Self-assembly of heptameric nanoparticles derived from tag-functionalized Phi29 connectors

The structure of an induced macromolecular assembly was characterized and found to consist of an ordered heptameric arrangement of recombinant phi29 gp10 connector molecules. Insertion of an N-terminal Strep-II/His₆ tag to the connectors led to the spontaneous formation of large nanoparticles that were distinct from free, wild-type phi29 connectors in both size and symmetry elements. The determination of single-molecule tomograms and image-averaged reconstructions allowed for the stoichiometric and topological characterization of the ordered assemblage, revealing that the nanoparticle is composed of five equatorial connectors arranged with pseudo-5-fold rotational symmetry, capped on its ends by two polar connectors. Additionally, all seven connectors are oriented with their narrower N-terminal necks into the nanoparticle core and wider C-terminal ends out toward the nanoparticle surface, a geometric arrangement accommodated by the shape complementarity of the conical connector profiles. A significant amount of conformational heterogeneity was detected, ranging from changes in overall nanoparticle diameter, to tilting of individual connectors, to variations in connector stoichiometry. Nevertheless, a stable, heptameric nanoparticle was resolved, revealing the significant potential of guided, peptide-mediated supramolecular self-assembly. With this construct, we anticipate the further design of variable N-terminal tags to allow for the generation of nanoparticles with tailored connector stoichiometry and topological arrangements. By modifying the surface-exposed C-terminal ends with application-appropriate moieties, the consistent structure and compact nature of these nanoparticles may prove beneficial in nanotechnological and nanomedical approaches.