Reengineering of an Artificial Protein Cage for Efficient Packaging of Active Enzymes.

Protein cages that readily encapsulate active enzymes of interest present useful nanotools for delivery and catalysis, wherein those with programmable disassembly characteristics serve as particularly attractive platforms. Here, a general guest packaging system based on an artificial protein cage, TRAP-cage, the disassembly of which can be induced by the addition of reducing agents, is established. In this system, TRAP-cage with SpyCatcher moieties in the lumen is prepared using genetic modification of the protein building block and assembled into a cage structure with either monovalent gold ions or molecular crosslinkers. The resulting protein cage can efficiently capture guest proteins equipped with a SpyTag by simply mixing them in an aqueous solution. This post-assembly loading system, which circumvents the exposure of guests to thiol-reactive crosslinkers, enables the packaging of enzymes possessing a catalytic cysteine or a metal cofactor while retaining their catalytic activity.

An artificial protein cage made from a 12-membered ring.

Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings, i.e., hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (i.e., a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Production of Metallic Alloy Nanowires and Particles Templated Using Tomato Mosaic Virus (ToMV).

We demonstrate a simple, low-energy method whereby tomato mosaic virus (ToMV) particles can be used to template the production of nanowires and particles consisting of alloys of gold (Au), platinum (Pt) and palladium (Pd) in various combinations. Selective nanowire growth within the inner channel of the particles was achieved using the polymeric capping agent polyvinylpyrrolidone (PVPK30) and the reducing agent ascorbic acid. The reaction conditions also resulted in the deposition of alloy nanoparticles on the external surface of the rods in addition to the nanowire structures within the internal cavity. The resulting materials were characterized using a variety of electron microscopic and spectroscopic techniques, which revealed both the structural and chemical composition of the alloys within the nanomaterials.

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

Complementary charge-driven encapsulation of functional protein by engineered protein cages in cellulo.

Charge-driven inclusion complex formation in live cells was examined using a degradation-prone fluorescent protein and a series of protein cages. The results show that sufficiently strong host-guest ionic interaction and an intact shell-like structure are crucial for the protective guest encapsulation.

Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin.

The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between Escherichia coli topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs the crucial gyrase dimer interface, while the other intercalates between the fragments of cleaved DNA substrate. Thus, albicidin efficiently locks DNA gyrase, preventing it from religating DNA and completing its catalytic cycle. Two additional structures of this trapped state were determined using synthetic albicidin analogues that demonstrate improved solubility, and activity against a range of gyrase variants and E. coli topoisomerase IV. The extraordinary promiscuity of the DNA-intercalating region of albicidins and their excellent performance against fluoroquinolone-resistant bacteria holds great promise for the development of last-resort antibiotics.

Shape-Morphing of an Artificial Protein Cage with Unusual Geometry Induced by a Single Amino Acid Change.

Artificial protein cages are constructed from multiple protein subunits. The interaction between the subunits, notably the angle formed between them, controls the geometry of the resulting cage. Here, using the artificial protein cage, "TRAP-cage", we show that a simple alteration in the position of a single amino acid responsible for Au(I)-mediated subunit-subunit interactions in the constituent ring-shaped building blocks results in a more acute dihedral angle between them. In turn, this causes a dramatic shift in the structure from a 24-ring cage with an octahedral symmetry to a 20-ring cage with a C2 symmetry. This symmetry change is accompanied by a decrease in the number of Au(I)-mediated bonds between cysteines and a concomitant change in biophysical properties of the cage.

Characterization of near-miss connectivity-invariant homogeneous convex polyhedral cages.

Following the discovery of a nearly symmetric protein cage, we introduce the new mathematical concept of a near-miss polyhedral cage (p-cage) as an assembly of nearly regular polygons with holes between them. We then introduce the concept of the connectivity-invariant p-cage and show that they are related to the symmetry of uniform polyhedra. We use this relation, combined with a numerical optimization method, to characterize some classes of near-miss connectivity-invariant p-cages with a deformation below 10% and faces with up to 17 edges.

Artificial Protein Cage with Unusual Geometry and Regularly Embedded Gold Nanoparticles.

Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.

Programmable polymorphism of a virus-like particle.

Virus-like particles (VLPs) have significant potential as artificial vaccines and drug delivery systems. The ability to control their size has wide ranging utility but achieving such controlled polymorphism using a single protein subunit is challenging as it requires altering VLP geometry. Here we achieve size control of MS2 bacteriophage VLPs via insertion of amino acid sequences in an external loop to shift morphology to significantly larger forms. The resulting VLP size and geometry is controlled by altering the length and type of the insert. Cryo electron microscopy structures of the new VLPs, in combination with a kinetic model of their assembly, show that the abundance of wild type (T = 3), T = 4, D3 and D5 symmetrical VLPs can be biased in this way. We propose a mechanism whereby the insert leads to a change in the dynamic behavior of the capsid protein dimer, affecting the interconversion between the symmetric and asymmetric conformers and thus determining VLP size and morphology.

Topogami: Topologically Linked DNA Origami.

DNA origami is a widely used DNA nanotechnology that allows construction of two-dimensional and three-dimensional nanometric shapes. The designability and rigidity of DNA origami make it an ideal material for construction of topologically linked molecules such as catenanes, which are attractive for their potential as motors and molecular machines. However, a general method for production of topologically linked DNA origami has been lacking. Here, we show that catenated single-stranded DNA circles can be produced and used as a universal scaffold for the production of topologically linked (catenated) DNA origami structures where the individual linked structures can be of any arbitrary design. Assembly of these topologically linked DNA origami structures is achieved via a simple one-pot annealing protocol.

Chemically induced protein cage assembly with programmable opening and cargo release.

Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryo­electron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage.

Artificial Protein Cage Delivers Active Protein Cargos to the Cell Interior.

Artificial protein cages have potential as programmable, protective carriers of fragile macromolecules to cells. While natural cages and VLPs have been extensively exploited, the use of artificial cages to deliver active proteins to cells has not yet been shown. TRAP-cage is an artificial protein cage with an unusual geometry and extremely high stability, which can be triggered to break apart in the presence of cellular reducing agents. Here, we demonstrate that TRAP-cage can be filled with a protein cargo and decorated with a cell-penetrating peptide, allowing it to enter cells. Tracking of both the TRAP-cage and the cargo shows that the protein of interest can be successfully delivered intracellularly in the active form. These results provide a valuable proof of concept for the further development of TRAP-cage as a delivery platform.

A single residue can modulate nanocage assembly in salt dependent ferritin.

Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium Thermotoga maritima (TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like Archeoglobus fulgidus ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg2+). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes.

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.

FRET-Mediated Observation of Protein-Triggered Conformational Changes in DNA Nanostructures.

DNA origami is a powerful technique, which allows virtually limitless 2D or 3D nanostructure designs to be constructed from DNA strands. Such nanostructures can even include programmable nanorobots, which are able to respond to the environment in predetermined ways. DNA aptamers hold particular promise as interfaces, which can enable proteins, peptides, and other non-nucleic acid biomolecules to trigger conformational changes in DNA nanostructures for diagnostic, biosensing, or therapeutic applications. Here, we provide the methodology for FRET-mediated observation of aptamer-triggered conformational change in a DNA origami box nanostructure. The method described can, in principle, be adapted to a wide variety of experimental circumstances where the DNA nanostructure conformational change is mediated by molecular or environmental cues.

Electrostatic Self-Assembly of Protein Cage Arrays.

Protein and peptide cages are nanoscale containers, which are of particular interest in nanoscience due to their well-defined dimensions and enclosed central cavities that can be filled with material that is protected from the outside environment. Ferritin is a typical example of protein cage, formed by 24 polypeptide chains that self-assemble into a hollow, roughly spherical protein cage with external and internal diameters of approximately 12 nm and 8 nm, respectively. The interior cavity of ferritin provides a unique reaction vessel to carry out reactions separated from the exterior environment. In nature, the cavity is utilized for sequestration and biomineralization to render iron inert and safe by shielding from the external environment. Materials scientists have been inspired by this system and exploited a range of ferritin superfamily proteins as supramolecular templates to encapsulate cargoes ranging from cancer drugs to therapeutic proteins. Interesting possibilities arise if such containers can themselves be arranged into even higher-order structures such as crystalline arrays. Here, we describe how crystalline arrays of negatively charged ferritin protein cages can be built by taking advantage of electrostatic interactions with cationic gold nanoparticles.

A Peptide-Nucleic Acid Replicator Origin for Life.

Evolution requires self-replication. But, what was the very first self-replicator directly ancestral to all life? The currently favoured RNA World theory assigns this role to RNA alone but suffers from a number of seemingly intractable problems. Instead, we suggest that the self-replicator consisted of both peptides and nucleic acid strands. Such a nucleopeptide replicator is more feasible both in the light of the replication machinery currently found in cells and the complexity of the evolutionary path required to reach them. Recent theoretical and mathematical work supports this idea and provide a blueprint for future investigations.

A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.