Proteins disulfide linkage

The immunoglobulin structure in Figure 6.45 represents the confluence of all the details of protein structure that have been thus far discussed. As for all proteins, the primary structure determines other aspects of structure. There are numerous elements of secondary structure, including /3-sheets and tight turns. The tertiary structure consists of 12 distinct domains, and the protein adopts a heterotetrameric quaternary structure. To make matters more interesting, both intrasubunit and intersubunit disulfide linkages act to stabilize the discrete domains and to stabilize the tetramer itself. 

Conserved sequences that fold into large loops stabilized by three disulfide linkages. The name Kringle comes from the Scandinavian pastry that these structures resemble. They can mediate certain protein-protein interactions. 

Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

Fungal cutinases show no free SH groups but have 4 Cys residues, indicating that they are in disulfide linkage [119]. The reaction of the native enzyme with DTE was extremely slow but in the presence of SDS at its CMC rapid reduction could be observed [102]. Reduction of the disulfide bridge resulted in irreversible inactivation of the enzyme and the protein tended to become insoluble. CD spectra of cutinase in the 205-230 nm region, before and after DTE reduc-... 

Figure 6.3 Mts-Atf-Biotin can be used to label bait proteins at available thiol groups using the MTS group, which forms a disulfide linkage after reaction. The modified protein then is allowed to interact with a protein sample and photoactivated with UV light to cause a covalent crosslink with any interacting proteins. Cleavage of the disulfide bond effectively transfers the biotin label to the unknown interacting protein.

Alternatively, a thiol-containing protein may be directly conjugated to the SPDP-modified dendrimer to create a disulfide linkage. Add a sulfhydryl-containing protein... 

Figure 14.10 The crosslinker SPDP can be reacted with amine particles to create thiol-reactive pyridyl disulfide groups on the surface. Thiol-containing proteins or other thiol molecules can be reacted with these activated particles to result in disulfide linkages, which are reversible by reduction.

Figure 18.14 NHS-SS-PEG4-biotin can be used to label a primary antibody molecule that has specificity for a protein or interest. Incubation of the biotinylated antibody with a sample, such as a cell lysate, allows the antibody to bind to its target. Capture of the antibody-antigen complex on an immobilized streptavidin reagent effectively isolates the targeted protein from the other proteins in the sample. The disulfide linkage in the spacer arm of the biotin tag permits elution of the immune complex from the streptavidin support using DTT and without using the strong denaturing condition typically required to break the streptavidin-biotin interaction.

Figure 22.26 SPDP-activated liposomes can be used to couple sulfhydryl-containing proteins, forming disulfide linkages.

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