The molecular design of peptide-assembled nanostructures relies on extensive knowledge pertaining to the relationship between conformational features of peptide constituents and their behavior regarding self-assembly, and characterizing the conformational details of peptides during their self-assembly is experimentally challenging. Here, we demonstrate that a hybrid-resolution modeling method can be employed to investigate the role that conformation plays during the assembly of terminally capped diphenylalanines (FF) through microsecond simulations of hundreds or thousands of peptides. Our simulations discovered tubular or vesicular nanostructures that were consistent with experimental observation while reproducing critical self-assembly concentration and secondary structure contents in the assemblies that were measured in our experiments. The atomic details provided by our method allowed us to uncover diverse FF conformations and conformation dependence of assembled nanostructures. We found that the assembled morphologies and the molecular packing of FFs in the observed assemblies are linked closely with side-chain angle and peptide bond orientation, respectively. Of various conformations accessible to soluble FFs, only a select few are compatible with the assembled morphologies in water. A conformation resembling a FF crystal, in particular, became predominant due to its ability to permit highly ordered and energetically favorable FF packing in aqueous assemblies. Strikingly, several conformations incompatible with the assemblies arose transiently as intermediates, facilitating key steps of the assembly process. The molecular rationale behind the role of these intermediate conformations were further explained. Collectively, the structural details reported here advance the understanding of the FF self-assembly mechanism, and our method shows promise for studying peptide-assembled nanostructures and their rational design.
Summary Amyloid fibril surfaces can convert soluble proteins into toxic oligomers and are attractive targets for intervention of protein aggregation diseases. Thus far, molecules identified with inhibitory activity are either large proteins or flat cyclic compounds lacking in specificity. The main design difficulty is flatness of amyloid surfaces and the lack of knowledge on binding interfaces. Here, we demonstrate, for the first time, a rational design of alpha-helical peptide inhibitors targeting the amyloid-beta 40 (Aβ40) fibril surfaces, based on our in silico finding that a helical fragment of Aβ40 interacts in a unique way with side-chain arrays on the fibril surface. We strengthen the fragment's binding capability through mutations and helicity enhancement with our Terminal Aspartic acid strategy. The resulting inhibitor shows micromolar affinity for the fibril surface, effectively impedes the surface-mediated oligomerization of Aβ40, and mitigates its cytotoxicity. This work opens up an avenue to designing aggregation modulators for amyloid diseases.
Peptide self-assembly inspired by natural superhelical coiled coils has been actively pursued but remains challenging due to limited helicity of short peptides. Side chain stapling can strengthen short helices but is unexplored in design of self-assembled helical nanofibers as it is unknown how staples could be adapted to coiled coil architecture. Here, we demonstrate the feasibility of this design for pentapeptides using a computational method capable of predicting helicity and fiber-forming tendency of stapled peptides containing noncoded amino acids. Experiments showed that the best candidates, which carried an aromatically substituted staple and phenylalanine analogs, displayed exceptional helicity and assembled into nanofibers via specific head-to-tail hydrogen bonding and packing between staple and noncoded side chains. The fibers exhibited sheet-of-helix structures resembling the recently found collapsed coiled coils whose formation was sensitive to side chain flexibility. This study expands the chemical space of coiled coil assemblies and provides guidance for their design.
Inhibition of amyloid‐β peptide (Aβ) aggregation is a promising therapeutic strategy for Alzheimer's disease (AD), as Aβ aggregation is generally believed to trigger AD pathology. Pre‐fibril Aβ‐oligomers induce membrane disruption and are crucial to neurotoxicity. We have previously designed a short peptide called cyclic helical amyloid surface inhibitor (cHASI) that can selectively bind to the Aβ fibril surface. Here, we use cHASI to efficiently inhibit the surface‐catalysed secondary nucleation process of Aβ in a lipid membrane environment. By incubating Aβ monomers with lipid vesicles, we show that during the assembly of Aβ into amyloid fibrils, oligomers are formed that markedly disrupt the lipid bilayer. Remarkably, when Aβ monomers are incubated with cHASI, although Aβ forms amyloid fibrils via primary nucleation and elongation, this pathway to fibrils does not damage the lipid bilayer. This indicates that only oligomers produced during secondary surface nucleation disrupt membrane integrity. The protective effect of cHASI is confirmed by cytotoxicity assays. Our study highlights the therapeutic potential for inhibiting the secondary nucleation process in Aβ aggregation, rather than inhibiting all pathways to fibril formation.
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