Methionine Position Research Articles

On the electrochemical oxidation of methionine residues of proteins

The oxidation of methionine side chain residues in proteins provides a great diversity of possible oxidation mechanisms dictated, among others, by the oxidizing species, solvent properties, and protein structure. The oxidation behavior of a series of short acetylated synthetic peptides, Ac-GMG, Ac-GGMGG and Ac-GGGMGGG, was investigated by differential pulse and square wave voltammetry, in a wide pH range, at glassy carbon electrode. It was found that always the first oxidation step represents the one-electron oxidation of thioether moiety with the formation of a radical cation. Following this, depending on the experimental conditions and side chain position of methionine, the radical is stabilized by the nucleophilic attack of water or by catalytic support of the neighboring carbonyl and amide groups in an intermediate structure, finally converted in methionine sulfoxide which can be further oxidized, at more positive potential, into methionine sulfone. For Ac-GGGMGGG, at pH 8.0, the amino and amide groups are active involved in the oxidation process and the electrode reaction takes place with proton transfer

Metabolic Consequences of Supplemented Methionine in a Clinical Context

The central position of methionine (Met) in protein metabolism indicates the importance of this essential amino acid for growth and maintenance of lean body mass. Therefore, Met might be a tempting candidate for supplementation. However, because Met is also the precursor of homocysteine (Hcy), a deficient intake of B vitamins or excessive intake of Met may result in hyperhomocysteinemia (HHcy), which is a risk factor for cardiovascular disease. This review discusses the evidence generated in preclinical and clinical studies on the importance and potentially harmful effects of Met supplementation and elaborates on potential clinical applications of supplemental Met with reference to clinical studies performed over the past 20 y. Recently acquired knowledge about the NOAEL (no observed adverse effect level) of 46.3mg · kg-1 · d-1 and the LOAEL (lowest observed adverse effect level) of 91mg · kg-1 · d-1 of supplemented Met will guide the design of future studies to further establish the role of Met as a potential (safe) candidate for nutritional supplementation in clinical applications.

Forced Degradation Testing as Complementary Tool for Biosimilarity Assessment.

Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore represent critical quality attributes (CQA) that require evaluation. To complement classical CQA, bevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to probe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2 subunit was completely oxidized. Additional oxidations were found in the light chain (LC) and in the Fd’ subunit of infliximab, but not in bevacizumab. By direct comparison of methionine positions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fd’. The forced oxidation approach was further exploited for comparison of respective biosimilar products. Both for bevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high similarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab, comparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP. It may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment, as well as for quality control of protein drugs.

Influence of methionine residue position on oxidative stability of glucose oxidase from Aspergillus niger

Glucose oxidase (GOx) is a promising candidate for construction of implantable miniature biofuel cells and biosensors for continuous glucose monitoring. The main drawback that limits current application of GOx in these devices is its low stability, especially sensitivity to reactive oxygen species. In order to address this problem, we performed saturation mutagenesis at all 11 methionine residues as their interaction with reactive oxygen species inactivates enzymes. For successful screening of these libraries a method based on yeast surface display (YSD) systems was developed. Mutations at methionine positions close to the GOx active site contributed the most to the oxidative stability, and combinations of the four best single mutations were tested. Combined mutants did not show higher stability or activity compared to the parental single mutants. To confirm oxidative stability of YSD expressed GOx mutants they were re-cloned in Pichia pastoris, purified and immobilized on macroporous copolymer. The additional kinetic analysis of immobilized GOx mutants confirmed that the best mutant with only one mutation close to the active site (M561S) has 2.5 times increased half-life in the presence of hydrogen peroxide compared to the wild-type variant.

Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

BackgroundThe alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region.ResultsTo explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme.ConclusionAlthough invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease.

Activation of the LicT Transcriptional Antiterminator Involves a Domain Swing/Lock Mechanism Provoking Massive Structural Changes

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 A resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ' interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.

Neurotoxicity and oxidative stress in D1M-substituted Alzheimer's Aβ(1-42): relevance to N-terminal methionine chemistry in small model peptides

Small model peptides containing N-terminal methionine are reported to form sulfur-centered-free radicals that are stabilized by the terminal N atom. To test whether a similar chemistry would apply to a disease-relevant longer peptide, Alzheimer's disease (AD)-associated amyloid beta-peptide 1-42 was employed. Methionine at residue 35 of this 42-mer has been shown to be a key amino acid residue involved in amyloid beta-peptide 1-42 [Aβ1-42]-mediated toxicity and therefore, the pathogenesis of AD. Previous studies have shown that mutation of the methionine residue to norleucine abrogates the oxidative stress and neurotoxic properties of Aβ(1-42). In the current study, we examined if the position of methionine at residue 35 is a criterion for toxicity. In doing so, we tested the effects of moving methionine to the N-terminus of the peptide in a synthetic peptide, Aβ(1-42)D1M, in which methionine was substituted for aspartic acid at the N-terminus of the peptide and all subsequent residues from D1 to L34 were shifted one position towards the carboxy-terminus. Aβ(1-42)D1M exhibited oxidative stress and neurotoxicity properties similar to those of the native peptide, Aβ(1-42), all of which are inhibited by the free radical scavenger Vitamin E, suggesting that reactive oxygen species may play a role in the Aβ-mediated toxicity. Additionally, substitution of methionine at the N-terminus by norleucine, Aβ(1-42)D1Nle, completely abrograted the oxidative stress and neurotoxicity associated with the Aβ(1-42)D1M peptide. The results of this study validate the chemistry reported for short peptides with N-terminal methionines in a disease-relevant peptide.

IMPORTANCE OF HELICES A AND H IN OXYGEN BINDING DIFFERENCES BETWEEN BOVINE AND HUMAN HEMOGLOBINS*

Human and bovine hemoglobins (Hbs) exhibit several functional differences. They have a similar oxygen affinity in the presence of 2,3-diphosphoglycerate (2,3-DPG); however, bovine Hb has a greatly diminished 2,3-DPG effect, which itself is chloride dependent. The question is to determine whether these differences have a common structural origin, or whether they evolved in an independent fashion. The decreased 2,3-DPG effect can be partially reproduced by mutations at the effector binding sites, substituting the βNA1 valine–NA2 histidine present in human Hb with a methionine. While changes of human Hb at these sites could provoke the bovine characteristic of the lower 2,3-DPG effect, the oxygen affinities of these mutated Hbs were not as low as that of the bovine Hb. Modifications responsible for tertiary structural modifications of helix A in human Hb might help shift the N-terminal methionine position, thereby locking helix A in place. We replaced the residues proline β5(A2), arginine β104(G6), and tyrosine β130(H8) of human Hb by the residues present in bovine β-globin, namely alanine, lysine, and phenylalanine, respectively. These mutations did not allow us to obtain a low oxygen affinity recombinant Hb (rHb). This indicates that other factors also influence oxygen binding and the effects are only partially coupled.

Difluoromethionine as a Novel 19F NMR Structural Probe for Internal Amino Acid Packing in Proteins

The successful incorporation of difluoromethionine (DFM), a novel 19F NMR probe of internal amino acid packing, into the three methionine positions (1, 14, and 107) of a recombinant protein, the ly...

Incorporation of trifluoromethionine into a phage lysozyme: implications and a new marker for use in protein 19F NMR.

Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function. Although the use of site-directed mutagenesis has greatly contributed to this goal, the approach is limited to the standard repertoire of twenty amino acids. Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics as well as point to the importance of specific residues to biological function. In our continuing investigations on the importance of the amino acid methionine in biological systems, the successful incorporation of L-S-(trifluoromethyl)homocysteine (L-trifluoromethionine; L-TFM) into bacteriophage lambda lysozyme (LaL), an enzyme containing three methionine residues, is reported. The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoromethyl iodide. An expression plasmid giving strong overproduction of LaL was prepared and transformed into an Escherichia coli strain auxotrophic for methionine permitting the expression of LaL in the presence of L-TFM. The analogue would not support growth of the auxotroph and was found to be inhibitory to cell growth. However, cells that were initially grown in a Met-rich media followed by protein induction under careful control of the respective concentrations of L-Met and L-TFM in the media, were able to overexpress TFM-labeled LaL (TFM-LaL) at both high (70%) and low (31%) levels of TFM incorporation. TFM-LaL at both levels of incorporation exhibited analogous activity to the wild type enzyme and were inhibited by chitooligosaccharides indicating that incorporation of the analogue did not hinder enzyme function. Interestingly, the 19F solution NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chemical shift range of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of chitopentaose. The 19F NMR analysis of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine positions gives rise to two separate resonances. The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure are induced by incorporated TFM. The similarities and differences between Met and TFM were analyzed using ab initio molecular orbital calculations. The methodology presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.

Structure of Metal Site in Azurin, Met121 Mutants of Azurin, and Stellacyanin Investigated by 111mCd Perturbed Angular Correlation (PAC)

The geometries of the metal sites in cadmium-substituted azurins have been investigated by 111mCd perturbed angular correlation (PAC). The study includes wild type azurin as well as Met121 mutants of azurin, where methionine has been substituted by Ala, Asn, Asp, Gln, Glu, and Leu. The nuclear quadrupole interaction of wild type azurin analyzed in the angular overlap model is well described as coordination of His46, His117, and Cys112 and cannot be described by coordination of Met121 and/or Gly45. For most of the mutants, there exist two coordination geometries of the cadmium ion. With the exception of the Glu and Asp mutants, one of the conformations is similar to the wild type conformation. The other coordination geometries are either best described by a coordinating water molecule close to the original methionine position or by coordination by the substituting amino acid. These experiments show that even though the methionine does not coordinate it plays an important role for the geometry of the metal site. The nuclear quadrupole interaction of stellacyanin was also measured. The value resembles the most prominent nuclear quadrupole interaction of the Met121-->Gln mutant of Alcaligenes denitrificans azurin, indicating that the structures of the two metal sites are similar.

Incorporation of Norleucine at Methionine Positions in Recombinant Human Macrophage Colony Stimulating Factor (M-CSF, 4-153) Expressed in Escherichia coli: Structural Analysis

Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.

Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins.

A yeast genetic system was developed to study how the basic regions of basic-helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG. The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis. Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG. The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region. However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function. In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position. The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites.

Discrimination of collagen types by methionine-specific stain

Chloroglycyl-L-methioninatoplatinum (II) (Pt-GLM) specifically labels methionine residues in collagen. Rat Type I and calf Type III collagen segment long spacing aggregates show differences in Pt-GLM bands, in agreement with known methionine positions. Reconstituted fibers formed from the same two collagens again demonstrate some differences in band pattern, which can be understood in terms of the amino acid sequences and the known packing of molecules in the fiber. Native collagen fibers have been extracted from rat and chick tendon (predominantly Type I collagen). These have been stained with Pt-GLM and studied in the Johns Hopkins STEM and a CTEM. Density profiles along the collagen fibers have been obtained, and those for successive 670-A repeats averaged, by computer programs. The position of peaks in the average density profiles for these various fibers show very good correlation with the known locations of Met residues. The band patterns and density profiles for native and reconstituted Type I fibers are very similar and differ from those of reconstituted Type III fibers.

Amino acid sequence of a cyanogen bromide cleavage peptide from hens egg phosvitin

1. 1. An almond phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) has been found to be an effective means of hen phosvitin dephosphorylation. 2. 2. A homoserine-peptide from the cyanogen bromide cleavage of the methionine-phosphoprotein isolated from hens egg phosvitin, on treatment with almond phosphatase followed by trypsin, afforded a number of peptides which were resolved by Sephadex fractionation and paper electrophoresis. 3. 3. Use of manual and automated Edman degradation methods, mass spectrometry and mild acid hydrolysis have provided information which establishes the sequence of the first 38 residues from the amino terminus to the methionine position in the original phosphoprotein.