Engineering Increased Stability into Self‐Assembled Protein Fibers

Abstract

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.

Engineering nanoscale order into a designed protein fiber

“…Curiously, however, the fibers measure Ϸ45 Ϯ 10 nm across, which is approximately 20 times thicker than expected for leucine-zipper-based dimers (24). Also, fiber thickening appears to stop to give a finite distribution of fiber widths (11,25,26). At present, we do not understand how fiber thickening occurs or how it is arrested, although mechanisms proposed for the assembly of fibrous collagens (27) and ␤-tape structures (28) may be applicable.…”

“…To discuss the work presented here and place it in context of our previously published work (26), it is necessary to summarize the latest work in the next two subsections. The previously published work centers on improving the stability of the SAFs through rational design.…”

“…As described below, the design peptides are given in ref. 26. However, here we present additional structural data and focus on using this information to develop detailed molecular models for the SAF structure.…”

“…Inspired by proposed mechanisms for the higher-order assembly of natural fibrous proteins (29)(30)(31)(32), we argued that this might best be achieved by optimizing potential interprotofibril Coulombic (charge-charge) interactions, that is, rather than by introducing hydrogen-bonding interactions, which would require specific orientation of the partnering protofibrils. To this end, we modeled contiguous copies of the SAF-p1 and SAF-p2 sequences (Table 1) as an extended dimeric coiled coil (26).…”

“…Whatever the kinetic mechanism for thickening, this lateral assembly is likely driven by the free energy associated with the formation of many low-affinity interactions between features that are brought together on neighboring component two-stranded coiled coils (protofibrils) in the thickened fiber, in effect, an avidity effect (11). As we have described (26), the changes to the peptides further thicken the assembled fibers and also stabilize them with respect to thermal denaturation. Here, we describe detailed structural studies of the rationally redesigned fibers, which illustrate that peptide assemblies with nanoscale order can be engineered from first principles and from the bottom up.…”