Characterizing aggregate growth and morphology of alanine-rich polypeptides as a function of sequence chemistry and solution temperature from scattering, spectroscopy, and microscopy.

The aggregation behavior and stability of a series of alanine-rich peptides, which are included as components of peptide-polymer conjugates, were characterized using a combination of biophysical techniques. Light scattering techniques were used to monitor changes in peptide morphology and size distributions as a function of time and temperature. The results show large particles immediately upon dissolution in buffer. At room temperature, these particles relaxed to reach a mostly monomeric peptide state, while at higher temperatures, they grew to form aggregates. Circular dichroism spectroscopy (CD) was used to monitor temperature- and time-dependent conformational changes as a function of peptide sequence and incubation time. CD measurements reveal that all of the sequences are helical at low temperatures with transitions to non-helical conformation with increased temperature. Samples incubated at room temperature were able to recover their original helicity. At increased temperature, the shorter and longer peptide sequences showed notable changes in conformation, and were not able to recover their original helicity after 72 h. After incubation for up to one week, β-sheet conformations were observed in these two cases, while only α-helical conformation loss was observed for the peptide of intermediate molecular weight. Transmission electron microscopy measurements reveal the formation of fibrils after 72 h of incubation at 60 °C for all samples, in agreement with the scattering measurements. Additional quenching experiments show that peptide aggregation can be stalled when solutions are cooled to room temperature.