Coiled-Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability

Abstract

The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Here we describe the symmetrydirected design of a hollow protein cage with dimensions similar to those of many icosahedral viruses. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain and cryo EM, and by atomic force and IR-photo-induced force microscopy established that Ico8 assembles into a flexible hollow cage with comprising 60-subunits with overall icosahedral geometry. Unexpectedly, the cages were found to encapsulate DNA, even though neither protein component binds nucleic acids on its own. Notably, the cages formed by Ico8 proved to be extremely stable towards thermal and chemical denaturation: whereas TriEst was unfolded by heating (T m ~75 ºC) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 ºC or in 8 M guanidine hydrochloride. The encapsulation of DNA and increased stability of the cages are new properties that emerge from the higher order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.

Connectability of protein cages

“…38,39 Along the same lines, coiled-coil forming peptides afford ideal glues for connecting protein building blocks with designed symmetries. 40,41 For example, a trimeric esterase equipped with three-, four-or ve-helix coiled coils forms tetrahedral, 42 octahedral, 43 or icosahedral 44 cages, respectively ( Fig. 3II).…”

De NovoDesigned Peptide and Protein Hairpins Self-assemble into Sheets and Nanoparticles

“…[26][27][28][29][30] In a different concept Marsh and colleagues combine de novo coiled-coil units and natural oligomeric proteins to render defined protein nanocages. [41][42][43][44] Here we present a self-assembling hairpin designs that form two distinct suporamolecular assemblies, namely, closed nanoparticles and extended sheets. These designs combine ideas from the SAGEs and Burkhard's nanoparticles.…”

Controlling the Assembly of Protein Cages towards Functional Supramolecular Materials

“…Apart from the electrostatic interaction and metal coordination mentioned in the previous two sections, other interactions such as hydrophobic interaction, 49,[69][70][71] coilcoil interaction [72][73][74][75][76] and specific icosahedral symmetry protein-protein interactions 77,78 can also be used to induce assembly of VLPs at neutral pH.…”

“…Another approach to induce assembly of higher ordered nanoscale cages is to incorporate de novo-designed coiled-coil domain into the subunit of protein cages (Figure 2.6 B) which results in symmetrical icosahedral VLPs. 72 With the development of the understanding of assembly pathway and mechanism, computational design of proteins based on symmetry principles, have been widely used in the last decades to create ordered assembly of icosahedral protein cages 79-81 ( Figure 2.6 C).…”

“…Self-assembly of Virus-like Particles Induced by Supramolecular Interactions trimers genetically fusioned with pentameric coiled-coil assemble into an icosahedral cage. 72 Copyright © 2019, with permission from American Chemical Society. C: atomic structure ilustration of a designed protein cage.…”