Engineering the filamentous molecular chaperone gamma prefoldin for construction of protein nanostructures

Proteins have the potential to direct the assembly of nanostructured materials and impart unique functions to such systems owing to their size, shape, and bioactivity. However, a current problem with the use of proteins as structural scaffolds is the controllable design of new and robust protein shapes, as well as generating stable oligomeric precursors. An extraordinarily stable filamentous protein, gamma prefoldin (gamma PFD), from the hyperthermophile Methanocaldococcus jannaschii was discovered and characterized. Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. gamma PFD was subcloned and expressed in Eschericia coli along with its homologs alpha and beta PFD. gamma PFD would not assemble with either protein, instead forming long filaments of defined dimensions with lengths exceeding 1 microm. A possible molecular model for filament assembly is discussed.;For the overall purpose of devising a controllable template for the construction of biomaterials, a capping protein was rationally designed to control the filament length over multiple length scales. A step-wise polymerization model of filament formation was developed that quantitatively describes the resulting distributions of filaments for lengths ranging from less than 10 nm to over 100 nm. The versatility of the gamma PFD was further demonstrated by generating a series of narrowly-sized hybrid filaments containing functional peptide fusion partners, illustrating that structure and function can be designed into the gamma PFD as independent and separable components. Implications of the design of the capping protein with regard to evolution of protein-protein association sites are also discussed.;Protein design has been used to develop new macromolecular protein structures like bundled filaments and micelles, but the structures are fundamentally limited by complexity, functionality, and recombinant expression levels. A combinatorial protein engineering approach was undertaken to design specifically interlocking proteins derived from the natural filamentous protein gamma prefoldin. Included with the rational design of two cross-linked PFD monomers, this approach enables creation of a diverse array of one dimensional and two-dimensional structures like square, hexagonal, and octagonal pores, and protein lattices of various repeat lengths.