The development of systematic approaches to explore protein-protein interactions and dynamic protein networks is at the forefront of biological sciences. Nanopatterned protein arrays offer significant advantages for sensing applications, including short diffusion times, parallel detection of multiple targets and the requirement for only tiny amounts of sample. Atomic force microscopy (AFM) based techniques have successfully demonstrated patterning of molecules, including stable proteins, with submicrometre resolution. Here, we introduce native protein nanolithography for the nanostructured assembly of even fragile proteins or multiprotein complexes under native conditions. Immobilized proteins are detached by a novel vibrational AFM mode (contact oscillation mode) and replaced by other proteins, which are selectively self-assembled from the bulk. This nanolithography permits rapid writing, reading and erasing of protein arrays in a versatile manner. Functional protein complexes may be assembled with uniform orientation at dimensions down to 50 nm. Such fabrication of two-dimensionally arranged nano-objects with biological activity will prove powerful for proteome-wide interaction screens and single molecule/virus/cell analyses.

The processing of propeptides and the maturation of 20S proteasomes require the association of beta rings from two half proteasomes. We propose an assembly-dependent activation model in which interactions between helix (H3 and H4) residues of the opposing half proteasomes are prerequisite for appropriate positioning of the S2-S3 loop; such positioning enables correct coordination of the active-site residue needed for propeptide cleavage. Mutations of H3 or H4 residues that participate in the association of two half proteasomes inhibit activation and prevent, in nearly all cases, the formation of full proteasomes. In contrast, mutations affecting interactions with residues of the S2-S3 loop allow the assembly of full, but activity impacted, proteasomes. The crystal structure of the inactive H3 mutant, Phe145Ala, shows that the S2-S3 loop is displaced from the position observed in wild-type proteasomes. These data support the proposed assembly-dependent activation model in which the S2-S3 loop acts as an activation switch.