3D Cryo-Electron\nMicroscopy Reveals the Structure\nof a 3‑Fluorenylmethyloxycarbonyl Zipper Motif Ensuring the\nSelf-Assembly of Tripeptide Nanofibers

Short peptide-based supramolecular hydrogels appeared\nas highly\ninteresting materials for applications in many fields. The optimization\nof their properties relies mainly on the design of a suitable hydrogelator\nthrough an empirical trial-and-error strategy based on the synthesis\nof various types of peptides. This approach is in part due to the\nlack of prior structural knowledge of the molecular architecture of\nthe various families of nanofibers. The 3D structure of the nanofibers\ndetermines their ability to interact with entities present in their\nsurrounding environment. Thus, it is important to resolve the internal\nstructural organization of the material. Herein, using Fmoc-FFY tripeptide\nas a model amphiphilic hydrogelator and cryo-EM reconstruction approach,\nwe succeeded to obtain a 3.8 Å resolution 3D structure of a self-assembled\nnanofiber with a diameter of approximately 4.1 nm and with apparently\n“infinite” length. The elucidation of the spatial organization\nof such nano-objects addresses fundamental questions about the way\nshort amphiphilic <i>N</i>-Fmoc peptides lacking secondary\nstructure can self-assemble and ensure the cohesion of such a lengthy\nnanostructure. This nanofiber is organized into a triple-stranded\nhelix with an asymmetric unit composed of two Fmoc-FFY peptides per\nstrand. The three identical amphiphilic strands are maintained together\nby strong lateral interactions coming from a 3-Fmoc zipper motif.\nThis hydrophobic core of the nanofiber is surrounded by 12 phenyl\ngroups from phenylalanine residues, nonplanar with the six Fmoc groups.\nPolar tyrosine residues at the C-term position constitute the hydrophilic\nshell and are exposed all around the external part of the assembly.\nThis fiber has a highly hydrophobic central core with an internal\ndiameter of only 2.4 Å. Molecular dynamics simulations highlight\nvan der Waals and hydrogen bonds between peptides placed on top of\neach other. We demonstrate that the self-assembly of Fmoc-FFY, whether\ninduced by annealing or by the action of a phosphatase on the phosphorylated\nprecursor Fmoc-FF<i>p</i>Y, results in two nanostructures\nwith minor differences that we are unable to distinguish.