Polypeptides posttranslational modification

While the first 20-30 residues of a peptide can readily be determined by the Edman method, most polypeptides contain several hundred amino acids. Consequently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein s a-amino group blocked , or unreactive with the Edman reagent. 

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. 

The maturation of proteins into their final structural state often involves the cleavage or formation (or both) of covalent bonds, a process termed posttranslational modification. Many polypeptides are initially synthesized as larger precursors, called proproteins. The extra polypeptide segments in these proproteins often serve as leader sequences that target a polypeptide... 

Biosynthesis of the polypeptide chain is realised by a complicated process called translation. The basic polypeptide chain is subsequently chemically modified by the so-called posttranslational modifications. During this sequence of events the peptide chain can be cleaved by directed proteolysis, some of the amino acids can be covalently modified (hydroxylated, dehydrogenated, amidated, etc.) or different so-called prosthetic groups such as haem (haemoproteins), phosphate residues (phosphoproteins), metal ions (metal-loproteins) or (oligo)saccharide chains (glycoproteins) can be attached to the molecule by covalent bonds. Naturally, one protein molecule can be modified by more means. 

When chromatographic resolution of species based on modifications located at the protein surface is desired, it may be advisable to use conditions that favor retention of native conformation.17 Here, the standard acidic conditions described in the preceding text may be inappropriate, and mobile phases buffered near neutrality may be required. Buffers based on ammonium acetate, ammonium bicarbonate, and triethylammonium phosphate may prove more useful in resolving polypeptide variants with differing posttranslational modifications, amino acid substitutions, or oxidation and deamidation products. The addition of more hydro-phobic ion-pairing agents may be needed to obtain polypeptide retention, and a variety of alkyl sulfonates and alkyl amines have been described for specific applications.17... 

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

Prerequisite is the existence of a primary amino group at the N-terminal end of a polypeptide, i.e., chemical or posttranslational modifications of this amino group, e.g., by methylation or acetylation, prevents success. If the amino group is not protected or the amino acid chain is not branched, this method suits well for examination of the uniformity of a purified protein. 

Most of the other posttranslational modifications involving the N- or C-terminus (Table 1) as well as the side-chain functionalities (Table 2) of the polypeptide chains occur under the control of enzymes that also dictate the regioselectivity of such chemical transformations. This regioselectivity is difficult to attain by synthetic procedures. Sophisticated protection schemes are required when additional chemistry must be performed on preassembled peptides, unless enzymatic methods can be used to supplement the synthetic strategies. As a consequence, the use of suitably modified amino acids as synthons is generally the preferred approach as will be discussed in the following sections. 

Polypeptides fold into their active, three-dimensional forms. Many proteins are further processed by posttranslational modification reactions. 

Hydroxyproline and hydroxylysine Collagen contains hydroxy proline (hyp) and hydroxylysine (hyl), which are not present in most other proteins. These residues result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains (Figure 4.6). The hydroxylation is, thus, an example of posttranslational modification (see p. 440). Hydroxy proline is important in stabilizing the triple-helical structure of colla gen because it maximizes interchain hydrogen bond formation. 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

Many polypeptide chains are covalently modified, either while they ae still attached to the ribosome or after their synthesis has been completed. Because the modifications occur after translation is initiated they are called posttranslational modifications. These modifications maj include removal of part of the translated sequence, or the covalent add-tion of one or more chemical groups required for protein activity. Som< types of posttranslational modifications are listed below. 

Posttranslational modification Examples of posttranslational modification POSTTRANSLATIONAL MODIFICATION OF POLYPEPTIDE CHAINS (p. 440) Many polypeptide chains are covalently modified after translation. Such modifications include trimming excess amino acids, phosphorylation which may activate or inactivate the protein, glycosylation which targets a protein to become part of a plasma membrane or lysosome or be secreted from the cell, or hydroxylation such as that seen in collagen. 

Bacteria, protozoa, and venomous animals synthesize numerous toxins that are used to kill their prey or to defend themselves. Sea anemones, jellyfish, cone snails, insects, spiders, scorpions, and snakes all make potent and highly specific neurotoxins. Plants form a host of alkaloids and other specialized products, some of which are specifically neurotoxic and able to deter predators. More than 500 species of marine cone snails of the genus Conus synthesize a vast array of polypeptide toxins (conotoxins), 487-489 some with unusual posttranslational modifications.490 491 The slow-moving snails are voracious predators that use their toxins, which they inject with a disposible harpoonlike tooth,492 to paralyze fish, molluscs, or worms.493... 

Posttranslational modifications include many covalent alterations Polypeptide processing, attachment of carbohydrate or lipid groups to specific side chains, and addition of many other low-molecular-weight ligands to side chains. 

In the life cycle of HIV, its RNA is translated into a polypeptide chain that is composed of several individual proteins including protease, integrase and reverse transcriptase, but in this form these enzymes are not functional. They must be cleaved by viral proteases from the assembled sequence in order for them to become functional. These posttranslational modifications allow the enzymes to facilitate the production of new viruses. The protease itself is made up of two 99-amino-acid monomers, and an aspartic acid residue in the monomer is required for the cleavage. The protease inhibitors inhibit the enzyme protease and consequently interfere with viral replication and maturation by preventing proteases from cleaving proteins into peptides. In humans, these drugs inhibit cleavage of HIV gag and pol polyproteins, which are part of the essential viral structural components, P7, P9, P17 and P24, and... 

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. 

Figure 4.2. Model polypeptide of 20 amino acids. The three-letter and one-letter abbreviations for amino acids are shown. Ionizable amino acids (-R) are shown as positively or negatively charged in bold. Note that the amino-terminal and carboxy-terminal ends are charged. All charges contribute to an overall charge state of the protein at a specific pH. The pi (isoelectric point) is the pH at which a proteins carries no net charge. The pi is helpful in classifying proteins as acidic, neutral, or basic. The addition of posttranslational modifications can also add charges to proteins and greatly affect the pi value.

Common posttranslational modifications of proteins. Frequently modified amino adds for each moiety are described. Most modifications involve low-mass substituents (<500amu), but some modifications are small polypeptides of considerable mass such as ubiquitin or highly related peptide isomers like SUMO-1, SUMO-2, and SUMO 3. SUMO is the abbreviation for similar to ubiquitin methyl organizer , SUMO has different family members (SUMO 1, 2, and 3 that can combine with proteins). 

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