Cryo-transmission electron microscopy structure of a gigadalton peptide fiber of de novo design

Abstract

Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of μm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using singleparticle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.fibrous proteins | protein design | helical reconstruction | X-ray crystallography

Modular Design of Self-Assembling Peptide-Based Nanotubes

“…The superhelices then pack unperturbed on a square lattice to form the highly ordered fibrous assemblies of peptide nanotubes. By contrast, in the well-characterized self-assembled peptide fiber (SAF) system highly ordered fibers result from remodeling of the component dimeric coiled-coil units to give an altered pitch compatible with 3 1 superhelices and hexagonal packing, corresponding to a 12 % compression of the coiled-coil pitch 13 . We propose that where the peptide fibers and nanotubes formed by other α-helical coiled-coil and barrel peptides explored herein do not form highly ordered arrays it is because: (1) their symmetries are incompatible with the required highly symmetric fibril-fibril packing; and (2) the building blocks are less flexible or compressible and are not readily remodeled.…”

“…S5). Such ripening is common in α-helical fibrous systems, and arises because symmetric assembly of identical building blocks creates patches on the fibril surfaces that foster fibril-fibril interactions and lead to thick-fiber formation 13,28 . Given the spontaneous and rapid assembly of the fibers, it is possible that these states are kinetically trapped structures, rather than global thermodynamic minima.…”

“…Of direct relevance to this report, several groups have employed sticky ended α-helical coiled coils to make peptide-based fibers [10][11][12] . In many of these cases, coiled-coil dimers, trimers and occasionally tetramers are used, which results in largely solid fibers 13 . Extending this to the use of α-helical barrels 14 -that is, coiled-coil pentamers and above that have central, solvent-accessible lumens (Fig.…”

Tunable and modular assembly of polypeptides and polypeptide-hybrid biomaterials

“…More elaborate superstructures can consist of more than two structural levels. Examples include, but are not limited to, ber and nanotube forming coiled coil peptides, 71,134 and spherically shaped nanoparticle assemblies. 135 Furthermore, in paper III the terminology is used since the HLH-polypeptides rst form four-helix bundles, then bers that in turn bundles up into spheres (described more in detail in section 6.3)…”

“…Furthermore, by employing well-established sequence-to-structure relationship rules it is possible to design α-helices that allow for precise self-assembly and oligomerization. 68 Using such rules, α-helical polypeptides have been used to create well-de ned bers, [69][70][71][72][73] and hydrogels, 70,74,75 and even as well-de ned structures as nanometer-sized tetrahedons. 76 Two α-helical folding motifs that have been used in this thesis are the coiled coil motif and the helix-loop-helix motif.…”