The effects of conformational constraints on aspartic acid racemization

In short polypeptides, L-aspartic acid (Asp) and L-asparagine (Asn) can undergo isomerization/deamidation reactions via a succinimide (Asu) intermediate resulting in the formation of D-aspartic acid. Some experimental evidence suggests that this reaction, which has been widely used in age determinations, is severely repressed in structured regions of proteins, like for example the triple-helix of collagen, indicating that conformational constraints imposed on the local peptide geometry by the peptide superstructure may inhibit the formation of Asu-containing sequences relative to Asp- or Asn-containing sequences. To examine this, molecular mechanics and molecular dynamics calculations have been performed on constrained and unconstrained Gly–Pro–Asn–Gly–Pro and Gly–Pro–Asu–Gly–Pro pentapeptides. In the constrained calculations, the influence of the protein helix is simulated by fixing the distance between the backbone-nitrogens in the first and last residue at 11.80 Å, a distance similar to that between comparable atoms in the collagen triple-helix. The results from these simulations show that the constraint significantly destabilizes the Asu-containing pentapeptide relative to the Asn-containing sequence; the molecular dynamics simulations, in which the influence of water was specifically taken into account, predict that the constraint raises the energy difference between these two pentapeptides by 8.8±3.8 kcal/mol. This shows that a protein helical structure may severely repress Asp racemization and Asn deamidation. The conservation of such structures should therefore be taken into consideration when using D/L Asp ratios for age determination.