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

Abstract

The precise assembly and engineering of molecular machines capable of handling biomolecules play crucial roles in most single-molecule methods. In this work we use 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, 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. 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.

Fig. 1. Design of a proteasome nanopore.

a, Structure of mouse REG (PDB ID: 5MSJ). b, Sticks diagram of the structure of serine-serine-glycine linker. c, Ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C). The transmembrane region of the protective antigen is in red. d, Structure of the REG-nanopore enhanced by molecular dynamics simulations. REG (a) was genetically fused to the transmembrane region of the protective antigen (c) via a short linker (b). e and f, Structure of the T. acidophilum proteasome α-and β-subunit (PDB ID: 1YA7). g, REG-nanopore was genetically fused to α-subunit of T. acidophilum proteasome. h, Structure of the designed proteasome-nanopore refined by MD simulations. i, Structure of VATΔN (PDB ID: 5G4G).

Fig. 2. Fabrication and optimization of the artificial nanopores.

a, Structural representation of the designed nanopores. The heptameric transmembrane part of protective antigen (PA) replaced an unstructured loop (red) in REG (cyan). One subunit in the transmembrane region and REG is highlighted in blue and showed in isolation. The hydrophobic residues anchoring the nanopore to the membrane are indicated in green. The REG-nanopore was generated by molecular dynamics simulations. b, Effects of linker length on the nanopore expression in E. coli. cells, insertion efficiency and nanopore stability. The side chains that point towards the outside and inside of the barrel are highlighted with grey and black lines, respectively. The first designed nanopore (0) is highlighted with a wider arrow. One deletion mutant (Δ2) and five insertion mutants (∇2, ∇4, ∇8, ∇12, and ∇16) were tested. The sequence of the protective antigen was used as template for the linker. REG is shown as a cyan rectangle. c, d Electrical properties of the functional ∇4 (c) and ∇2 (d) mutants. On the left is sequence of the mutant, in the middle a typical current trace, and on the right the current histogram corresponding the insertions of multiple pores at +35 mV. e, top, linker optimization tested by substituting several residues. Bottom, Electrical properties of a REG-nanopore with homo-polymeric serine linkers. f, ATPase-nanopore formed by introducing the transmembrane barrel elongated with the homo-polymeric serine linkers within a loop (red) at the AAA+ ATPase domain of Trypanosoma brucei σ54-RNA polymerase. g, Formation of a PA26-nanopore by introducing the β-barrel elongated by the serine linker into a loop in the top face of PA26 from Aquifex aeolicus. Electrical data were collected at ±35 mV in 1 M NaCl, 15 mM Tris, pH 7.5, using 10 kHz sampling rate and a 2 kHz low-pass Bessel filter.

Fig. 3. Electrical properties of REG-nanopore.

a, Cut-through of a surface representation of REG-nanopore. The pore is coloured according to the vacuum electrostatic potential as calculated by PyMOL on the final snapshot of the multiscale MD model. b, A typical current trace recorded of a single REG-nanopore at +35 and -35 mV. c, Current–voltage (I−V) characteristics of three different nanopores. d, Reversal potential measured using asymmetric ion concentrations (trans:cis, 0.5 M NaCl: 2.0 M NaCl), showing that the pore is cation-selective, as expected from the electrostatic potentials of the nanopore lumen. The REG-nanopore was added to the trans side. e-f, Chemical structure of β-CD (e) and γ-CD (f) (left), scatter plots of I _res% versus dwell time (right), and representative trace of 20 μM β-CD (e) and 20 μM γ-CD (f) blockades (below). g, Chemical structure of imipramine and a representative trace of γ-CD cyclodextrin blockades (20 μM) in the presence of 100 μM of imipramine. h-j, typical peptide blockades (bottom) and resulting scatter plots of I _res% versus dwell time (top) for 4 μM of angiotensin I (h) 4 μM dynorphin A (i) and a 2 μM equimolar mixture of the two (j). The REG-nanopore and all analytes were added to the cis side. Electrical recordings were collected at -35 mV in 1 M NaCl, 15 mM Tris, pH 7.5. All traces except (b) were sampled at 50 kHz sampling rate and a 10 kHz low-pass Bessel filter, and an additional Gaussian low-pass filter with a 5 kHz cut-off was digitally applied. In b, data were collected at 10 kHz sampling rate and a 2 kHz low-pass Bessel filter.

Fig. 4. Design of the artificial proteasome-nanopore.

a, Structure of the T. acidophilum proteasome (the α-subunit is orange and magenta, the β-subunit is green) in complex with PA26 (cyan). The C-terminal of PA26 (S231) is near L21 of the proteasome α-subunit. b, A representation of the reconstitution of the artificial proteasomal nanopore. To obtain complex 3, two separate vectors were used to express the three proteins. The REG-nanopore was fused to the proteasome α-subunit and contained a His-tag. The protein was co-expressed with the untagged β-subunits and a second α-subunit containing a Strep-tag. His-tag affinity chromatography was used to co-purify complex 1 and 3. Then a Strep-Tag affinity chromatography was used to purify 3. c, Electrical behaviour of a single pore at ±35 mV in 1 M NaCl, 15 mM Tris, pH 7.5, using 10 kHz sampling rate and a 2 kHz low-pass Bessel filter. d, Native PAGE (left, ~5 μg) and SDS-PAGE (right, ~2 μg) analyses of the purified complex 3. SDS-PAGE revealed the presence of three unique bands corresponding well the molecular weights of REGαΔ20, proteasome αΔ12-subunit, and proteasome β-subunit (51.8, 25.9, and 22.3 kDa). The native PAGE showed that REGαΔ20, proteasome αΔ12-subunit, and proteasome β-subunit form a stable complex 3. e, TEM image of the proteasome-nanopore. f, Cut-through of a surface representation of proteasome-nanopore enhanced by molecular dynamics simulations and coloured (blue, positive; red, negative) according to the vacuum electrostatic potential as calculated by PyMOL.

Fig. 5. Controlled translocation through the proteasome-nanopore. a-c, Thread-and-read.

Typical current traces and relative scatter plots showing the average I _res% versus dwell time provoked by the translocation of S1 (a, 20.0 μM S1 and 20.0 μM VAT, 23 independent nanopore experiments), GFP (b, 5.0 μM and 5.0 μM VATΔN, 42 independent experiments), and GFP in 1 M urea (c, 5.0 μM and 5.0 μM VATΔN, 13 independent experiments) through an inactive proteasome-nanopore mediated by VATΔN in the presence of 2.0 mM ATP. The proteasome-nanopore and substrates were added to the cis side. d-f, Chop-and-drop. Typical current traces provoked by the transport of oligopeptides across an activated proteasome. d, Unassisted S1 translocation (>50 independent experiments). e, VATΔN and ATP assisted S1 transport induce fragmentation into small peptides, which fast transport is seldomly observed (23 independent experiments). f, VATΔN assisted GFP-ssrA proteolytic cleavages produce peptides that are too short to be detected by the nanopore (15 independent experiments). Data were collected at 40°C and -30 mV in 1 M NaCl, 15 mM Tris, pH 7.5, using a 10 kHz low-pass Bessel filter with a 50 kHz sampling rate. The traces were then filtered digitally with a Gaussian low-pass filter with a 5 kHz cutoff.