Engineering nanoscale order into a designed protein fiber

Abstract

We have established a designed system comprising two peptides that coassemble to form long, thickened protein fibers in water. This system can be rationally engineered to alter fiber assembly, stability, and morphology. Here, we show that rational mutations to our original peptide designs lead to structures with a remarkable level of order on the nanoscale that mimics certain natural fibrous assemblies. In the engineered system, the peptides assemble into two-stranded ␣-helical coiled-coil rods, which pack in axial register in a 3D hexagonal lattice of size 1.824 nm, and with a periodicity of 4.2 nm along the fiber axis. This model is supported by both electron microscopy and x-ray diffraction. Specifically, the fibers display surface striations separated by nanoscale distances that precisely match the 4.2-nm length expected for peptides configured as ␣-helices as designed. These patterns extend unbroken across the widths (>50 nm) and lengths (>10 m) of the fibers. Furthermore, the spacing of the striations can be altered predictably by changing the length of the peptides. These features reflect a high level of internal order within the fibers introduced by the peptidedesign process. To our knowledge, this exceptional order, and its persistence along and across the fibers, is unique in a biomimetic system. This work represents a step toward rational bottom-up assembly of nanostructured fibrous biomaterials for potential applications in synthetic biology and nanobiotechnology.bionanoscience ͉ nanofibers ͉ peptide assembly ͉ rational protein design ͉ self-assembly

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

“…2A, Upper). These had not been seen before, but relate to the hexagonal packing of the coiled coils as observed by wide-angle X-ray fiber diffraction (26). It is important to note here that the images are formed from the projected electron density of the protein A B C only, so these striations are a direct visualization of the supramolecular structure of the SAFs.…”

“…As we have noted before (26), this corresponds almost precisely to the length of each SAF peptide when configured as a coiled coil-i.e., 41.44 Å (¼28 · 1.48 Å, the rise per residue in an α-helical coiled coil). Thus, coiled-coil peptides are aligned along the long axis of the fibers as per our original design (24) and subsequent structural analyses of stained and dried fibers (26).…”

“…There is considerable order in both dimensions of the fibers (Fig. 1C), which we have suggested is imparted by periodic interfibril side-chain interactions both laterally and longitudinally through the fibers (24)(25)(26). Although this model is helpful, our understanding remains incomplete, and any further design and engineering studies of the SAFs are limited by this low-resolution model.…”

“…1B). We have presented several designs on this theme (24)(25)(26) and explored the detailed mechanism of assembly (27). Thus far, we have probed the structure of the fibers to low resolution (approximately 2 nm) by combined negative-stain TEM and X-ray fiber diffraction (26).…”

Structural specificity in coiled-coil interactions

“…A peptide with similar properties was designed by hand in 2005 [24], and both designs show solution properties consistent with the intended folds. Coiled-coil peptides have also been designed to form fibers (both amyloid and non-amyloid) upon external triggering by pH or temperature, or simply upon mixing [25][26][27][28].…”