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)
Shao
Y
Y
Boston Biomedical Research Institute, Massachusetts 02114, USA.
Xu
M Q
MQ
Paulus
H
H
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D003488:Cyanogen Bromide; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000032:analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013388:Succinimides / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D006877:Hydroxamic Acids; D010455:Peptides; D013388:Succinimides; D001216:Asparagine; D004259:DNA-Directed DNA Polymerase; D003488:Cyanogen Bromide