Bottom-up fabrication of a proteasome-nanopore that unravels and processes single proteins

The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work, using components from all three domains of life, we fabricate an integrated multi-protein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, we designed a stable and low-noise β-barrel nanopore sensor by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. Then, an archaeal 20S proteasome was built into the artificial nanopore to control the unfolding and linearised transport of proteins across the nanopore. This multi-component molecular machine opens the door to two approaches in single-molecule protein analysis, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then addressed either as fragmented peptides or intact polypeptides.