1.D.253. The Proteosomal Beta-Barrel NanoPore (P-BB-NP) Family
Zhang et al. 2021 used bottom-up fabrication to synthesize 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. Zhang et al. 2021 used components from all three domains of life to fabricate an integrated multiprotein complex that controls the unfolding and threading of individual proteins across a nanopore. This 900 kDa multicomponent device was made in two steps. First, a stable and low-noise β-barrel nanopore sensor was designed by linking the transmembrane region of bacterial protective antigen to a mammalian proteasome activator. An archaeal 20S proteasome was then built into the artificial nanopore to control the unfolding and linearized transport of proteins across the nanopore. This multicomponent 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. In the descriptions presented below, the numbered references and figures referred to are cited in Zhang et al. 2021 and can be found there.
Membrane-spanning channels and pores have key roles in cellular processes and in biotechnological applications such as nanopore DNA sequencing1. A nanopore protein sequencing device requires the unravelling of a protein and the recognition of individual amino acids by ionic currents as the polypeptide is transported across the nanopore. Several studies have revealed that tiny differences between molecules can be identified by nanopore currents2–7, suggesting that amino acid recognition should be a tractable problem. One of the main remaining challenges is to design a nanopore sensor capable of unfolding proteins without influencing the ionic signal8,9. Small transmembrane proteins have been designed to control the passive transport of ions across membranes, including a synthetic ion channel10, a four-helix divalent metal-ion transporter11, membrane-spanning pores12,13 and a DNA-scaffolded pore14. However, the ability to design nanopores with an integrated biopolymer handling unit had not previously been designed. Such devices would add a new dimension to the protein engineering field, and allow designing next-generation nanopore sensors for biopolymer analysis. Advances in this field, nonetheless, have been hampered for a number of reasons. Usually, molecular machines form multimeric complexes that require complex post- and co-translational assembly15. The latter is particularly challenging because all components must be soluble, unprocessed by proteases, and co-expressed at similar levels. The introduction of artificial transmembrane regions provides an additional challenge as it reduces the solubility of the individual component and it can prevent proper assembly. Moreover, the design of the interface between the hydrophobic transmembrane polypeptides and the hydrophilic components remained unexplored prior to the study of Zhang et al. 2021. Finally, in order to obtain a functional device, the nanopore must remain constantly open, and the operation of the molecular machine should not occlude the nanopore sensor.
Zhang et al. 2021 addressed these challenges and fabricated in two steps a 42-component 900 kDa integrated nanopore sensor that consists of three co-assembled proteins. In the first step, aided by molecular dynamic simulations, a strategy to design artificial nanopores from a soluble protein with a toroid shape (Fig. 1a-d). The designed synthetic nanopores showed an activity and electrical proprieties identical to the nanopores found in nature. In the second step, the multiprotein 20S proteasome from Thermoplasma acidophilum 16 was incorporated into the artificial nanopore (Fig. 1e-h). This design allowed two approaches to single-molecule protein analysis including sequencing. In the chop-and-drop mode, unfolded proteins are first degraded by the proteasome, and the resulting fragments are delivered to the nanopore. In the thread-and-read mode, intact substrates are detected as they translocate across the nanopore. Notably, the activity of the proteasome and the protein unfolding step did not have an influence on the ionic signal (Zhang et al. 2021).