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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151280/1/pro3676_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151280/2/pro3676-sup-0001-supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151280/3/pro3676.pd

Elaborating a coiledâ coilâ assembled octahedral protein cage with additional protein domains

De novo design of protein nanoâ cages has potential applications in medicine, synthetic biology, and materials science. We recently developed a modular, symmetryâ based strategy for protein assembly in which short, coiledâ 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 coiledâ coil and protein building block. We have used this approach to design wellâ 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 coiledâ coil: in this case by fusing maltoseâ 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 ~ 60â fold greater yield of purified protein compared to the original cage design. This study shows the potential of using small, coiledâ coil motifs as offâ theâ shelf components to design MDaâ sized protein cages to which additional structural or functional elements can be added in a modular manner.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146469/1/pro3497.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146469/2/pro3497_am.pd

Symmetryâ Directed Selfâ Assembly of a Tetrahedral Protein Cage Mediated by de Novoâ 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 proteinâ protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiledâ coil domains to assemble proteins into higherâ order structures. Here, we demonstrate the assembly of a trimeric enzyme into a wellâ defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiledâ coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiledâ coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.Geometry lesson: A modular approach for assembling proteins into largeâ scale geometric structures was developed in which coiledâ coil domains acted as â twist tiesâ to facilitate assembly. The geometry of the cage was specified primarily by the rotational symmetries of the coiled coil and building block protein and was largely independent of protein structural details.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/1/cbic201700406_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/2/cbic201700406.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/3/cbic201700406-sup-0001-misc_information.pd

Invited Review Meeting Report: SMART Timing-Principles of Single Molecule Techniques Course at the University of Michigan 2014

ABSTRACT: Four days after the announcement of the 2014 Nobe

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.A novel protein nanocage has been developed capable of on- demand reversible disassembly via simple buffer exchanges under mild conditions. Data presented herein also show the nanocage is capable of in vivo and in vitro cargo loading, suggesting a broad range of possible applications in biocatalysis, bionanotechnology, and biomedicine. Additional findings include structure determination and protein design verification via cryo- electron microscopy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170959/1/ange202110318_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170959/2/ange202110318-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170959/3/ange202110318.pd

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.A novel protein nanocage has been developed capable of on- demand reversible disassembly via simple buffer exchanges under mild conditions. Data presented herein also show the nanocage is capable of in vivo and in vitro cargo loading, suggesting a broad range of possible applications in biocatalysis, bionanotechnology, and biomedicine. Additional findings include structure determination and protein design verification via cryo- electron microscopy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170980/1/anie202110318_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170980/2/anie202110318-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170980/3/anie202110318.pd

Front Cover: Symmetryâ Directed Selfâ Assembly of a Tetrahedral Protein Cage Mediated by de Novoâ Designed Coiled Coils (ChemBioChem 19/2017)

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138873/1/cbic201700481.pd