Triggered Reversible Disassembly of an Engineered Protein Nanocage*.

Protein nanocages play crucial roles in sub-cellular compartmentalization and spatial control in all domains of life and have been used as biomolecular tools for applications in biocatalysis, drug delivery, and bionanotechnology. The ability to control their assembly state under physiological conditions would further expand their practical utility. To gain such control, we introduced a peptide capable of triggering conformational change at a key structural position in the largest known encapsulin nanocompartment. We report the structure of the resulting engineered nanocage and demonstrate its ability to disassemble and reassemble on demand under physiological conditions. We demonstrate its capacity for in vivo encapsulation of proteins of choice while also demonstrating in vitro cargo loading capabilities. Our results represent a functionally robust addition to the nanocage toolbox and a novel approach for controlling protein nanocage disassembly and reassembly under mild conditions.

Metal-dependent assembly of a protein nano-cage.

Short, alpha-helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo-designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion-dependent assembly of a tetrahedral protein cage. Ni2+ , Co2+ , Cu2+ , and Zn2+ ions were evaluated, with Ni2+ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion-protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.

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

The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Furthermore, the stabilization of enzyme activity has many practical applications in biotechnology and medicine. Here we describe the symmetry-directed design of an extremely stable, enzymatically active, hollow protein cage of Mr ≈ 2.1 MDa with dimensions similar to those of a small icosahedral virus. 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-photoinduced force microscopy established that Ico8 assembles into a flexible hollow cage comprising 20 copies of the esterase trimer, 60 protein subunits in total, with overall icosahedral geometry. Notably, the cages formed by Ico8 proved to be extremely stable toward thermal and chemical denaturation: whereas TriEst was unfolded by heating ( Tm ≈ 75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The increased stability of the cages is a new property that emerges from the higher-order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.

Elaborating a coiled-coil-assembled octahedral protein cage with additional protein domains.

De novo design of protein nano-cages has potential applications in medicine, synthetic biology, and materials science. We recently developed a modular, symmetry-based strategy for protein assembly in which short, coiled-coil sequences mediate the assembly of a protein building block into a cage. The geometry of the cage is specified by the combination of rotational symmetries associated with the coiled-coil and protein building block. We have used this approach to design well-defined octahedral and tetrahedral cages. Here, we show that the cages can be further elaborated and functionalized by the addition of another protein domain to the free end of the coiled-coil: in this case by fusing maltose-binding protein to an octahedral protein cage to produce a structure with a designed molecular weight of ~1.8 MDa. Importantly, the addition of the maltose binding protein domain dramatically improved the efficiency of assembly, resulting in ~ 60-fold greater yield of purified protein compared to the original cage design. This study shows the potential of using small, coiled-coil motifs as off-the-shelf components to design MDa-sized protein cages to which additional structural or functional elements can be added in a modular manner.

Symmetry-Directed Self-Assembly of a Tetrahedral Protein Cage Mediated by de Novo-Designed Coiled Coils.

The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of protein-protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiled-coil domains to assemble proteins into higher-order structures. Here, we demonstrate the assembly of a trimeric enzyme into a well-defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiled-coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiled-coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.

Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.

Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines.