Deterministic chaos in the self-assembly of β sheet nanotubes from an amphipathic oligopeptide

Abstract

The self-assembly of designed peptides into filaments and other higher-order structures has been the focus of intense interest because of the potential for creating new biomaterials and biomedical devices. These peptide assemblies have also been used as models for understanding biological processes, such as the pathological formation of amyloid. We investigate the assembly of an octapeptide sequence, Ac-FKFEFKFE-NH₂, motivated by prior studies that demonstrated that this amphipathic β strand peptide self-assembled into fibrils and biocompatible hydrogels. Using high-resolution cryoelectron microscopy (cryo-EM), we are able to determine the atomic structure for two different coexisting forms of the fibrils, containing four and five β sandwich protofilaments, respectively. Surprisingly, the inner walls in both forms are parallel β sheets, while the outer walls are antiparallel β sheets. Our results demonstrate the chaotic nature of peptide self-assembly and illustrate the importance of cryo-EM structural analysis to understand the complex phase behavior of these materials at near-atomic resolution.

Figure 1.. Cryo-EM of KFE8

(A) Cryoelectron micrograph of the KFE8 tubes assembled at ambient temperature. Scale bar, 20 nm. Black arrow points to the thinner tube and red arrow points to the ribbon. (B) Cryoelectron micrograph of the annealed KFE8 tubes. The peptide was heated immediately after solubilization to 95°C and slowly cooled to 25°C. Scale bar, 20 nm. Black arrows point to the thinner tubes and white arrows point to the thicker tubes. (C–E) Top view of the ribbon, thinner tube and thicker tube. (F–H) Side views of the 3D reconstructions of the ribbon (F), thinner tube (G), and the thicker tube (H). The outer surface is shown on the left for each of these, while a cutaway view revealing the surface of the lumen is shown on the right.

Figure 2.. The thinner tube is made of two ribbons

(A) The cryo-EM structure of one of two protofilaments that form the thinner tube. The surface of this protofilament from the helical reconstruction is on the left. The high-resolution cross-sectional density map is on the right, with the atomic model built into the map shown. (B) The ASU of the thinner tube contains eight strands: the parallel inner strands are in yellow, while the antiparallel outer strands are in green and light green. The hydrophobic interactions within the ASU involve the interdigitated phenylalanine residues. (C) The thinner tubes can be sorted into two classes, in which the two protofilaments are related by either a C₂ symmetry (class 1) or a screw symmetry (class 2). (D) The protofilament interface exists in the inner wall only. The hydrogen bonds are highlighted between protofilaments and within parallel sheets.

Figure 3.. The thicker tube forms after annealing

(A) The thicker tube cryo-EM structure. The 5-fold averaged helical reconstruction is on the left, with a single protofilament shown in cyan. The cross-sectional density map is on the right, with an atomic model built into the map. (B) The ASU of the thicker tube. The hydrophobic interactions within the ASU arise from the interdigitated phenylalanine residues. (C) The protofilament interface exists only in the inner wall. The hydrogen bonds between protofilaments are shown as dotted lines.

Figure 4.. The different helical packings of the same eight-residue peptide

(A) One stack containing one sheet of the inner wall and one sheet of the outer wall (thinner tube on the left and thicker tube on the right). The tilt angles of the sheet from the plane normal to the helical axis are indicated on top. (B) Structural alignment of a single inner β sheet containing 2 strands (left) and 10 strands (right). (C) Structural comparison of two adjacent inner β sheets containing six strands. The alignment was made on the left sheets, resulting in the twist differences on the right. (D and E) The inner-outer strand pairs in the thinner and thicker tube. A cartoon illustration is shown in (E). The N terminus of the peptide is labeled. Structurally similar interfaces within the thinner and thicker tubes are highlighted in the same color. The ASU of protofilaments within the thicker tube have the same pairwise arrangement of interfaces (magenta and orange) under the imposed helical symmetry as one pair of interfaces within the thinner tube. The ASU of the protofilaments within the thinner tubes shows an additional pair of interfacial interactions (blue and green).