Proteins peptide cleavage, methionine residues

Selective cleavage of peptides and proteins is an important procedure in biochemistry and molecular biology. The half-life for the uncatalyzed hydrolysis of amide bonds is 350 500 years at room temperature and pH 4 8. Clearly, efficient methods of cleavage are needed. Despite their great catalytic power and selectivity to sequence, proteinases have some disadvantages. Peptides 420,423,424,426 an(j proteins428 429 can be hydrolytically cleaved near histidine and methionine residues with several palladium(II) aqua complexes, often with catalytic turnover. 

Modification by performic acid oxidation Treatment of proteins with performic acid leads to the oxidation of cysteine and cystine residues to cysteic acid residues (Sanger 1949). Methionine residues are quantitatively converted to the sulfone (Hirs 1956), and tryptophan undergoes oxidative destruction (Toennies and Homiller 1942 Benassi et al. 1965). Other amino acids are not modified, provided that precautions are taken to avoid chlorination (Thompson 1954 Hirs 1956), or bromination (Sanger and Thompson 1963) of tyrosine residues. Cleavage of peptide bonds does not occur on performic acid oxidation at low temperature. 

Myristoylation is typically considered to be an early co-translational event that occurs in the cytoplasm as soon as -60 amino acids of the nascent peptide emerge from the ribo-somal tunnel (I. Deichaite, 1988), and after the N-terminal glycine residue is made available by cellular methionyl-aminopeptidases that remove the initiator methionine residue. However, myristate can be attached post-translationally to N-terminal glycine in synthetic peptides of the appropriate sequence, and also to N-terminal glycines that are newly generated after caspase-mediated cleavage of proteins during apoptotic cell death. The latter process is termed morbid myristoylation (J. Zha, 2000 M. Mishkind, 2001). 

Cyanogen bromide (BrC=N) causes the hydrolysis of the amide bond on the C-side of a methionine residue. Cyanogen bromide is more specific than the endopeptidases about what peptide bonds it cleaves, so it provides more reliable information about the primary structure (the sequence of amino acids). Because cyanogen bromide is not a protein and therefore does not recognize the substrate by its shape, cyanogen bromide will still cleave the peptide bond if proline is at the cleavage site. 

A protein that has 10 methionine residues will usually yield 11 peptides on cleavage with CNBr. Highly specific cleavage is also obtained with trypsin, a proteolytic enzyme from pancreatic juice. Trypsin cleaves polypeptide chains on the carboxyl side of arginine and lysine residues (Figure 4.24 and Section 9.1.4). A protein that contains 9 lysine and 7 arginine residues will usually yield 17 peptides on digestion with trypsin. Each of these tryptic... 

Analyses of the structure and modifications of the collagen molecule/chain require solubilization and fragmentation of the protein to smaller peptides. In principle, two methods can be used—non-enzymatic (chemical) or enzymatic treatment. The chemical method is cleavage by CNBr. Cyanogen bromide splits the protein molecule at specific locations—at the methionines (in this case toward the C-terminal end). In the collagen molecule, methionine is a relatively rare amino acid (some 10-20 amino acids per collagen molecule). The small number of methionine residues leads to a rather limited number of cleavage products (CNBr peptides). The profile of CNBr peptides is typical. 

In the treatment of RNase-A with cyanogen bromide, chain cleavage occurs at Met 13. The peptide comprising residues 1-13, where 13 is now homoserine or homoserine lactone rather than methionine, is designated C-peptide (139). This derivative added to S-protein at a molar ratio of 600 1 gave between 50 and 80% of the maximum activity. 

Figure 1 Strategy for cloning a peptide-coding sequence (CDS) as tandem repeats in the vector pET31b. The resulting fusion protein, comprising the ketosteroid isomerase (KSI), peptide repeats, and His-tag, is targeted to inclusion bodies. The fusion protein can be recovered and cleaved, in this case, with cyanogen bromide (CNBr) which acts at the methionine (M) residues allowing further separation of pure peptide from the other fusion components. The cleavage by CNBr results in a C-terminal homoserine lactone (hsl) on each peptide monomer.

Treatment of proteins with cyanogen bromide results in cleavage of the peptide chain COOH-terminal to methionyl residues with concomitant conversion of the methionine to homoserine lactone which is in equilibrium with homoserine. In certain instances (e.g. -Met-Ser-or -Met-Thr-) some of the methionine is converted to homoserine without peptide bond cleavage (see Schroeder et al. 1969). Homoserine and its lactone are also products of the breakdown of the carboxymethylsulfonium salts of methionine ( 2.5.10). 

Recently, 2-(2-nitrophenylsulfenyI)-3-methyl-3-bromoindolenine (BNPS-skatole) has replaced N-bromosuccinimide, which had been used frequently in earlier studies. At low reagent to protein tryptophan ratios, in 50% aqueous acetic acid, BNPS-skatole reacts selectively with tryptophan residues converting these to the oxindole derivative. Methionine is concommitantly converted to the sulfoxide. At high concentrations of reagent, slow selective cleavage (to the extent of 15-60%) of the peptide bonds involving tryptophanyl residues is... 

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