Acetone-induced structural variant of insulin amyloid fibrils.

Self-propagating polymorphism of amyloid fibrils is a distinct manifestation of non-equilibrium conditions under which protein aggregation typically occurs. Structural variants of fibrils can often be accessed through physicochemical perturbations of the de novo aggregation process. On the other hand, tiny changes in the amino acid sequence of the parent protein may also result in structurally distinguishable amyloid fibrils. Here, we show that in the presence of acetone, the low-pH fibrillization pathway of bovine insulin (BI) leads to a new type of amyloid with the infrared features (split amide I' band with the maximum at 1623 cm-1) bearing a striking resemblance to those of the previously reported fibrils from recombinant LysB31-ArgB32 human insulin analog formed in the absence of the co-solvent. Insulin fibrils formed in the presence ([BI-ace]) and absence ([BI]) of acetone cross-seed each other and pass their infrared features to the daughter generations of fibrils. We have used dimethyl sulfoxide (DMSO) coupled to in situ infrared spectroscopy measurements to probe the stability of fibrils against chemical denaturation. While both types of fibrils eventually undergo DMSO-induced disassembly coupled to a ß-sheet→coil transition, in the case of [BI-ace] amyloid, the denaturation is preceded by the fibrils transiently acquiring the [BI]-like infrared characteristics. We argue that this effect is caused by DMSO-induced dehydration of [BI-ace]. In support to this hypothesis, we show that, even in the absence of DMSO, the infrared features of [BI-ace] disappear upon drying. We discuss this very peculiar aspect of [BI-ace] fibrils in the context of recently accessed in silico models of plausible structural variants of insulin protofilaments.

Self-Assembly of Insulin-Derived Chimeric Peptides into Two-Component Amyloid Fibrils: The Role of Coulombic Interactions.

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 ß-sheets as the self-assembly of free poly-E and poly-K chains under similar conditions results in amorphous antiparallel ß-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.

Reduction of a disulfide-constrained oligo-glutamate peptide triggers self-assembly of ß2-type amyloid fibrils with the chiroptical properties determined by supramolecular chirality.

Disulfide bonds prevent aggregation of globular proteins by stabilizing the native state. However, a disulfide bond within a disordered state may accelerate amyloidogenic nucleation by navigating fluctuating polypeptide chains towards an orderly assembly of ß-sheets. Here, the self-assembly behavior of Glu-Cys-(Glu)4-Cys-Glu peptide (E6C2), in which an intrachain disulfide bond is engineered into an amyloidogenic homopolypeptide motif, is investigated. To this end, the Thioflavin T (ThT) fluorescence kinetic assay is combined with infrared spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and Raman scattering measurements. Regardless of whether the disulfide bond is intact or reduced, E6C2 monomers remain disordered within a broad range of pH. On the other hand, only reduced E6C2 self-assembles into amyloid fibrils with the unique infrared traits indicative of three-center hydrogen bonds involving main-chain carbonyl as a bifurcating acceptor and main-chain NH and side-chain -COOH groups as hydrogen donors: the bonding pattern observed in so-called ß2-fibrils. AFM analysis of ß2-E6C2 reveals tightly packed rectangular superstructures whose presence coincides with strong chiroptical properties. Our findings suggest that formation of chiral amyloid superstructures may be a generic process accessible to various substrates, and that the fully extended conformation of a poly-Glu chain is a condition sine qua non for self-assembly of ß2-fibrils.

Reversible Freeze-Induced ß-Sheet-to-Disorder Transition in Aggregated Homopolypeptide System.

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 ß-sheet-rich ionic coaggregate of poly(l-lysine), PLL, and poly(l-glutamic acid), PLGA. Freezing aqueous suspensions of the PLL-PLGA ß-aggregate in the presence of low concentrations of salt (NaBr) induces an instantaneous ß-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-ß-sheet "refolding". However, in the absence of traces of soluble salts, the ß-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 ß-sheet-rich aggregates. Possible scenarios of freeze-induced salt-bridge rupture and removal of water from nanocanals are discussed.