The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65–72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn → isoAsp where the local structure of the loop 65–72 has been modified keeping intact the C65–C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65–72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic l-Asp/l-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of l-Asp by l-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (l-Asp/l-isoAsp)-X bond and of the X-(l-Asp/l-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either l-Asp or l-isoAsp, these identify one or the other form. Characterization and relative quantification of l-Asp/l-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1–7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41–46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

Conserved deamidation of PKA catalytic subunit isozymes Cα and Cβ—more than 25% at Asn2 in vivo in both cases—has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457–469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the “repair” of isoAsp to Asp by the action of protein l-isoaspartyl (d-aspartyl) O-methyl transferase (PIMT). l-Succinimide is prone to racemization to d-succinimide, which may hydrolyze to d-isoAsp- and d-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of d-isoAsp- and d-Asp-containing T1 fragments in addition to the l-isomers. The small amount of the l-isoAsp isomer, representing only part of the d-isoAsp isomer, and the relatively large amounts of the l-Asp and d-Asp isomers argues for an effective action of PIMT present in cardiac tissue.

Asn deamidation was monitored in Ala-based octadecapeptides of varying α-helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala → Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed-phase high-performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of α-helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.