Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis

Abstract

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.

Keywords: coupled catalysis; enzyme encapsulation; hierarchical structure; nanoreactor; self-assembly; superlattice; virus-like particle.

Figure 1

Schematic illustration of hierarchical self-assembly from protein subunit building blocks to a superlattice of catalytically active virus-like particles (VLPs). (a) Illustration of directed assembly of a P22 VLP with encapsulated cargo. Scaffolding proteins (SPs) direct the assembly of coat proteins (CPs) into the capsid structure. Cargos are guided towards co-encapsulation inside of the VLP via SP fusion. (b) Individual CPs self-assemble to form a VLP, templated by a SP-cargo fusion protein. The green protein represents a SP fusion protein with KivD, which catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde, and the purple protein represents a SP fusion protein with AdhA, which catalyzes the conversion of isobutyraldehyde to isobutanol. (c) VLPs self-assemble into higher ordered superlattice materials, mediated by positively charged PAMAM dendrimers (blue). The array material, which is comprised of the two separately encapsulated enzymes, retains catalytic communication after assembly and performs a two-step reaction to produce isobutanol.

Figure 2

Assembly of P22 VLPs mediated by G6 PAMAM dendrimer. (a) Assembly of wtP22 and P22-E2 variant in solution under a range of ionic strengths (I) was assessed by monitoring light scattering at 800 nm. A rapid increase in optical density was observed upon addition of G6 PAMAM dendrimer to both wtP22 and P22-E2 under I = 0 – 247 mM due to the formation of large aggregates. In the case of P22-E2 + G6, an increase in optical density was observed upon addition of G6 under the same I range as wtP22 + G6. (Inset) A solution of P22-E2 mixed with G6 in I = 206 mM became turbid (left), whereas when mixed in I = 329 mM remained clear (right). (b) The zeta potential of each P22 VLP at pH 7.0 and the I_T for assembly with G6.

Figure 3

SAXS analysis of P22 VLP assemblies mediated by G6 PAMAM dendrimers. (a, b) The structure of P22 assemblies formed under various ionic strengths (I) was investigated by SAXS. SAXS data are plots of structure factor S(q) versus scattering vector q (plots are vertically offset for clarity). In the case of P22-E2 (b) new peaks attributed to a structure factor of the assembly became more prominent with increasing ionic strength up to the threshold ionic strength (I_T) for each construct. This indicates that the array materials with longer-range order are achieved near the I_T. Conversely, the structure of the wtP22 assembly (a) shows broad peaks, suggesting some short-range order of wtP22 VLPs in the assembly, however no prominent peaks due to long-range order were observed. (c) 2D scattering patterns of individual P22-E2 VLPs and P22-E2 + G6 assembled at I = 206 mM. (d) Comparison of the experimental scattering profile of P22-E2 + G6 assembled at I = 206 mM (black) matched with the simulated scattering pattern of an FCC structure with lattice parameter a = 87.0 nm and a nearest neighbor distance between VLPs of 61.5 nm (blue).

Figure 4

TEM characterization and structural model of P22-E2 superlattice. (a) The structure of the P22-E2 + G6 superlattice assembled at I = 206 mM was observed by TEM following Pt-Pd shadowing of the sample. An expanded image (bottom right) and an image of VLP arrangement in the corresponding area (top right) are also presented. (a1) An image of the assembly showing hexagonal close packing of P22-E2 VLPs. The FFT image (inset) indicates the inter-row spacing of about 54 nm, which corresponds to a nearest neighbor distance of 62 nm, which matches well with the SAXS data. (a2) An image of the assembly showing a square lattice arrangement of P22-E2 VLPs. The FFT image (inset) indicates the period that corresponds to the center-to-center distance between the nearest neighbor VLPs, is about 61 nm. The blue-line square in the top right illustration corresponds to a unit cell on the {100} face. (b) Structural model of an FCC unit cell, consisting of 14 icosahedrons. The yellow spheres associated with each face of the icosahedron represent the dendrimer molecules. Because a sphere at each lattice point in an FCC structure is surrounded by 12 nearest neighbor spheres and eight tetrahedral coordination sites, 12 faces of an icosahedron are in close proximity to the nearest neighbors and the other 8 faces are exposed to tetrahedral coordination sites. (c) Structural model of the tetrahedral coordination site in the FCC lattice. Here, 12 dendrimers on each icosahedron, which bridge to the 12 nearest neighbor icosahedra are shown in blue. The other eight dendrimers are shown in yellow. These eight dendrimers are located at the tetrahedral coordination sites in the FCC lattice.

Figure 5

Catalytic activity of co-assembled array composed of P22-E2-KivD and P22-E2-AdhA and corresponding non-assembled mixture. (a) Reaction scheme of the sequential conversion of α-ketoisovalerate to isobutyraldehyde by Ketoisovalerate Decarboxylase (KivD) followed by the reduction of isobutyraldehyde to isobutanol by Alcohol Dehydrogenase A (AdhA) with the concomitant oxidation of NADH. (b, c) The progress of the two-step reaction catalyzed by the mixture of free nanoreactors, co-assembled nanoreactor superlattices, or condensed superlattices was monitored with gas chromatography-mass spectrometry (GC-MS) (production of isobutyraldehyde and isobutanol) and UV-Vis spectroscopy (conversion of NADH to NAD⁺). Catalytic activity of the co-assembled superlattices and the condensed material, in which the superlattice was resuspended in one-tenth of initial buffer volume, was compared with mixture of free P22-E2-KivD and P22-E2-AdhA under the same conditions of enzyme and substrate concentration. The net conversion profiles were similar between the free nanoreactors and the non-condensed superlattices (b), whereas the conversion was significantly accelerated in the condensed superlattice sample (c). The dotted line in the NADH depletion and isobutanol production indicates 100% conversion of supplied substrate to the final product.