Disulfide bridges in proteins are formed by the oxidation of cysteine residues. These cross-links play a key role in stabilizing the 3D-structure of disulfide rich polypeptides. The particular arrangement of the multiple disulfide bonds form distinct structural motifs such as the inhibitor cystine knot (ICK), found in many spider venom peptides. This arrangement has proven to be a very stable fold attracting much interest in bioengineering efforts. Recently, a new class of two domain disulfide rich peptides have been reported. This new class has been shown to contain an unusual tandem repeat of the inhibitor cystine knot (ICK) motif, where two ICK motifs are attached via a linker. The remarkable property of these peptides is that they elicit a pharmacological response that is different to that caused by either of the constituent ICK motifs individually (or as a mixture). The bivalency of these peptides therefore appears to be responsible for the observed activity. The low yield of natively folded peptide when expressed recombinantly in a bacterial host and the occurrence of homologous domains present further technical challenges for structural studies by NMR.
Here, these two-domain disulfide rich peptides are considered founding members of a broader class of bivalent peptides that are named secreted cysteine-rich repeat proteins (SCREPs). To further investigate this emerging class of peptides a general method to recombinantly produce SCREPs was developed by expressing the individual domains of SCREPs as exteins in modified intein systems, this was followed by in vivo protein ligation. This approach was tested on DkTx, the first reported SCREP, and showed a 10-fold increase in yield compared to when the peptide is expressed as a single gene. Further, segmental isotope labelling was demonstrated by selectively labeling one of the domains of DkTx. Finally, the high-resolution solution structure of DkTx was determined by multidimensional NMR spectroscopy.