Ravi Raghav Sonani

Contrast-matched neutron scattering shows that co-assembly and self-sorting are not mutually exclusive, revealing internally self-sorted domains within a single co-assembled peptide nanotube architecture.

It has been known for more than half a century that enzymes can polymerize to either activate or repress their catalytic functions. Using electron cryo-microscopy (cryo-EM), we serendipitously found that two commonly used enzymes in molecular microbiology research, alkaline phosphatase and proteinase K, can unexpectedly form filaments. While the physiological significance of fibrillation in alkaline phosphatase and proteinase K is unknown, the atomic models of these polymers show the ability of cryo-EM to discover and determine high-resolution structures of macromolecular assemblies before they have been characterized or purified. IMPORTANCE: Electron cryo-microscopy (cryo-EM) has emerged over the past decade as the dominant technique in structural biology for determining the atomic structures of biological macromolecular complexes. One less appreciated feature of cryo-EM is that, in many cases, complexes do not need to be purified. Thus, the atomic structures of assemblies that can be found in biological samples as “contaminants” can be readily determined. In this paper, the structures of filaments formed by a phosphatase and a protease are described.

Electron-rich and electron-deficient naphthalene-capped dipeptides co-assemble into a light-responsive network that shows nanoscale reorganisation and a reversible yellow–to–red response.

Controlled assembly of DNA expands its utility in materials science. Most DNA-based architectures rely on Watson–Crick base pairing and stacking. Identifying additional programmable interactions could widen the design space. Single-stranded DNA (ssDNA) is known to adsorb on carbon nanotubes (CNTs) as sequence- and chirality-dependent helices, but direct structural evidence for a nanotube lattice-templating mechanism has been limited. Here we use cryo-electron microscopy to compare the assembly of the ssDNA sequence TTA TAT TAT ATT (ss65) on enantiomeric (6,5) CNTs. On the left-handed (+) (6,5) CNT, ss65 forms stacked, ring-like wraps with an axial repeat of 15.3 Å and with micrometer-scale coherence length. In contrast, on the right-handed (−) (6,5) CNT, ss65 adopts a conventional 1-start helical wrap with a helical pitch of ∼16.4 Å. These results indicate that the handedness of the underlying chiral lattice can bias ssDNA into distinct topologies (helix versus rings) and suggest a strategy for DNA assembly based on CNT lattice recognition, in addition to base pairing and stacking.

Peroxisomes are membrane enclosed organelles hosting diverse metabolic processes in eukaryotic cells. Having no protein synthetic abilities, peroxisomes import all required enzymes from the cytosol through a peroxin (Pex) import system. Peroxisome targeting sequence 1 (PTS1)-tagged cargo is recognized by cytosolic receptor, Pex5. The cargo-Pex5 complex docks at the peroxisomal membrane translocon, composed of Pex14 and Pex13, facilitating translocation into the peroxisomal lumen. Despite its significance, the structural basis of the process is only partially understood. Here, we characterize the cargo-Pex5-Pex14 _NTD ternary complex from Trypanosoma cruzi . Cryo-electron microscopy maps enabled model building for Pex5 (residues 327–462 and 487–653) bound to malate dehydrogenase (MDH; residues 1–323) cargo tetramer and Pex14 _NTD (residues 21–85). The model provides insight into conformational heterogeneity and identifies secondary interfaces. Specifically, we observe that orientations of Pex5 relative to MDH span a 17° angle. Additionally, PTS1- and Wxxx(F/Y)-independent contact surfaces are observed at MDH-Pex5 and Pex5-Pex14 _NTD interfaces, respectively. Mutational analysis indicates that the non-PTS1 MDH-Pex5 interface does not significantly contribute to the affinity, but limits the conformational heterogeneity of MDH-Pex5 interface. The Pex5-Pex14 _NTD interface constitutes an extended binding site of Pex14 _NTD over Pex5. We discuss the implications of these findings for understanding peroxisomal import mechanism.

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.

Many useful materials can be prepared by the self-assembly of oligopeptides. The design rules around such peptides are relatively established and they assume well-defined and pure materials. In many cases however, the purity of the peptide is less than 95%, and the ability of likely impurities to self-assemble is an open question. Here, we discuss the self-assembly of the gel-forming octapeptide FEFEFKFK and two analogues, EFEFKFK and FEFEfKFK, to examine the effect of an amino acid deletion and of epimerization at one position. Both the truncated peptide and epimerized peptide can still form gels. Mixing these peptides with the parent FEFEFKFK leads to the formation of new, but different, self-assembled structures. This has direct implications for our understanding of the necessary design rules for self-assembly regarding the influence of potential impurity in these systems, as well as demonstrating that much remains to be learned about sequence to structure relationships in self-assembling peptide systems.

Benzophenone-based materials remain widely used as photoinitiators for ultraviolet light-induced free radical polymerizations. Traditionally, polymerization is spatially controlled using top-down techniques such as photomasks, which produce well-defined polymeric films. In contrast, we present an alternative method for controlling polymerization by employing supramolecular materials to localize the photoinitiator. This approach uses benzophenone-functionalized dipeptides that are specifically tuned to enable supramolecular gel noodle formation, which act as structural templates. We show that polymerization of acrylate monomers around the gel noodles can increase the Young's modulus by up to 2 orders of magnitude and produce mechanically robust structures that can be handled. The self-assembly of the supramolecular photoinitiators is also explored using viscosity and SAXS measurements, providing an understanding of why only 4BPAcFF successfully forms gel noodles. Our method offers a simple yet effective technique for localizing polymerization, enabling fine-tuning of mechanical properties and the fabrication of intricate designs such as hollow-core structures.