Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence.

Protein splicing is a self-catalyzed, posttranslational process which converts a precursor polypeptide into two new proteins by the excision of an internal polypeptide segment and the ligation of the flanking polypeptides. Evidence has been presented that protein splicing involves a branched intermediate, which is resolved into the two protein products by the cyclization of an asparagine residue to aminosuccinimide [Xu, M. Q., Comb, D. G., Paulus, H., Noren, C. J., Shao, Y., & Perler, F. (1994) EMBO J. 13, 5517-5522]. This report describes the chemical synthesis of a peptide with a C-terminal aminosuccinimide residue, corresponding to the putative C-terminus of the excised intervening sequence (intein) derived from the thermostable DNA polymerase of Pyrococcus species GB-D. The synthetic aminosuccinimide peptide was compared with the C-terminal cyanogen bromide peptide of the excised intein and found to be indistinguishable in terms of its chromatographic properties, high-resolution mass spectrum, and colorimetric assay involving reaction with hydroxylamine. This establishes definitively that protein splicing is accompanied by the cyclization of asparagine to yield an aminosuccinimide residue at the C-terminus of the excised intein and that this unusual residue is therefore a natural constituent of spliced proteins. The effects of pH and temperature on the stability of the synthetic aminosuccinimide peptide are described.(ABSTRACT TRUNCATED AT 250 WORDS)