Met B(4) and Met B(25), does not result in the pH-dependent aggregation/precipitation of the protein; and (ii) metal-catalyzed oxidation of porcine relaxin (pRlx), which does not contain His at position A(12), leads to chemical degradation of the protein [e.g., Met A(2) is oxidized] but produces only slight pH-dependent aggregation/precipitation of the protein. In addition, experimental evidence is provided to show that the physical instability of rhRlx observed at pH >6.0 does not appear to be related to the pH-dependent solubility of a common protein degradant. Instead, it appears that several oxidation products of His A(12) are produced in a pH-dependent manner and that these oxidation products produce different effects on the physical stability of the protein. Evidence in support of this conclusion includes the observation that the soluble degradants of rhRlx showed reduced levels of His, reduced levels of the T(2)-T(7) tryptic fragment that contained His A(12), and the presence of 2-oxo-His. Similarly, the precipitated degradants of rhRlx showed reduced levels of His but no 2-oxo-His. In addition, the soluble degradants, which contain 2-oxo-His, appear to exist as monomers having an average molecular weight similar to that of rhRlx. These results suggest that the metal-catalyzed oxidation of His A(12) leads to other, as yet unidentified oxidation products of His A(12) that affect the secondary/tertiary structure of the protein more significantly than does 2-oxo-His and ultimately lead to the physical instability of the protein observed at higher pH values.     

Some properties of human small heat shock protein Hsp22 (H11 or HspB8)

Untagged recombinant human small heat shock protein with apparent molecular mass 22 kDa (Hsp22) was obtained in homogeneous state. Size exclusion chromatography and chemical crosslinking with dimethylsuberimidate indicate that Hsp22 forms stable dimers. Being highly susceptible to oxidation Hsp22 forms disulfide crosslinked dimers and poorly soluble high molecular mass oligomers. According to CD spectroscopy oxidation of Hsp22 results in disturbing of both secondary and tertiary structure. Hsp22 possesses a negligibly low autophosphorylation activity and under the conditions used is unable to phosphorylate casein or histone. Hsp22 effectively prevents heat-induced aggregation of yeast alcohol dehydrogenase and bovine liver rhodanese with chaperone activity comparable to that of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20).     

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.     

An alkaline pH control strategy for methionine adenosyltransferase production in Pichia pastoris fermentation

Pichia pastoris is a successful system for expressing heterologous proteins and its fermentation pH is always maintained below 7.0. However, particular proteins are unstable under acidic conditions, such as methionine adenosyltransferase (MAT), and thus fermentation under acidic pH conditions is unsuitable because protein activity is lost owing to denaturation. Here, a strategy employing alkaline pH in the late fermentation period was developed to improve MAT production. Initially, P. pastoris KM71 was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, the expressed MAT displayed highest stability at pH 8.0; however, pH 8.0 inhibited cell growth and induced cell rupture, thus affecting protein production. To balance MAT stability and Pichia cell viability, different pH control strategies were compared. In strategy A (reference), the induction pH was maintained at 6.0, whereas in strategy B, it was gradually elevated to 8.0 through a 25 h transition period (80 ～ 105 h). MAT activity was 0.86 U/mg (twofold higher than the control). However, MAT content was reduced by 50% when compared with strategy A, because of proteases released upon cell lysis. To improve cell viability under alkaline conditions, glycerol was added in addition to methanol (strategy C). When compared with strategy B, the MAT-specific activity remained nearly constant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MAT production represents an excellent alternative for expressing proteins that are stable only under alkaline conditions.     

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.