Cysteine disulfide bridge formation

Disulfide bridges formation ChEs contain 8-10 cysteines six of these form three internal disulfide bridges. The cysteine that is located four amino acids upstream the carboxyl terminus forms a disulfide bridge with a cysteine of an identical subunit, creating an interchain disulfide bridge, which stabilizes the dimeric structure. 

Remarkably, the mutation of the type 1 copper site Cys-310 to Ser in the Al domain resulted in an inactive enzyme that was partially defective for secretion from the cell (Tagliavacca et al., 1997). The occurrence of intact type 1 copper sites in F8 is also supported by the fact that their putative cysteine ligand residues were shown not to be involved in disulfide bridge formation (McMullen et al., 1995). Thus the F8 has hve halfcysteines in each A domain. Analysis of the disulhde bond pattern in F8 showed that four half-cysteines are involved in the formation of two disulfide bonds, while the fifth remains free. The free Cys residues have been identified as Cys-310 in domain Al, Cys-692 in domain A2, and Cys-2000 in domain A3. All these Cys residues match analogous residues involved in type 1 copper binding in ceruloplasmin. However, as mentioned above, only two of the domains, Al and A3, contain the complete set of ligands for the type 1 copper. 

Different protecting groups (S-Trityl, S-Acetamidomethyl...) have been introduced on the cysteine side chains to optimise the reaction conditions of disulfide bridge formation. The conditions developed by Kamber and his group have been used to make the disulfide bridge (ref. 5). The best results have been obtained when we use S trityl on the L-cysteine and S-acetamidomethyl on the 7 cysteine in N,N-dimethylformamide in the presence of an excess of iodine (4 equivalents). The excess of iodine is eliminated with ascorbic acid. The final peptide is isolated by precipitation with water. 

Spider venom peptides, toxic peptides from spiders that mainly modulate neurotransmission. Spider venoms are rich in neurotoxins that influence ion channels, interfere with neurotransmitter exocytosis, or affect neurotransmitter binding. The most important families are the atracotoxins (36-68 amino acids) and the latrotoxins. Many spider venom peptides are translated as prepropeptides and post-translationally modified, e.g., by disulfide bridge formation and C- or N-terminal modification. Because of the high diversity of its constituents, the spider venom is sometimes regarded as a biogenic structurally constrained combinatorial peptide library where nearly all amino acids of the mature sequence may be mutated, with the exception of a few strictly conserved cysteine residues responsible for the three-dimensional fold of the toxin [G. Estrada etal, Nat. Prod. Rep. 2007, 24,145]. 

Also important for stabilizing a protein s tertiary stmcture are the formation of disulfide bridges between cysteine residues, the formation of hydrogen bonds between nearby amino acid residues, and the presence of ionic attractions, called salt bridges, between positively and negatively charged sites on various amino acid side chains within the protein. 

Insect OBPs are secretory proteins whose only posttranslational modification is the formation of three disulfide bridges [39,45] from six cysteine residues. That six cysteine residues are well conserved in OBPs from species of the same order is a hallmark of these proteins. The disulfide links of OBPs in a few species have been determined by analytical methods, first in the OBPs from B. mori [45,46]. As part of our attempt to get better insight into the structural biology of pheromone-binding proteins, we have determined the disulfide linkages... 

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. 

The ability of coordinated NO to react with thiols has led to the suggestion of an alternative mechanism for activating guanylate cyclase. This involves nitroprusside oxidation of protein sulfhydryls to cross-link the protein with a disulfide bridge. For example, papain, which has an essential cysteine (cys-25) and glyceradehyde-3-phosphate dehydrogenase (cys-149) are both inhibited by nitroprusside with formation of [Fe(CN)5(NO)] and [Fe(CN)4NO] [132]. The suggested anaerobic reaction is ... 

For the synthesis of double-stranded symmetrical and unsymmetrical monocystine peptides the formation of an intermolecular disulfide bridge is required. For homodimerization of cysteine peptides all the methods discussed in Section 6.1.1 can be applied taking into account the reactivity of the different oxidative agents toward sensitive amino acid residues present in the peptide sequences. Synthetic approaches based on the direct use of suitable cystine derivatives can be envisaged, at least for small-size peptides since disproportionation would in all cases retain the homodimeric structure 241... 

A further development of the DMSO/H+ method for oxidation of cysteine peptides led to the cysteine-sulfoxide acid-catalyzed intermolecular disulfide formation with a second S-unprotected or acid-labile protected cysteine component as shown in Scheme 19. 1471 The protonation of the sulfoxide by TfOH in the case of 5(0)-Mob or TFA in the case of 5(0)-Acm derivatives provides electrophilicity to the sulfur atom to allow attack by the second S-unprotected cysteine component (formed by the fast deprotection of the 5-Mob group with TfOH in presence of dimethylsulfide) to generate in a site-directed manner the interchain disulfide bond. Although extensive experience with this method has not been accumulated for interchain disulfide bridging, it has been successfully applied for intrachain site-directed disulfide bond formation in chicken calcitonin-gene-related peptide.1 79 ... 

Alternatively, both peptide chains could be protected at one cysteine residue as a 5-Acm derivative and at the second cysteine residue by an acid-labile [Trt, Mob, Xan, or Bzl(4-Me)], base-labile (Fm), or reduction-labile (5-tBu) group. Both peptide chains may then be separately converted into the free thiol/Acm-protected form for selective activation of one chain as S-SPy or. S -Npys derivatives by reaction with di(2-pyridyl)disulfide or di[5-nitro(2-pyridyl)]disulfide, or as a sulfenohydrazide derivative by reaction with azodicarbocylic acid derivatives for formation of the first interchain disulfide bridge. 

Cleavage of the Trt group of one chain 54 with a weak acid to give 55 and its subsequent thiolysis of the. S -SPy derivative of the second chain 57 directs the formation of the first interchain disulfide bond in 58. The second interchain disulfide bridge is formed between the two Acm-protected cysteine residues of the [bis(Acm), bis(tBu), mono-disulfide]-hetero-dimer 58 by treatment with iodine. Finally, treatment of 59 with chlorosilane/sulfoxide produces the third disulfide bond between the two tBu-protected cysteine residues yielding human insulin (42). 

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