Proteins disulfide formation

Freedman, R.B. The formation of protein disulfide bonds. Curr. Opin. Struct. Biol. 5 85-91, 1995. 

This thiol-disulfide interconversion is a key part of numerous biological processes. WeTJ see in Chapter 26, for instance, that disulfide formation is involved in defining the structure and three-dimensional conformations of proteins, where disulfide "bridges" often form cross-links between q steine amino acid units in the protein chains. Disulfide formation is also involved in the process by which cells protect themselves from oxidative degradation. A cellular component called glutathione removes potentially harmful oxidants and is itself oxidized to glutathione disulfide in the process. Reduction back to the thiol requires the coenzyme flavin adenine dinucleotide (reduced), abbreviated FADH2. 

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. 

Proteins modified with 2-iminothiolane are subject to disulfide formation upon sulfhydryl oxidation. This can cause unwanted conjugation, potentially precipitating the protein. The addition of a metal-chelating agent such as EDTA (0.01-0.1M) will prevent metal-catalyzed oxidation and maintain sulfhydryl stability. In the presence of some serum proteins (i.e., BSA) a 0.1M concentration of EDTA may be necessary to prevent metal-catalyzed oxidation, presumably due to the high contamination of iron from hemolyzed blood. 

Purify the thiolated protein from unreacted Traut s reagent by gel filtration using your buffer of choice (i.e., 20mM sodium phosphate, 0.15M NaCl, ImM EDTA, pH 7.2). The addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and the resultant disulfide formation. After purification, use the thiolated protein immediately... 

The deacetylated protein should be used immediately to prevent loss of sulfhydryl content through disulfide formation. The degree of—SH modification may be determined by performing an Ellman s assay (Section 4.1, this chapter). 

Figure 19.19 shows a plot of the results of such an assay done to determine the maleimide content of activated BSA. This particular assay used 2-mercaptoethanol which is relatively unaffected by metal-catalyzed oxidation. For the use of cysteine or cysteine-containing peptides in the assay, however, the addition of EDTA is required to prevent disulfide formation. Without the presence of EDTA at 0.1 M, the metal contamination of some proteins (especially serum proteins such as BSA) is so great that disulfide formation proceeds preferential to maleimide coupling. Figure 19.20 shows a similar assay for maleimide-activated BSA using the more innocuous cysteine as the sulfhydryl-containing compound. 

Essex, D. W., Miller, A., Swiatkowska, M., Feinman, R. D., Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin. Biochemistry 38 (1999), p. 10398-10405... 

N. F. Tabachnik, P. Blackburn, C. M. Peterson, A. Cerami, Protein Binding of JV-2-Mer-captoethyl-2,3-diaminopropane via Mixed Disulfide Formation after Oral Administration of WR 2721 , J. Pharmacol. Exp. Ther. 1982, 220, 243 - 246. 

The extent of formation of protein disulfides with time was determined by withdrawing aliquots which were acidified to pH 5.5 and alkylated with N-ethylmaleimide. The disulfide content of the peptide was determined after its isolation. Formation of two intrapeptide disulfide bonds proceeded at the same rate (within experimental error) as formation of the first two disulfides in reduced lysozyme. The first-order rate constant for these two processes (0.5 min-1) was eight times that describing the rate of oxidation of reduced lysozyme in the presence of 6 M guanidinium chloride, suggesting substantial specificity in the process in absence of denaturant. An additional indication of specificity was the finding that 13-105 reached its maximum of two —S—S— bonds in less than 20 minutes, retaining one reduced thiol from 20 to 240 minutes. For subsequent studies this material was S-alkylated with N-ethylmaleimide. 

Disulfide bridges are, of course, true covalent bonds (between the sulfurs of two cysteine side chains) and are thus considered part of the primary structure of a protein by most definitions. Experimentally they also belong there, since they can be determined as part of, or an extension of, an amino acid sequence determination. However, proteins normally can fold up correctly without or before disulfide formation, and those SS links appear to influence the structure more in the manner of secondary-structural elements, by providing local specificity and stabilization. Therefore, it seems appropriate to consider them here along with the other basic elements making up three-dimensional protein structure. 

Anfinsen, C. B., and E. Haber Studies on the reduction and re-formation of protein disulfide bonds. Journ. Biol. Chem. 236, 1361—1.363 (1961). 

The most common posttranslational modifications, discussed in the following sections, include phosphorylation, sulfation, disulfide formation, N-methylation, O-methylation, S-methylation, N-acetylation, hydroxylation, glycosylation, ADP-ribosylation, prenylation, biotinylation, lipoylation, and phosphopan-tetheine tethering. Many of the posttranslational modifications are proven to be cross talks. Other modifications exist in a smaller extent and include oxidation of methionine, C-methylation, ubiquitylation, carboxylation, and amidation. These topics will not be covered in this chapter which is meant to focus primarily on the recent literature (2005-08). For a more complete coverage of all posttranslational modifications and earlier literature (up to 2005), the reader is referred to Professor Christopher T. Walsh s book Posttranslational Modification of Proteins Expanding Nature s Inventory ... 

Cysteine disulfide formation is one of the most important posttranslational modifications involved in protein structure. Disulfides play a crucial role in maintaining the structure of many proteins including insulin, keratin, and many other structurally important proteins. While the cytoplasm and nucleus are reducing microenvironments, the Golgi and other organelles can have oxidizing environments and process proteins to contain disulfide bonds (Scheme 5). 

Researchers at Sunesis pharmaceuticals have developed a fragment-based drug discovery method termed tethering [25]. The approach, which is illustrated in Scheme 2.6, shares a number of features with DCC. Whereas protein-directed DCLs equilibrate small molecules via disulfide formation, say, in the presence of a protein that acts as a thermodynamic trap, tethering uses a cysteine residue on the protein surface to reversibly capture small-molecule thiol fragments from solution. Tethering is designed... 

The formation of mixed enzyme-CoA or enzyme-acyl carrier protein disulfides was supported by the fact that the activation by these compounds was readily reversed by treatment with glutathione or cysteine (49). Preliminary experiments (50) with radioactive CoA or by titration of sulfhydryl groups in the protein suggest that maximum activation is associated with the incorporation of 4 equivalents of CoA per mole of protein (one per subunit). 

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