Disulfide bridge in proteins

Peroxides, too, are Raman active they exhibit a band at 860 cm (Fig. 4.1-16B). Disulfides are characterized by the very strong Raman band of the S-S stretching vibration at 500 cm (Figs. 4.1-16C, 4.1-17C). This band, together with the a(C-S) vibration at 500 - 750 cm, strong in the Raman spectrum, is employed to elucidate the conformation of disulfide bridges in proteins (Lord, 1977). 

Finally there is evidence of interaction between sulfur metabolism, silicon metabolism, copper, and cell division. Copper is known to interfere with the formation of disulfide bonds and it can also denature proteins. Sulfur is generally involved in nuclear and cellular division. One possible effect of copper may be the disruption of disulfide bridges in proteins. Another is through general disruption of other pathways involving covalently bonded sulfur. In addition, silicon is a required compound in division of diatoms. Following... 

Metabolic oxidation reactions may occur to the side chains of sulfor-containing residues, similar to that observed for in vitro chemical instability. Methionine can be oxidized to the sulfoxide, whereas metabolic oxidation of cysteine residues forms a disulfide. Metabolic reductive cleavage of disulfide bridges in proteins may occur, yielding free sulfhydryl groups. 

Figure 41 shows characteristic group frequencies of sulfides and disulfides. The stretching vibration of C-S bonds gives rise to a weak infrared but a strong Raman signal at 730-570 cm . Similarly, the S-S stretching vibration at 500 cm is a very strong Raman line but very weak infrared band (Fig. 42), Both Raman signals are very diagnostic in the conformational analysis of disulfide bridges in proteins.

Free cystine is relatively stable at temperatures around 100 °C. The reaction that is observable during thermal processing of foods at temperatures of approximately 100 °C is partial elimination of hydrogen sulfide from bound cystine, which is often called protein desulfuration (Figure 2.51). This reaction splits the disulfide bridges in protein with the formation of cysteine and sulfenic acid... 

In a recent study, Thornton (1981) analyzed in detail the distribution and topology of disulfide bridges in proteins of known sequence and structure (28 proteins) and in proteins of known structure and disulfide connectivities... 

Wame, N.W., and Laskowski, M., 1990, All fifteen possible arrangements of three disulfide bridges in proteins are known. Biochem. Biophys. Res. Com. 172 1364-1370. 

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

The three-dimensional shapes of many proteins are governed and stabilized by S—S bonds connecting what would ordinarily be remote segments of the molecule. We ll have more to say about these disulfide bridges in Chapter 27. 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

In summary, it appears that the protein has to adopt the correct fold before the Rieske cluster can be inserted. The correct folding will depend on the stability of the protein the Rieske protein from the thermoacidophilic archaebacterium Sulfolobus seems to be more stable than Rieske proteins from other bacteria so that the Rieske cluster can be inserted into the soluble form of the protein during expression with the help of the chaperonins. If the protein cannot adopt the correct fold, the result will be either no cluster or a distorted iron sulfur cluster, perhaps using the two cysteines that form the disulfide bridge in correctly assembled Rieske proteins. 

With the advent of monoclonal antibodies, the search for tumour-specific antigens became the biggest cottage industry since unemployment. It rapidly became apparent that a 90 kD disulfide-bridged transmembrane protein was present in many tumour cells - it was the transferrin receptor, and as they say, the rest is history. It has become a standard procedure to determine the in vivo growth potential of tumours by measuring transferrin receptor expression. 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

Amino acid variants of IL-2 have been used to investigate the relationship between retention and protein structure in gradient RPLC.22 The protein contains three cysteine residues in its primary sequence at positions 58, 105, and 125. The two located at positions 58 and 105 are linked in a disulfide bridge in the native molecule. A series of variants in which the three cysteinyl residues were replaced with serines were compared. Substitution with serine at positions 58 or 105 forces the molecule to form an unnatural disulfide between positions 125 and 58 or 105. A methionine residue located at position 104 can also be oxidized to the sulfoxide... 

The original NMR studies of fruit brazzein indicated that the protein adopts a cysteine-stabilized (CSa ) fold in which the a-helix and P-strands are stabilized by the presence of four disulfide bridges.Other proteins with this fold include members of the rapeseed family of serine... 

These complex macrobicycles were assembled by the incorporation of eight more building blocks, forming 12-24 new bonds in a one-pot reaction. The more complex three-dimensional multi-macrocyclic pseudopeptides like the igloos might in principle be considered mimics of internally disulfide bridged crumpled proteins like the knottins (Scheme 27) [101]. 

Since an increased number of disulfide bridges in relatively short polypeptide chains leads to compact globular structures with the disulfides mainly buried in the nonpolar core, such excised protein fragments should represent, even in the precursor molecules, stable subdomains. Therefore, sufficient structural information can be retained for a correct refolding at least to some extent, if appropriate experimental conditions are applied in terms of peptide concentration, redox reagents, temperature and/or reaction buffers. A great deal of... 

One may stress that in proteins clefts are built by coiling back and forth a linear thread maintained by intermolecular binding forces (and eventually a few disulfide bridges). In the case of smaller synthetic molecules, organic synthesis provides means for building the molecular architecture using more highly connected cornerstones. 

Almost all cells contain a high concentration (3-9 mM) of the thiol-containing tripeptide glutathione (G-SH, Box 11-B). In its disulfide form it participates in forming disulfide bridges in secreted extracellular proteins (Eq. 10-9) via intermediate mixed disulfides. Mixed disulfides with glutathione as well as with other thiols can also be formed within cells by oxidative... 

The disulfide bridges in some proteins are between different peptide chains. Insulin, for instance, has two interchain as well as one intrachain S—S bridges (Figure 25-8). 

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