Conformational transitions involving aggregated proteins or peptides are of paramount biomedical and biotechnological importance. Here, we report an unusual freeze-induced structural reorganization within a β-sheet-rich ionic coaggregate of poly­(l-lysine), PLL, and poly­(l-glutamic acid), PLGA. Freezing aqueous suspensions of the PLL–PLGA β-aggregate in the presence of low concentrations of salt (NaBr) induces an instantaneous β-sheet-to-disorder transition, as probed by infrared spectroscopy in the amide I′ band region. The conformational rearrangement of polypeptide chains appears to be fully synchronized with the global liquid-to-ice phase transition. In contrast to the known freeze-induced transitions, the process described here is fully reversible: the subsequent thawing results in an instantaneous disorder-to-β-sheet “refolding”. However, in the absence of traces of soluble salts, the β-sheet framework of the PLL–PLGA aggregate remains resistant to freezing as no transition is observed. We note that the occurrence of the transition depends on the type of salt present in the sample. Our results highlight a hidden dimension of the structural dynamics within β-sheet-rich aggregates. Possible scenarios of freeze-induced salt-bridge rupture and removal of water from nanocanals are discussed.

Canonical amyloid fibrils are composed of covalently identical polypeptide chains. Here, we employ kinetic assays, atomic force microscopy, infrared spectroscopy, circular dichroism, and molecular dynamics simulations to study fibrillization patterns of two chimeric peptides, ACC1–13E8 and ACC1–13K8, in which a potent amyloidogenic stretch derived from the N-terminal segment of the insulin A-chain (ACC1–13) is coupled to octaglutamate or octalysine segments, respectively. While large electric charges prevent aggregation of either peptide at neutral pH, stoichiometric mixing of ACC1–13E8 and ACC1–13K8 triggers rapid self-assembly of two-component fibrils driven by favorable Coulombic interactions. The low-symmetry nonpolar ACC1–13 pilot sequence is crucial in enforcing the fibrillar structure consisting of parallel β-sheets as the self-assembly of free poly-E and poly-K chains under similar conditions results in amorphous antiparallel β-sheets. Interestingly, ACC1–13E8 forms highly ordered fibrils also when paired with nonpolypeptide polycationic amines such as branched polyethylenimine, instead of ACC1–13K8. Such synthetic polycations are more effective in triggering the fibrillization of ACC1–13E8 than poly-K (or poly-E in the case of ACC1–13K8). The high conformational flexibility of these polyamines makes up for the apparent mismatch in periodicity of charged groups. The results are discussed in the context of mechanisms of heterogeneous disease-related amyloidogenesis.

Canonical amyloid fibrils are composed of covalently identical polypeptide chains. Here, we employ kinetic assays, atomic force microscopy (AFM), infrared spectroscopy, circular dichroism (CD), and molecular dynamics (MD) to study fibrillization patterns of two chimeric peptides, ACC1-13E8 and ACC1-13K8, in which potent amyloidogenic stretch derived from the N-terminal segment of insulin A-chain (ACC1-13) is coupled to octaglutamate or octalysine segments, respectively. While the large electric charges on monomers of either peptide prevent aggregation at neutral pH, stoichiometric mixing of ACC1-13E8 and ACC1-13K8 triggers rapid self-assembly of two-component fibrils driven by favorable Coulombic interactions. The role of low-symmetry non-polar ACC1-13 pilot sequence is crucial in enforcing the amyloidal parallel -sheet motif as self-assembly of free poly-E and poly-K chains under similar conditions results in amorphous antiparallel -sheet conformation. Interestingly, the pathway to highly ordered fibrils is accessible to ACC1-13E8 also when paired with non-polypeptide polycationic amines such as branched poly-ethylenimine, PEI, instead of ACC1-13K8. Remarkably, such synthetic polycations are more effective in triggering fibrillization of ACC1-13E8 than poly-K (or poly-E in the case of ACC1-13K8). High conformational flexibility of these polyamines makes up for the apparent mismatch in periodicity of charged groups. The results are discussed in the context of mechanisms of heterogenous disease-related amyloidogenesis.

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