Peptide Chain Termination

Caskey CT, Forrester WC, Tate W (1984) Peptide chain termination. In Clark BFC, Petersen HU (eds) Gene expression the translational step and its control. Munksgaard, Copenhagen... 

The protein truncation test is a way of testing large genes (e.g., NEl) for which an antibody is available. PTT can detect nonsense mutations that are peptide chain terminating. These show up, after reverse transcription/cell-free translation, as shorter-than-normal peptides in an electrophoretic gel (Eig. 3C). 

The answer is b. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101-120.) The genetic code uses three-nucleotide words, or codons, to specify the 20 different amino acids (see the chart below). There are 64 different three-base pair codons (three positions with four possible nucleotides at each). It follows that the genetic code must be degenerate, i.e., different codons can specify the same amino acid. Three codons are reserved as stop signals that result in peptide chain termination. The linear correspondence of codons in DNA and of amino acids in protein domains is interrupted by the presence of introns in DNA. Codons differ from the dinucleotide tandem repeats that provide useful DNA polymorphisms, or the trinucleotide repeats that can be responsible for disease. The genetic code is universal in the sense that codon-amino acid relationships are the same in all organisms. 

Beaudet, a. L. and Caskey, C. T. (1971) Mammalian peptide chain termination, II. Codon specific and GTPase activity of release factor. Proc. Nat. Acad. Sci. U.S.A. 68, 619-624. 

Caskey, C. T., 1977, Peptide chain termination, in Molecular Mechanisms of Protein Biosynthesis (H. Weissbach and S. Pestka, eds.), pp. 443-465, Academic Press, New York. 

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

These methodologies have been reviewed (22). In both methods, synthesis involves assembly of protected peptide chains, deprotection, purification, and characterization. However, the soHd-phase method, pioneered by Merrifield, dominates the field of peptide chemistry (23). In SPPS, the C-terminal amino acid of the desired peptide is attached to a polymeric soHd support. The addition of amino acids (qv) requires a number of relatively simple steps that are easily automated. Therefore, SPPS contains a number of advantages compared to the solution approach, including fewer solubiUty problems, use of less specialized chemistry, potential for automation, and requirement of relatively less skilled operators (22). Additionally, intermediates are not isolated and purified, and therefore the steps can be carried out more rapidly. Moreover, the SPPS method has been shown to proceed without racemization, whereas in fragment synthesis there is always a potential for racemization. Solution synthesis provides peptides of relatively higher purity however, the addition of hplc methodologies allows for pure peptide products from SPPS as well. 

A disulfide bond between cysteine residues in different peptide chains links the otherwise separate chains together, while a disulfide bond between cysteine residues in the same chain forms a loop. Such is the case, for instance, with vasopressin, an antidiuretic hormone found in the pituitary gland. Note that the C-terminal end of vasopressin occurs as the primary amide, -CONHz, rather than as the free acid. 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

Edman degradation (Section 26.6) A method for N-terminal sequencing of peptide chains by treatment with Af-phenylisothiocyanate. 

Figure 6.8 Pendllins are similar to the bacterial peptidogiycan terminal alanylalanine moiety. Because of this similarity, the enzyme transpeptidase recognizes 8-lactam antibiotics as substrate. As a result of this the 8-lactam is incorporated in the peptide chain thereby making peptide-peptide cross-linking impossible. The occurrence of this phenomenon stops the construction of the bacterial cell wall.

A nonsense codon may appear that would then result in the premature termination of amino acid incorporation into a peptide chain and the production of only a fragment of the intended protein molecule. The probabihty is high that a premamrely terminated protein molecule or peptide fragment will not function in its assigned role. 

Glycopeptides containing a 2-acetamido-N-(L-aspart-4-oyl)-2-deoxy-/ -D-glucopyranosylamine residue (11) may be synthesized either by direct addition of the carbohydrate residue to the peptide chain, or by elongation of the peptide chain from 11 in the direction of the carboxylic, or the amino, terminal. 

Elongation of the peptide chain in the direction of the N-terminal by a solid-phase procedure was introduced by Lavielle and coworkers,132 who prepared the glycopeptide [3-0-(2-acetamido-2-deoxy-/ -D-gluco-pyranosyl)-L-serine]-L-glycyl-L-alanine (189) by first coupling 168, de-protected by hydrogenolysis, to an L-alanyl resin (188), and, after re-... 

Peptide synthesis was amenable to solid-phase techniques since the process was repetitive. The C-terminal amino acid is attached to polymeric surface and the peptide chain is assembled via a two-step process coupling of the incoming amino acid that has the alpha-amino group protected... 

The insertion of histidine in position two of the peptide chain results in the simultaneous binding of the N-terminal amine, the imidazole, and the intervening amide-N to the Ni11 ion. The major complex [NiH iL]+ with NH2,N am,Nim binding mode is pseudo-octahedral and in... 

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