Synthesis and disaggregation of asparagine repeat-containing peptides.

ABSTRACT

Of all amino acid repeats in eukaryotes, polyglutamine (polyQ) is the most frequent, followed by polyasparagine (polyN). Glutamine repeats are expanded in proteins associated with several neurodegenerative disorders. The expanded polyQ domain is known to induce aggregation, and it is hypothesized that aggregation is directly causative of pathology. Despite the widespread presence of asparagine repeats in invertebrate eukaryotes, polyN is curiously quite rare in vertebrates. Several investigators have characterized the conformational and aggregation properties of polyQ-containing peptides and proteins, and to a lesser extent, peptides containing mixed glutamine and asparagine, but to our knowledge, there is no detailed characterization of polyN-containing peptides. Such a comparison could elucidate reasons for the paucity of asparagine repeats in humans. In this study, we synthesized a peptide containing a 24-asparagine repeat (N24). For aggregation studies, it is critical to start with monomeric unaggregated peptide. A protocol involving dissolution in mixed trifluoroacetic acid and hexafluoroisopropanol (TFA + HFIP) solvents is widely used for disaggregation of polyQ peptides. We used the same protocol for N24 but discovered that there was both oxidative damage and insufficient disaggregation. Oxidation of tryptophan, used as a flanking residue, was common. Moreover, we found evidence of Förster resonance energy transfer between Trp and its oxidation product N-formylkynurenine, even in chemical denaturants. This suggested that N24 was insufficiently disaggregated, a conclusion that was further supported by gel electrophoresis analysis. Oxidation was reduced, but not eliminated, by addition of methionine to the buffer. Formic acid proved to be a better disaggregator and caused no oxidative damage. The glutamine repeat peptide Q24 also underwent some oxidation after extended incubation in TFA + HFIP, but there was no evidence of Förster resonance energy transfer, and samples appeared monomeric by gel electrophoresis. This result indicates that polyN-containing peptides self-associate more strongly than polyQ-containing peptides. Circular dichroism spectra reveal a greater propensity for ß-turn formation in polyN than polyQ, providing an explanation for the increased stability of polyN aggregates relative to polyQ.