(27a) Competition of Folding-Induced Assembly and Liquid-Liquid Phase Separation Produces Diverse Morphologies of Homochiral Peptide Mixtures

The delicate balance of electrostatic and hydrophobic forces are critical during protein folding events and misfolding has been linked to many diseases. An important example is that of Alzheimer’s disease involving formation of pathological amyloid fibers of misfolded tau proteins that are intrinsically disordered proteins (IDPs). Similarly, during complex coacervation of simple peptide mixtures such as polylysine and polyglutamic acid of the same chirality (predominantly L-form in nature), solid precipitates form instead of liquid-like coacervate droplets. The formation of solid precipitates is mitigated by using synthetic peptides containing alternating D,L-amino acids in the backbone indicating that hydrogen bonding driven beta-sheet formation is a competing factor; however, the misfolding phenomena requires a deeper understanding of propensities to form irreversible beta sheet rich solid precipitates versus reversible random-coil rich liquid-like droplets.

Our investigation involving coacervation of 1:1 charge ratio of oppositely charged peptides containing L-amino acids (polylysine and polyglutamic acid) reveals that this phase behavior is complicated by the influence of peptide concentration on intra- and intermolecular interactions. Specifically, at low ionic strengths, whereas mixtures above the overlap concentration C* of peptides form solid-like precipitates consistent with published literature, mixtures form liquid-like coacervate droplets to dense networks of partially coalesced droplets at concentrations below C*. Molecular dynamics simulations suggest that this difference can be traced to extended peptide complexes with higher beta-sheet content that form at elevated concentration, which are prone to non-equilibrium self-assembly into bulk precipitates. Furthermore, we show that the pathway of precipitate formation is different below and above C* in the presence of added salt, resulting in distinct precipitate morphologies that are sensitive to thermal history and mixing pathways. Finally, at high ionic strengths (2-5 M NaCl), we observe a re-entrant transition to coacervate droplets, indicating that release of counter-ions and low beta sheet content can facilitate liquid-liquid phase instability under these conditions. This complex phase behavior suggests a subtle balance between non-equilibrium hydrogen bonding-driven beta-sheet assembly and electrostatically-driven coacervate formation results in a diversity of morphologies; the new results and its implications for protein folding and assembly will be discussed in this talk.