Half-cystin

The correct pairing of half-cystine residues is shown to be dependent upon specific noncovalent bonds 17). With this finding in mind, oxidation of a pair of associating thiols (7 and 2) was chosen as a model reaction. Thiol 7 has the same group as cysteine side chain (HSCH2), 2 being a derivative of cysteamine. 

Comparisons between these toxins allow delineation of the variability of each position in the sequence. For instance, the residues which are extremely invariant (conservative) for both types of sea anemone toxin are the half-cystines, certain glycyl residues which are expected to be involved in )9-turns, and only a few other residues - Asp 5 or 6, Arg 13 or 14, and Tryp 30 or 31 (the numbering depends upon the toxin type) — expected to be important for folding or receptor binding. Rather surprising is the variation in the residues which NMR studies (22,23) have shown are involved in formation of the four stranded )9-pleated sheet. 

There are four disulfide bonds in short-chain (Type I) neurotoxins. This means that there are eight half-cystines. However, all Hydrophiinae toxins have nine halfcystines with one cysteine residue. An extra cysteine residue can be readily detected from the Raman spectrum as the sulfhydryl group shows a distinct S-H stretching vibration at 2578 cm" Some Laticaudinae toxins do not have a free cysteine residue as in the cases of L. laticaudata and L. semifasciata toxins. In long toxins (Type II) there are five disulfide bonds (Table III). 

The A and B peptide chains in insulin are linked through disulfide bridges. Their presence was suspected from the change in molecular weight which followed the reduction of insulin. For quantitative analyses the S-S bridges had to be broken. Sanger, following the approach used by Toennies and Homiller (1942), oxidized the protein with performic acid, so that the half-cystines were converted to cysteic acid. After oxidation, insulin could be separated into its A and B chains, the A peptide with 20 amino acid residues and the B with 30. 

Now let us examine the distribution and position of disulfides in proteins. The simplest consideration is distribution in the sequence (see Fig. 51), which is apparently quite random, except that there must be at least two residues in between connected half-cystines. Even rather conspicuous patterns such as two consecutive halfcystines in separate disulfides turn out, when the distribution is plotted for the solved structures (Fig. 51), to occur at only about the random expected frequency. The sequence distribution of halfcystines is influenced by the statistics of close contacts in the three-dimensional structures, but apparently there are no strong preferences of the cystines that could influence the three-dimensional structure. 

Fig. 51. Number of residues between sequence-neighbor half-cystines. Pairs which are in the same disulfide are shown by asterisks and those which are not by circles.

There is a correlation between the backbone conformations which commonly flank disulfides and the frequency with which disulfides occur in the different types of overall protein structure (see Section III,A for explanation of structure types), although it is unclear which preference is the cause and which the effect. There are very few disulfides in the antiparallel helical bundle proteins and none in proteins based on pure parallel /3 sheet (except for active-site disulfides such as in glutathione reductase). Antiparallel /3 sheet, mixed /8 sheet, and the miscellaneous a proteins have a half-cystine content of 0-5%. Small proteins with low secondary-structure content often have up to 15-20% half-cystine. Figure 52 shows the structure of insulin, one of the small proteins in which disulfides appear to play a major role in the organization and stability of the overall structure. 

BgK K+ channel-acting toxin from sea anemone Bunodosoma granulifera C (Cys) cysteine (reduced form) or half-cystine residue (oxidized form)... 

Some of the multiple-cystine peptides are resistant to enzymatic and/or chemical cleavage, and thus the more simple methods described in Section 6.1.6.2 cannot be applied. In these cases assignment of their disulfide connectivities is generally attempted by NMR structural analysis. Thereby, if the two half-cystine residues are located on opposite sides of the NMR-derived 3D structure, a disulfide bond between them is improbable. 

Peptide bonds are cleaved in a nonselective, but not in a completely random manner. Based on anchimeric side-chain assistance, steric factors, and bond strains, acid-labile peptide bonds are predicted to include sites containing Asp, Glu, Ser, Thr, Asn, Gin, Gly, and ProJ22l The disulfide topologies of circulin B and cyclopsychotride, backbone-cyclized peptides with three disulfide bonds, were determined by partial hydrolysis for 5 hours.[22 Occasionally, the bond between adjacent half-cystine residues is cleaved due to the nonselective nature of the mechanism of partial acid hydrolysis.[21] By this procedure, in all cases, a complex mixture of peptide fragments is produced which requires careful chromatographic separation by RP-HPLC for subsequent analysis by mass spectrometry (see Section 6.1.6.2.7). 

Adjacent half-cystine residues are present in many peptides and proteins. In most of the cases they form two disulfide bonds with other cysteines in the molecule, unless the peptide bond between them is cis (see Section 6.1.5.1 ).t40 41 Specific enzymes that cleave the Cys-Xaa bond have not yet been discovered, although there are a few reports of cleavage of the Cys-Cys bond by enzymes such as elastase and pepsinJ42-43 For peptides with Cys-Cys bonds the cleavage method in Section 6.1.6.2.4 is recommended. 

The absence of half-cystine residues in collagens with chain compositions [ai(I)]2a2 and [ 2(11)13 exclude cystine disulfide bridges from participation in cross-linking in these collagens. However, half-cystine residues have been identified in [ai(III)]3 collagen and disulfide bridges may serve as cross-links in this type of collagen191). 

The molecular weight of 320,000 obtained for the muscle enzyme from sedimentation-diffusion data at 2-6 mg/ml and v = 0.75 (132) is to be compared with 270,000 obtained by Wolfenden et al. from s20,w = 11.1 S and D2 ,w = 3.75 X 10 7 cm2 sec1, and v = 0.731 calculated from the amino acid content (92). The rabbit muscle enzyme has a normal amino acid content, that is, no unusually low or large amount of a particular amino acid was found. Of the 32 cysteine/half-cystine residues per mole based on a molecular weight of 270,000, 6.2 were rapidly titrated with p-mercuribenzoate (92). Typical protein absorption spectra were reported for elasmobranch fish (126), carp (125), rat (127), and rabbit muscle enzyme (68). An E m at 280 nm = 9.13 has been reported for the rabbit muscle enzyme (133). The atypical absorption spectrum with a maximum at 275-276 nm observed by Lee (132) is indicative of contaminating bound nucleotides. 

The amino acid composition of RNases Ti, Nl( and Ui, which are classified as guanyloribonucleases, are similar to each other, as shown in Table III. It is remarkable that all three RNases have four residues of half-cystine. From this finding, it may be expected that these three en-... 

Methionine and half-cystine were determined in duplicate as methionine sulfone and cysteic acid on the performic acid-oxidized protein [8. Moore, JBC 238, 235 (1963)). 

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