(66e) Engineering the Self-Assembly of Ultrastable Protein Filaments Into 2D and 3D Multifunctional Nanostructures

Self-assembling protein templates have enormous potential as biomaterials for the fabrication of multifunctional nanostructures that require precise positioning of individual molecules, such as enzymes, in regular patterns. We are endeavouring to expand the realm of multidimensional protein assembly by generating new proteins that assemble into 2D and 3D shapes of controllable size and symmetry for template-based construction of advanced biomaterials. One of the central protein building blocks enabling these efforts is the g-prefoldin (g-PFD), a chaperone protein isolated from the hyperthermophilic archaeon Methanocaldococcus jannaschii. The g-PFD oligomerizes into filaments several microns in length and exhibit remarkable stability up to at least 94°C. The malleable and distinct modular architecture of the g-PFD filament provide an ideal starting point to construct novel protein architectures. Our current aim is to develop a standardized biomolecular construction set comprised of g-PFD filaments of specific size, and two- and three-way connectors that can be assembled into diverse structures of controllable size and shape.

We have demonstrated the creation of a three-way connector containing the g-PFD fused with a trimerization domain that is capable of assembling with filaments into 2D “pinwheel-like” structures, and self-closing loops using two-way connectors. Furthermore, we are able to position nanoparticles at specific locations upon the pinwheel assembly. Based on our initial success to incorporate a two- and three-way connector into the γ-PFD subunit we are working to create tunable binding partners to control the assembly and dimensions of our biomoleculer construction kit. In particular, we used simple but well-understood structural elements such as coiled-coils to create strong hydrophobic interactions between two self-associating helical bundles. As a consequence, two g-PFD subunits containing opposing α-helical stretches (so-called “bait” and “prey” sequences) form a tight heterodimeric coil-coiled bundle. These bait-prey proteins are specific for each other, and are capable of capping filament length. Subsequently, the bait-prey pair will be used to control de novo assembly of the three-way connector into various 3D structures. Ultimately, the ability to control the position and orientation of functional molecules at the nanoscale using self-assembling supramolecular protein templates, such as the g-PFD, will greatly expand opportunities for the template-based construction of biomaterials.