Sticky-End Assembly of a Designed Peptide Fiber Provides Insight into Protein Fibrillogenesis

Abstract

Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had "sticky-ends" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with "blunt-ends" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred micrometers long and 20 times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.

Engineering nanoscale order into a designed protein fiber

“…Predominantly, such work has used ␤-structured peptides that form amyloid-like structures (3, 5, 6, 10). Relatively less has been attempted with ␣-helix-based assemblies, however (7,(11)(12)(13)(14). The development of ␣-helical systems would provide useful comparisons with the more-explored amyloid-like systems and also allows the application of the considerable body of protein design and engineering knowledge for ␣-helical structures and assemblies (15)(16)(17).…”

“…In particular, considerable attention has been paid to one type of coiled-coil architecture, namely the leucine-zipper motif, which is accepted as the archetypal two-stranded, parallel coiled coil. Previously, we have combined established rules for leucine-zipper assembly in the first-generation design of a selfassembling-fiber (SAF) system (7,11,17).…”

Self-assembly of synthetic collagen triple helices

“…Selfassembling peptides and proteins have been designed to serve as materials for biological and nanotechnological applications (16,17). Using the self-assembly approach, Woolfson and coworkers (18,19) have produced fibers with a design based on a dimeric coiled-coil structure. Likewise, fibrous peptides based on the natural protein elastin (20) and de novo building blocks (15,(21)(22)(23)(24)(25)(26) have been assembled and implicated as biomaterial candidates.…”

Controlling the Assembly of Coiled–Coil Peptide Nanotubes

“…Circular dichroism (CD) spectroscopy confirmed this in solution. Owing to chiral scattering, broadened fibrous α‐helical systems, such as CC‐Hex‐T at pH 7.4, give red‐shifted CD spectra of reduced intensity, Figure 2 b, compared with typical α‐helical spectra 11. However, decreasing the pH for CC‐Hex‐T samples gave increased signal and loss of the red shift, with the transition complete by pH 5.6 (Figure 2 d).…”