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A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues

: Güttler, Bert H.-O.; Cynis, Holger; Seifert, Franziska; Ludwig, Hans-Henning; Porzel, Andrea; Schilling, Stephan


Amino acids 44 (2013), No.4, pp.1205-1214
ISSN: 0939-4451 (Print)
ISSN: 1438-2199 (Online)
Journal Article
Fraunhofer IZI ()
Alzheimer's disease; PIMT; capillary electrophoresis; 1H NMR; isoaspartate

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer’s disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day−1, peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (k D2O/k H2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.