The gene sequences of several other peptides, predicted to contain ICK motifs, and present in the Cucurbitaceae family, have been determined recently. These precursor proteins have similar architectures to MCh-1 and MCh-2, with the mature peptides at the terminal region of the precursor proteins. However, the cleavage sites that yield the mature peptide vary for these different disulfide-rich peptides. Known cleavage sites include after an alanine residue for a trypsin inhibitor TI-I from Trichosanthes kirilowii and cleavage after a glycine residue to yield TGTI-II from the towel gourd, as shown in Figure 6. Both MCh-1 and MCh-2 appear to require cleavage after a leucine residue to yield the mature peptides. The diversity in the cleavage sites suggests that a range of proteases are involved in the maturation of plant ICK peptides. The oxidative folding of MCh-1 was analyzed using RP-HPLC and MS. MCh-1 represents an ideal peptide for investigating the oxidative SJN 2511 ALK inhibitor Refolding process of the ICK motif since its in vitro oxidation is slow enough to allow the isolation and characterization of intermediates formed during folding. Although IIa was the major intermediate, numerous other intermediates were present in the oxidative refolding of MCh-1, both in the presence and in the absence of the shuffling reagent glutathione. Refolding of purified IIa resulted in numerous species, including two-disulfide and three-disulfide isomers. This complexity in the folding pathway indicates that IIa does not convert directly to the native form. By contrast with the oxidative refolding process, the reductive unfolding is very simple and IIa was the only intermediate observed. It is interesting to note the differences in retention times of the intermediates during the selective reduction and the stepwise alkylation, or the oxidative refolding and the stepwise alkylation. The intermediate IIa eluted just before the native peptide and the similarity of the retention times of the intermediate with that of the native peptide indicates that the three-dimensional structures are similar and that the intermediate also contains a compact structure. The retention time of NEM-alkylated IIa was almost the same as that of IIa. This similarity in retention times suggests that the NEM replacement did not affect the conformation of IIa. By contrast, the retention times of NEM-alkylated Ia, the fully alkylated MCh-1 and intermediate Ia decreased dramatically relative to the native peptide, which indicates that those structures are not compact. Different folds of MCh-1 intermediates are shown in Figure 7. The two-disulfide intermediate observed during the folding of MCh-1 is equivalent to the major intermediates previously reported for the plant ICK peptides EETI-II, MCoTI-II and kalata B1. The structures of these intermediates have been analyzed using NMR spectroscopy and shown to contain the native fold but lack the CysI-CysIV disulfide bond. EETI-II and MCoTI-II are both squash trypsin inhibitors with similar sequences, and do not share sequence similarity with kalata B1, with the exception of the six cysteine residues. However, both MCoTI-II and kalata B1 contain a cyclic backbone in addition to the ICK motif. Therefore this intermediate accumulates during folding in both cyclic and acyclic peptides and does not appear to have stringent sequence requirements given the diversity of sequences across these different peptides. Despite the conservation of the intermediate IIa in the folding of various ICK peptides, the pathways involved in the formation of the native peptide vary. The intermediate IIa observed during the folding of MCoTI-II appears to be the direct precursor to the native peptide, in contrast to the intermediate observed during the folding of kalata B1, which requires rearrangement of the disulfide bonds to form the native peptide. The MCh-1 IIa intermediate is also Gefitinib likely to require rearrangement of the disulfide bonds based on the additional intermediates observed in the analysis of the folding of purified intermediate IIa. The conservation of the two-disulfide intermediate implies an integral role in the folding of the ICK motif. Indeed, it is tempting to speculate that this intermediate is involved with the evolution of the ICK given it is equivalent to the proposed ancestral fold. The disulfide-directed b-hairpin comprising two-disulfide bonds equivalent to the CysII-CysV and CysIII-CysVI bonds in the ICK motif has been proposed to be the ancestral fold of the ICK.