Polypeptide bonds, formation

Solid phase peptide synthesis (Section 27 18) Method for peptide synthesis m which the C terminal ammo acid is co valently attached to an inert solid support and successive ammo acids are attached via peptide bond formation At the completion of the synthesis the polypeptide is removed from the support... 

Gelatin stmctures have been studied with the aid of an electron microscope (23). The stmcture of the gel is a combination of fine and coarse interchain networks the ratio depends on the temperature during the polymer-polymer and polymer-solvent interaction lea ding to bond formation. The rigidity of the gel is approximately proportional to the square of the gelatin concentration. Crystallites, indicated by x-ray diffraction pattern, are beUeved to be at the junctions of the polypeptide chains (24). 

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. 

Disulfide bonds between and within polypeptides stabilize tertiary and quaternary structure. However, disulfide bond formation is nonspecific. Under oxidizing conditions, a given cysteine can form a disulfide bond with the —SH of any accessible cysteinyl residue. By catalyzing disulfide exchange, the rupture of an S— bond and its reformation with a different partner cysteine, protein disulfide isomerase facilitates the formation of disulfide bonds that stabilize their native conformation. 

Figure 2.2 (a) Peptide bond formation, (b) Polypeptides consist of a linear chain of amino acids successively... 

Successive amino acids are joined together during protein synthesis via a peptide (i.e. amide) bond (Figure 2.2). This is a condensation reaction, as a water molecule is eliminated during bond formation. Each amino acid in the resultant polypeptide is termed a residue , and the polypeptide chain will display a free amino (NH2) group at one end and a free carboxyl (COOH) group at the other end. These are termed the amino and carboxyl termini respectively. 

Analogous techniques facilitating sequencing from a polypeptide s C-terminus remain to be satisfactorily developed. The enzyme carboxypeptidase C sequentially removes amino acids from the C-terminus, but often only removes the first few such amino acids. Furthermore, the rate at which it hydrolyses bonds can vary, depending on what amino acids have contributed to bond formation. Chemical approaches based on principles similar to the Edman procedure have been attempted. However, poor yields of derivatized product and the occurrence of side reactions have prevented widespread acceptance of this method. 

The peptidyl transferase centre of the ribosome is located in the 50S subunit, in a protein-free environment (there is no protein within 15 A of the active site), supporting biochemical evidence that the ribosomal RNA, rather than the ribosomal proteins, plays a key role in the catalysis of peptide bond formation. This confirms that the ribosome is the largest known RNA catalyst (ribozyme) and, to date, the only one with synthetic activity. Adjacent to the peptidyl transferase centre is the entrance to the protein exit tunnel, through which the growing polypeptide chain moves out of the ribosome. 

Through the formation of polypeptide bonds between amino acids, very long chains of sequences are obtained. Generally, proteins consist of hundreds and thousands of amino acids. For example, human hemoglobin has four polypeptide chains, of which two are cf-chains and two are j3-chains. There are 141 amino acids in each a-chain with a sequence of... 

A reaction between two identical molecular entities of chemical species to give a new species. Dimerization need not require covalent bond formation. Formation of a complex between two polypeptide chains or formation of an excimer are processes that do not require covalent bond formation. If the two polypeptide chains are identical, the complex is usually referred to as a homodimer otherwise, the resulting complex is referred to as heterodimer. 

The stereochemistry of step polymerization is considered now. Bond formation during step polymerization almost never results in the formation of a stereocenter. For example, neither the ester nor the amide groups in polyesters and polyamides, respectively, possess stereocenters. Stereoregular polymers are possible when there is a chiral stereocenter in the monomer(s) [Oishi and Kawakami, 2000 Orgueira and Varela, 2001 Vanhaecht et al., 2001], An example would be the polymerization of (R) or (S)-H2NCHRCOOH. Naturally occurring polypeptides are stereoregular polymers formed from optically active a-amino acids. 

The inherent drawbacks of the oxidative refolding approach for synthetic polypeptides containing multiple cysteine residues is the individual behavior of each peptide that derives from the encoded sequence, more or less pronounced structural information which prevents general procedures to be elaborated and proposed. Nevertheless, this synthetic approach remains attractive because of its simplicity compared to the synthetic strategies for re-gioselective disulfide bond formation (Section 6.1.1-6.1.4), and it is certainly indispensable if the number of cysteine residues exceeds the presently available chemistry for site-directed cysteine pairings. 

Proofreading by Aminoacyl-tRNA Synthetases The amino-acylation of tRNA accomplishes two ends (1) activation of an amino acid for peptide bond formation and (2) attachment of the amino acid to an adaptor tRNA that ensures appropriate placement of the amino acid in a growing polypeptide. The identity of the amino acid attached to a tRNA is not checked on the ribosome, so attachment of the correct amino acid to the tRNA is essential to the fidelity of protein synthesis. 

Puromycin, made by the mold Streptomyces al-boniger, is one of the best-understood inhibitory antibiotics. Its structure is very similar to the 3 end of an aminoacyl-tRNA, enabling it to bind to the ribosomal A site and participate in peptide bond formation, producing peptidyl-puromycin (Fig. 27-31). However, because puromycin resembles only the 3 end of the tRNA, it does not engage in translocation and dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the peptide. This prematurely terminates polypeptide synthesis. 

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. 

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