Vincent P. Conticello

Can replacement of single atoms within amino acid sequences redirect peptide self-assembly into different supramolecular structures? To address this question, we investigated a compositionally similar class of bola-amphiphilic peptides that were designed to self-assemble into filamentous nanostructures. The chemical differences between these peptides were confined to minimally perturbative substitutions on a phenylalanine side chain at a single site within the sequence. Cryo-EM structural analysis of five peptide filaments at near-atomic resolution revealed the presence of distinct supramolecular architectures between the different peptides. Despite sharing a common cross-β framework, the filament structures displayed distinct helical symmetries, protofilament organizations, and steric zipper interfaces. We hypothesize that the observed structural divergence between filaments arises from subtle changes in side-chain polarity and solvent interactions that remodel peptide packing at structural interfaces within cross-sectional amyloid layers. These findings demonstrate that the supramolecular structural landscape of self-assembling peptides is sensitive to atomic-level substitution and suggest that chemical editing provides a strategy for interrogating and potentially controlling biomolecular assembly. These results further highlight a fundamental limitation in the predictive design of peptide-based materials in that subtle chemical modifications in local composition can produce disproportionate changes in higher-order structure.

The ability to redirect established protein design principles toward new supramolecular architectures remains a central challenge in peptide-based materials engineering. Here, we demonstrate that a highly designable coiled-coil motif can be reprogrammed to form cross-α filaments through inversion of the hydrophobic face of a type II heptad repeat sequence. AlphaFold-Multimer was employed to screen designed peptide to identify sequences capable of forming extended cross-α assemblies. A designed peptide, ROX-LL, self-assembled into monomorphic α-helical filaments. Cryo-electron microscopy, synchrotron SAXS, and solid-state NMR measurements revealed the presence of cross-α architecture assembled from antiparallel αhelical dimers. The resulting assembly preserved the canonical structural features of coiled-coil molecular design while adopting a fundamentally different supramolecular architecture. Individual protofilaments consisted of cross-α stacks of concatenated Alacoil-like four-helix bundles exhibiting canonical knobs-into-holes packing. These findings demonstrate that coiled-coil structural interfaces can be reprogrammed through molecular design to create a distinct filamentous protein fold and identify a novel strategy for engineering cross-α peptide assemblies.

Evolutionary pressure on microbial communities propagating under extreme environmental conditions often results in unique structural adaptations to promote cell survival. Here, we report an investigation of AbpX, a biomatrix protein identified in cultures of the hyperthermophilic archaeon Pyrodictium abyssi . Under ex vivo and in vitro conditions, AbpX assembles into a paracrystalline lattice composed of semiflexible fibrils. CryoEM analysis of recombinant AbpX fibrils reveals that the precursor protein polymerizes through donor strand complementation (DSC), a process previously reported for chaperone-usher fimbriae in Gram-negative bacteria. Unlike the latter DSC protein polymers, AbpX undergoes chaperone-free polymerization in the presence of calcium ions, which are sequestered at the donor strand-acceptor groove interface between protomers in the fibril. Using a combination of cryoEM and crystallographic information, a structural model is proposed for the AbpX lattice that provides insight into its potential role in biofilm formation. These findings suggest that calcium ion coordination may contribute to fibril assembly and preorganize fibrils for incorporation into the protein lattice. Bioinformatic analysis indicates that AbpX exemplifies a distinct and broadly distributed clade of calcium ion responsive biomatrix proteins within the TasA superfamily that can be fabricated into hydrogel biomaterials in vitro under environmentally benign conditions.

The lability of quaternary structure in sequence space is a well-known phenomenon in peptide self-assembly that directly impacts the reliable and predictive design of peptide-based nanomaterials. Semiconservative changes in peptide sequence can significantly alter supramolecular structure, but how subtle do those substitutions need to be to have a measurable structural impact? To address this question, a compositionally related series of bola-amphiphilic peptides was prepared that differ solely in the chemical identity of a phenylalanine derivative. Each peptide self-assembles into high aspect-ratio filaments based on a parallel cross-b architecture. Using cryo-EM helical reconstruction, the structures of five filaments were determined at near-atomic resolution. The supramolecular structures of filaments derived from these peptide sequences are remarkably distinct despite being based on similar cross-b folds that engage in amyloid-like steric zipper interactions. This study suggests that chemical editing of self-assembling peptide sequences, in which minimally perturbative atomic substitutions are introduced, can significantly impact supramolecular structure.

Helix-helix interactions are mediated through highly designable interfaces within tertiary and quaternary structures of proteins and protein assemblies. The structural regularity of these interfaces suggests that ordered self-assembled structures could be constructed from implementation of these interactions between appropriately designed helical protomers. This review summarizes the current understanding of helix-helix interactions within different classes of naturally occurring α-helical protein filaments. The implications of this structural information for the de novo design of synthetic filamentous nanomaterials will be discussed with reference to examples in which these principles have been successfully implemented. A specific case study will focus on the designability of cross-α helical filaments, a recently discovered structural class in which the helical protomers are arranged in a perpendicular orientation with respect to the protofilament axis. This discussion will include an evaluation of the frequency of occurrence of cross-α interfaces in the PDB, the effectiveness of structural prediction from sequence information, and the potential for de novo design of interfaces that promote cross-α interactions.

Evolutionary pressure on microbial communities propagating under extreme environmental conditions often results in unique structural adaptations to promote cell survival. Here, we report an investigation of AbpX, a biomatrix protein identified in cultures of the hyperthermophilic archaeon Pyrodictium abyssi. Under ex vivo and in vitro conditions, AbpX assembles into a para-crystalline lattice composed of semiflexible fibrils. CryoEM analysis of recombinant AbpX fibrils reveals that the precursor protein polymerizes through donor strand complementation (DSC), a process previously reported for chaperone-usher fimbriae in Gram-negative bacteria. Unlike the latter DSC protein polymers, AbpX undergoes chaperone-free polymerization in the presence of calcium ions, which are sequestered at the donor strand-acceptor groove interface between protomers in the fibril. Using a combination of cryoEM and crystallographic structural information, an atomic model is proposed for the AbpX lattice that provides insight into its potential role in biofilm formation. These findings suggest that calcium ion coordination triggers fibril assembly and pre-organizes the fibrils for incorporation into the protein lattice. Bioinformatic analysis indicates that AbpX exemplifies a distinct and broadly distributed clade of calcium ion responsive biomatrix proteins within the TasA superfamily that can be fabricated into hydrogel biomaterials in vitro under environmentally benign conditions.

Cannulae are structurally rigid tubular protein filaments that accumulate on the extracellular surface of archaea within the family Pyrodictiaceae during cell growth. These obligate anaerobes propagate under hyperthermophilic conditions in which cannulae form a biomatrix that interconnects and sustains cells. The persistence of cannulae in this environment suggests that these filaments display significant thermostability, which has attracted technological interest in their development as synthetic protein-based biomaterials. Here, we report cryoEM structural analyses of ex vivo and in vitro assembled recombinant cannulae. We demonstrate that the interactions between protomers in native and recombinant cannulae is based on donor strand complementation (DSC), a form of non-covalent polymerization previously observed for bacterial chaperone-usher pili. Unexpectedly, calcium ion coordination at the subunit interfaces reinforces the network of donor strand interactions in the cannulae. This study provides insight into the mechanism of assembly of cannulae and the structural origin of their high stability and rigidity.

Amphipathic character, encoded within the polar sequence patterns of antimicrobial peptides, is a critical structural feature that influences membrane disruptive behavior. Similarly, polar sequence patterns induce self‐assembly of amphipathic peptides, which results in the formation of ordered supramolecular structures. The relationship between self‐assembly and membrane activity remains an open question of relevance for the development of effective antimicrobial peptides. Here, we report the structural investigation of a class of lytic peptides that self‐assemble into filamentous nanomaterials. CryoEM analysis was employed to determine the structure of one of the filaments, which revealed that the peptides are self‐assembled into a bilayer nanotube, in which the interaction between layers of amphipathic α‐helices was mediated through hydrophobic interactions. The relative stability of the filament peptide assemblies depended on the influence of sequence modifications on the helical conformation. Antimicrobial assays indicated that cytolytic activity was associated with dynamic disassociation of the filamentous assemblies under the assay conditions. Structural modifications of the peptides that stabilized the filaments abrogated lytic activity. These results illuminate a reciprocal relationship between self‐assembly and antimicrobial activity in this class of amphipathic peptides and that reversible assembly was critical for the observation of biological activity.

We investigate the relationship between sequence and supramolecular structure for a pair of bola-amphiphilic peptides, Ac-KLIIIK-NH ₂ and Ac-KIIILK-NH ₂ , that self-assemble into supramolecular hydrogels based on cross-b amyloid fibrils.