title: Protein trans-splicing of an atypical split intein showing structural flexibility and cross-reactivity.
abstract: Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins) with a peptide bond. In a split intein, the intein fragments (I(N) and I(C)) can reassemble non-covalently to catalyze a trans-splicing reaction that joins the exteins from separate polypeptides. An atypical split intein having a very small I(N) and a large I(C) is particularly useful for joining synthetic peptides with recombinant proteins, which can be a generally useful method of introducing site-specific chemical labeling or modifications into proteins. However, a large I(C) derived from an Ssp DnaX intein was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. Here, we show that this I(C) could undergo trans-splicing in the presence of I(N), and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this I(C) could trans-splice with small I(N) sequences derived from two other inteins, showing a cross-reactivity of this atypical split intein. Furthermore, we found that this I(C) could trans-splice even when the I(N) sequence was embedded in a nearly complete intein sequence, suggesting that the small I(N) could project out of the central pocket of the intein to become accessible to the I(C). Overall, these findings uncovered a new atypical split intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein.
authors.affiliation: Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, People's Republic of China.
authors.forename: Huiling
authors.initials: H
authors.lastname: Song
authors.forename: Qing
authors.initials: Q
authors.lastname: Meng
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D047668:Inteins; D009154:Mutation; D019154:Protein Splicing / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins