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.

Two stages in the rational redesign of a peptide‐based, self‐assembling fiber (SAF) are described. The SAF system comprises two peptides designed to form an offset α‐helical coiled‐coil heterodimer. The “sticky‐ends” are complementary and promote longitudinal assembly. Alone, the two peptides are unstructured, but co‐assemble upon mixing to form α‐helical fibrils, which bundle to form fibers 40–50 nm wide and tens of micrometers long. Assembly is controllable and occurs at pH 7 in water, making SAFs a potential scaffold for 3D cell culture. The purposes of the redesigns were 1) to investigate the fiber‐thickening process, and 2) to increase fiber stability for potential biological and biomedical applications. First, mutations were made to the original peptide designs to increase fibril–fibril interactions and so produce thicker and more‐stable fibers. The second iteration aimed to increase the primary peptide–peptide interactions by increasing the overlap in the offset dimer and so promote the initial step in fiber formation. As judged by circular dichroism spectroscopy and transmission electron microscopy, both iterations improved fiber assembly and stability: the critical peptide concentration for assembly improved from 60 μM to 4 μM; the midpoint of thermal unfolding increased from 22 °C to 65 °C; and the salt tolerance improved from 75 mM to greater than 250 mM KCl. These improvements bring closer applications of the SAF system under physiological conditions, for example as a biocompatible material for 3D cell culture. In addition, ordered surface features were observed in the second‐ and third‐generation fibers compared with the original design. This indicates improved internal order in the redesigned fibers. In turn, this suggests a molecular mechanism for the improved stability and sheds light on the fiber‐assembly process.

No abstract

To improve our understanding of conformational transitions in proteins, we are attempting the de novo design of peptides that switch structural state. Here, we describe coiled-coil peptides with sequence and structural duality; that is, features compatible with two different coiled-coil motifs superimposed within the same sequence. Specifically, we promoted a parallel leucine-zipper dimer under reducing conditions, and a monomeric helical hairpin in an intramolecularly disulfide bridged state. Using an iterative process, we engineered peptides that formed stable structures consistent with both targets under the different conditions. Finally, for one of the designs, we demonstrated a one-way switch from the helical hairpin to the coiled-coil dimer upon addition of disulfide-reducing agents.