Proteins disulfide crosslinks

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. 

Iodoacetate derivatives have been used for decades to block or crosslink sulfhydryl groups in proteins and other molecules (Chapter 1, Section 5.2). At mildly alkaline pH values (pH 8-8.5), iodoacetyl derivatives are almost entirely selective toward the cysteine —SH groups in proteins. Disulfide reduction or thiolation reagents can be used to create the required sulfhydryl groups on proteins containing no free sulfhydryls. 

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.

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

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. 

Vulcanisation involves the crosslinking of the polymer chains by mono-, di-or polysulfide bridges rather like the disulfide crosslinks in natural proteins (see Chapter 4, p. 51). The process modifies the properties of the resultant polymer, and makes the product more suitable for the manufacture of footwear, hoses, raincoats, tyres and so on. 

The respiratory cytochrome c found in the mitochondria of all eukaryotes is a protein with one heme c (Fig. 1) and 103-113 amino acids in a single polypeptide chain with no disulfide crosslinks. It is small and easily extracted from ruptured mitochondria, in contrast to the other protein components of the respiratory chain, which are larger and generally membrane-bound. For this reason, cytochrome c was the first cytochrome to be studied extensively, and has received the most attention from biochemists. The story of its discovery by MacMunn in 1884, its violent rejection by chemist and editor Hoppe-Seyler, subsequent neglect until rediscovered by David Keilin in 1925, and extensive characterization by Hugo Theorell and his school, is a familiar story which need not be repeated here. Good accounts of this history are to be found in reviews... 

Wu ZM, Zhang XG et al (2009) Disulfide-crosslinked chitosan hydrogel for cell viability and controlled protein release. Eur J Pharm Sci 37 198-206... 

Figure 17-7 shows a portion of hair and disulfide crosslinks joining two protein chains. These crosslinks give hair its strength. (I say more about this disulfide bond in Permanents — that aren t, a little later in this section.)... 

Blrk family (Laskowski and Kato, 1980) are small proteins with disulfide crosslinks between the proteinase-binding regions. Bound trypsin or chymotrypsin was stabilized In Its 1 1 complex with LBI both enzymes were stabilized In the ternary (trypsin—LBI—chymotrypsin) complex (Fig. 7). With highly stable Inhibitors, such as LBI or BPTI, association produced no observable stabilization of the Inhibitor (Zahnley, 1979,1980). 

The wool protein chains are joined periodically through the disulfide crosslinked cystine, a diamino acid that is contained within two adjacent chains. About 40% of the protein chains spiral upon themselves and internally hydrogen bond to form an a-helix. Near the periodic cystine... 

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