Disulfide crosslinking

Numerous organisms, both marine and terrestrial, produce protein toxins. These are typically relatively small, and rich in disulfide crosslinks. Since they are often difficult to crystallize, relatively few structures from this class of proteins are known. In the past five years two dimensional NMR methods have developed to the point where they can be used to determine the solution structures of small proteins and nucleic acids. We have analyzed the structures of toxins II and III of RadiarUhus paumotensis using this approach. We find that the dominant structure is )9-sheet, with the strands connected by loops of irregular structure. Most of the residues which have been determined to be important for toxicity are contained in one of the loops. The general methods used for structure analysis will be described, and the structures of the toxins RpII and RpIII will be discussed and compared with homologous toxins from other anemone species. 

Purify the thiolated protein from excess DTT by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, ImM EDTA, pH 7.2. The modified protein should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

Figure 21.10 Cystamine may be used to make immunotoxin conjugates by a disulfide interchange reaction. Modification of antibody molecules using an EDC-mediated reaction creates a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol can be coupled to the cystamine-modified antibody to form disulfide crosslinks.

A final method of forming disulfide crosslinks between toxins and targeting molecules is the use of S-sulfonate formation using sodium sulfite (Na2SC>3) in the presence of sodium tetrathion-ate (Na2S40g). Tetrathionate reacts with sulfhydryls to form sulfenylthiosulfate intermediates (section 1.1.5.2). These derivatives are reactive toward other thiols to create disulfide linkages... 

This paper describes the successful synthesis and examination of polyfr-(amino /9-thiosulfate) ether] (PATE), a water soluble photolabile polymer. Evidence has been presented that the PATE polymer is zwitterionic and forms weak associations in aqueous solutions. Heat treatment of PATE films result in extensive crosslinking, presumably through a disulfide bond. This work presents strong evidence that PATE is activated by deep UV radiation, and that a disulfide crosslink is formed. Sensitization experiments demonstrate that the crosslinking reaction can be induced by a triplet sensitizer. Finally, preliminary results point out the potential for application of PATE films as active photoimaging systems. 

The large size of soluble glutenln molecules is due to limited disulfide bonds between polypeptide chains. The insolubility of residue protein is attributable to extensive Intermolecular disulfide crosslinks. 

Table II provides some examples of how the effects of disulfide crosslinks on Re are reflected in the observed Mapp s for a series of proteins of known molecular weight. The divergence between the true M and the Mapp observed for crosslinked random coils clearly demonstrates the dependence of the method on linear dimensions for the polymer under investigation. It also shows the need for coupling this method with exact methodology for molecular weight determination if incontrovertible data are required.

With an accelerated system, a simple network structure with dtalkenyl mono- and disulfide crosslinks and conjugated tnene units as main-chain modifications is obtained ... 

Elasticity Hydrophobic bonding, disulfide crosslinks Meats, baked goods Muscle proteins... 

Model compounds based on 2-methyl-2-pentene were studied to supplement the 13C chemical shift assignments of the products from accelerated sulfur vulcanisation of NR. It is observed in the model compound data that it may not be possible to distinguish between a 13C NMR resonance which is due to disulfidic crosslinks and a peak due to pendent accelerator groups, while a large chemical shift difference ( 3 ppm) is observed for the monosulfidic bonds. The MBS-accelerated sample shows similar new resonances as seen in the TMTD accelerated systems. In this comparison however, the quantitative aspects of the data might be obscured due to the differences in the state of cure among the different formulations. 

Elastic fibers form the network in skin and cardiovascular tissue (elastic arteries) that is associated with elastic recovery. Historically the recovery of skin and vessel wall on removal of mechanical loads at low strains has been attributed to elastic fibers. Elastic fibers are composed of a core of elastin surrounded by microfibrils 10 to 15 nm in diameter composed of a family of glycoproteins recently termed fibrillins. Fibrillins are a family of extracellular matrix glycoproteins (MW about 350,000) containing a large number of cysteine residues (cysteine residues form disulfide crosslinks). Several members of the family have been described. The common molecular features include N and C terminal ends with 47 tandemly repeated epi-... 

Elastin is a macromolecule synthesized as a 70,000 single peptide chain, termed tropoelastin and secreted into the extracellular matrix where it is rapidly crosslinked to form mature elastin. The carboxy-terminal end of elastin is highly conserved with the sequence Gly-Gly-Ala-Cys-Leu-Gly-Leu-Ala-Cys-Gly-Arg-Lys-Arg-Lys. The two Cys residues that form disulfide crosslinks are found in this region as well as a positively charged pocket of residues that is believed to be the site of interaction with microfibrillar protein residues. Hydrophobic alanine-rich sequences are known to form a helices in elastin these sequences are found near lysine residues that form crosslinks between two or more chains. Alanine residues not adjacent to lysine residues found near proline and other bulky hydrophobic amino acids inhibit a helix formation. Additional evidence exists for (3 structures and 3 turns within elastin thereby giving an overall model of the molecule that contains helical stiff segments connected by flexible segments. 

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