Posttranslational modifications disulfide bonds

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of prohne and lysine, and disulfide bond formation that accompany mamra-tion. While Edman sequencing can detect the presence of most posttranslational events, technical hmitations often prevent identification of a specific modification. 

Chemical changes include posttranslational modifications including glycosy-lation, phosphorylation, disulfide bond formation and exchange (scrambling), proteolysis or hydrolysis, and deamidation or oxidation of amino acids.4... 

Posttranslational modifications can be broken down into two main classes those that are reversible and those that are irreversible. Included in the large group of reversible posttranslational modifications are phosphorylation, acetylation, and disulfide formation. Irreversible posttranslational modifications include peptide bond cleavage as in intein splicing also irreversible is the introduction of a phosphopantetheinyl group during fatty acid, polyketide, and nonribosomal peptide biosyntheses. The current debate is whether to classify lysine N-methylation as reversible or irreversible. Recently, there have been reports of lysine demethylases. ... 

Cysteine disulfide formation is one of the most important posttranslational modifications involved in protein structure. Disulfides play a crucial role in maintaining the structure of many proteins including insulin, keratin, and many other structurally important proteins. While the cytoplasm and nucleus are reducing microenvironments, the Golgi and other organelles can have oxidizing environments and process proteins to contain disulfide bonds (Scheme 5). 

Prolyl 4-hydroxylation is the most abundant posttranslational modification of collagens. 4-Hydroxylation of proline residues increases the stability of the triple helix and is a key element in the folding of the collagen triple helix. " In vertebrates, almost all the Yaa position prolines of the Gly-Xaa-Yaa repeat are modified to 4(I( )-hydroxylproline by the enzyme P4H (EC 1.14.11.2), a member of Fe(II)- and 2-oxoglutarate-dependent dioxygenases. This enzyme is an 0 2/ b2-type heterotetramer in which the / subunit is PDI (EC 5.3.4.1), which is a ubiquitous disulfide bond catalyst. The P4H a subunit needs the 13 subunit for solubility however, the 13 subunit, PDI, is soluble by itself and is present in excess in the ER. Three isoforms of the a subunit have been identified and shown to combine with PDI to form [a(I)]2/ 2) [< (II)]2/32> or [a(III)]2/32 tetramers, called the type... 

Chemical modifications of proteins have been carried out for a long time prior to any interest in the understanding of the chemical basis of the process. Early studies were motivated by the interest in quantitative determination of proteins and amino acids that conform its structure [104]. Intramolecular reactions occur naturally in posttranslational modifications such as disulfide bonding, glycosylation, or terminal residue cyclization. These modifications are relevant in structure-function relationships. They can produce conformational changes in order to switch between... 

Cystine is composed of two molecules of cysteine linked through oxidation of their —SH groups to give a disulfide bond. Such oxidation, which is important in stabilizing the folded structure of some proteins, represents a posttranslational modification of a protein. Thus, cystine is never incorporated as such into a polypeptide during translation, and there is no codon that corresponds to it. 

The protein molecular mass is insufficient information for identification, but it is adequate to confirm identity therefore, MS is one of the preferred techniques for characterization and quality control of recombinant proteins and other biomolecules. In the same way, it has been used to study posttranslational modifications (like glycosylation and disulfide bonding pattern), and other processes that can modify protein mass.11... 

The formation of interchain disulfide linkages in the C-terminal propeptide. The latter cannot be formed until translation is nearly finished. The rate of disulfide bond and triple-helix formation varies greatly from one cell type to another—only minutes in tendon cells that synthesize type I collagen but an hour in cells that synthesize basement membrane collagen. These differences in synthesis time may account for the variations in hydroxylation and glycosylation. Thus, the extent of posttranslational modifications depends not only on levels of enzyme and cofactors but also on the time available. 

Posttranslational modification reactions prepare polypeptides to serve their specific functions and direct them to specific cellular or extracellular locations. Examples of these modifications include proteolytic processing (e.g., removal of signal proteins), glycosylation, methylation, phosphorylation, hydroxylation, lipophilic modifications (e.g., N-myristoylation and prenylation), and disulfide bond formation. 

However, in addition to the presence of disulfide bonds, most conopeptide sequences exhibit a high degree of additional posttranslational modifications (PTMs), which include carboxylation of glutamate to form carboxyglutamate and hydroxylation of proline, lysine, and D-valine to form 7-hydroxyproline (Hyp), -hydroxylysine, and D-7-hydroxyvaline, respectively. Other PTMs include epimerization of L-amino acids to form their respective D-amino acid counterparts, halogenation of tryptophan to produce 5-Br-tryptophan... 

Mass spectrometry, in addition to RP-HPLC, serves as a powerful technique for assessing disulfide bond formation. Additionally, tandem mass spectrometry (MS-MS) can in some instances be used to define the pairing of cysteines, like the non-conserved disulfide bond in CCL28 that finks C30 to C80 (Thomas et al., 2015), or confirm the locations of posttranslational modifications, like pyroglutamate formation in CCL2 as shown in Figs. 4 and 5, respectively. 

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