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 -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.
|Translated title of the contribution||Engineering Increased Stability into Self-Assembled Protein Fibers|
|Pages (from-to)||1022 - 1030|
|Number of pages||9|
|Journal||Advanced Functional Materials|
|Publication status||Published - May 2006|