N-terminal cysteine

Zinc complexes of peptides with N-terminal cysteine are monomeric of the form ZnL2 where the peptide is an anion with the SH deprotonated. Tetrahedral N2S2 coordination is observed with the chelating peptides and Zn(Cys-Gly-NH2)2 was structurally characterized.857... 

Peptides typically are prepared for this ligation process using a-alkyl thioesters, because they are simple to make at the time of peptide synthesis. However, due to the relatively slow reaction kinetics of alkyl thioesters, most native chemical ligation processes have been catalyzed through the use of thiol compound additives, such as benzyl mercaptan or thiophenol (Dawson et al., 1997). These compounds react with the initial a-alkyl thioester to form another intermediate, an aryl thioester, which is more reactive toward the N-terminal cysteine on the other peptide to be coupled. A study... 

Muir et al. (1998) realized that the intein reaction could be used to facilitate a native chemical ligation with a synthetic N-terminal cysteine-containing peptide or cysteine-containing molecule. With the discovery of a mutant intein that could form an intermediate thioester but not go on to complete the splice and ligation reaction (Xu and Perler, 1996 Chong et al.,... 

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

However, if the expressed protein is treated on the affinity support using thiophenol, this also will release the protein and result in a phenylthioester at its C-terminal, which is the reactive intermediate imminendy suitable for native chemical ligation. Treatment of this activated thioester protein with a N-terminal cysteine peptide induces the native chemical ligation reaction and couples the peptide to the expressed protein through an amide bond (Severinov and Muir, 1998) (Figure 17.27). 

Figure 17.28 EPL reactions can be used to couple a fusion protein to a surface containing a thioester derivative. After cells are grown and the fusion protein expressed, a pH and temperature shift causes intein cleavage with release of the expressed protein with an N-terminal cysteine residue. Reaction with the thioester surface results in a native chemical ligation reaction that forms an amide bond linkage with the expressed protein.

Figure 17.29 An expressed protein containing a mutant intein segment can undergo self cleavage to form an N-terminal cysteine residue, which then can be reacted with a thioester probe to label specifically the protein via an amide bond.

A major scaffolding protein of the PSD is PSD95. Two N-terminal cysteines of this protein bind palmitic acid residues, which anchor PSD95 to lipid rafts [30], PSD95 contains several domains that bind other proteins three so-called PDZ domains (short for PSD95/disc large/zona occludens-1), a src homology (SH3) domain, and a gua-nylate kinase (GK) domain. This family of proteins are... 

Scheme 34 Overview of native chemical ligation (NCL). Two unprotected segments react in a reversible thiol/thioester reaction only the thioester product between the C-terminal thioester and the N-terminal cysteine can react further to form the desired amide bond via nucleophilic attack of the cysteine amine group.

A full-length human csk DNA that codes for Csk, a 50-kDa protein that catalyzes the phosphorylation of a tyrosine within the C-terminal tail of Srk, was inserted in a plasmid and in-frame with a modified intein-chitin binding domain (CBD) encoding sequence, where intein is a protein-splicing element. 130 The expressed protein underwent a conversion into a thioester of the N-terminal cysteine of the intein, but the normal second step of an intein splicing did not occur. The protein was then bound to a chitin resin and washed. The resin containing the bound protein was treated with 2% benzenethiol to cleave the thioester and give the free phenyl thioester, which was immediately treated with one of the synthetic... 

Two approaches for solid-phase chemical ligation have been described. Canne et al. have developed an elegant system that utilizes an oxime forming ligation to attach the first peptide to the resin, a selectively cleavable ester link to remove the peptide from the resin as a C-terminal carboxylic acid, and the Acm group to protect the N-terminal cysteine residue)311 A complementary approach has been developed by Brik et al. that utilizes native chemical ligation to attach the first peptide to the solid support, a safety-catch acid labile linker to remove the final polypeptide from the support as a C-terminal amide and either Acm or Msc group for N-terminal cysteine protection)32 ... 

A lipoprotein present in the periplasmic space of E. coli is anchored to the outer bacterial membrane by a triacylated modified N-terminal cysteine containing a glyceryl group in thioether linkage as shown in the following structure (see also Section E,l). 

Other very important applications of C-terminal thioester-functionalized peptides include their usage in the condensation of large unprotected peptide fragments (ligation see Vol. E22a, Section 4.1.5). 4,5,74 In this process the thioester-modified unprotected peptide reacts with the N-terminal cysteine of a second unprotected peptide giving a thioester intermediate. This step is followed by a rapid intramolecular S—>N shift with formation of the thermodynamically favored amide bond at the ligation site. 

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