Bottom-up fabrication of a multi-component nanopore sensor that unfolds, processes and recognizes single proteins

Transmembrane channels and pores have many biotechnological applications, notably in the single-molecule sequencing of DNA. Small synthetic nanopores have been designed using amphipathic peptides, or by assembling computationally designed transmembrane helices. The fabrication of more complex transmembrane devices has yet to be reported. In this work, we fabricated in two steps a multi-protein transmembrane device that addresses some of the main challenges in nanopore protein sequencing. In the first step, artificial nanopores are created from soluble proteins with toroid shapes. This design principle will allow fabricating a variety of nanopores for single-molecule analysis. In the second step one subuinit of the 20S proteasome from Thermoplasma acidophilum is genetically integrated into the artificial nanopore, and a 28-component nanopore-proteasome is co-assembled in E. coli cells. This multi-component molecular machine opens the door to two new approaches in protein sequencing, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then identified by the nanopore sensor either as intact or fragmented polypeptides. The ability to integrate molecular devices directly onto a nanopore sensors allows creating next-generation protein sequencing devices, and will shed new lights on the fundamental processes of biological nanomachines.