Intein-mediated protein splicing is an autocatalytic process in which the intervening intein sequence is removed from a precursor protein and the adjoining extein segments are ligated with a native peptide bond. The residues proximal to the splice junction and residues within the intein direct these reactions¹ .  The splicing reaction has been shown to be efficient when the residue joining the two-protein domain is a cysteine. However, the presence of a free cysteine poses a problem while expressing disulfide rich proteins. We employed protein ligation using a serine residue to structurally characterize a two-domain disulfide rich peptide toxin from the earth tiger tarantula ² .

The double knot tarantula toxin  (DkTx) has been shown to be the most avid binder of TRPV1 channels and shown to consist of two homologous inhibitory cysteine-knot (ICK) motifs² . Each ICK domain contains three disulfide bonds resulting in a total of six disulfide bonds which can theoretically form > 10,000 isoforms. The low yield of natively folded DkTx when expressed recombinantly and the occurrence of homologous domains present technical challenges for structural studies by NMR. Here, we have expressed the individual ICK’s as exteins in two different intein systems and performed in vivo protein ligation to enhance the yield of natively folded DkTx. Further, segmental isotope labeling of the ICK motifs has been performed allowing the nuclear resonance signal from each individual domain (internal domain) to be analysed in the context of the full-length protein. Multidimensional heteronuclear NMR data is acquired using non-uniform sampling and processed using maximum entropy reconstruction, an approach that significantly reduces the data acquisition time and produces high-resolution data for facile analysis ³ .