Unfoldase-mediated protein translocation through an α-hemolysin nanopore

Abstract

Using nanopores to sequence biopolymers was proposed more than a decade ago1. Recent advances in enzyme-based control of DNA translocation2 and in DNA nucleotide resolution using modified biological pores3 have satisfied two technical requirements of a functional nanopore DNA sequencing device. Nanopore sequencing of proteins was also envisioned1. Although proteins have been shown to move through nanopores4, 5, 6, a technique to unfold proteins for processive translocation has yet to be demonstrated. Here we describe controlled unfolding and translocation of proteins through the α-hemolysin (α-HL) pore using the AAA+ unfoldase ClpX. Sequence-dependent features of individual engineered proteins were detected during translocation. These results demonstrate that molecular motors can reproducibly drive proteins through a model nanopore—a feature required for protein sequence analysis using this single-molecule technology.

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

“…This is an important task as the examination of proteins and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 possible future sequencing of polypeptides has received a major boost. 67,68 In order to adapt the biophysical model to polypeptides, the basal hopping rate would have to be varied along the sequence (currently it is a constant), and the free energy would have to include more terms such as the pore-biopolymer interactions. The varied hopping rate could be obtained following previous studies by extrapolating the translocation speed to zero voltage.…”

Single-molecule sensing with nanopores

“…11 Full de novo protein sequencing is a much harder problem than DNA sequencing, however, because it requires distinguishing between 20 different amino acids rather than just 4 bases. Still, early steps are being made in that direction.…”

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore

“…For example, pseudo-rotaxanes formed by a DNA thread capped by a DNA processing enzyme are used in nanopore and protein sequencing applications. [15][16][17][18][19][20][21][22][23] Nanopores might also be used to study single proteins. Ionic currents through nanopores are very sensitive to the environment of the nanopore and small differences between protein homodimers 24 or isomeric protein-DNA interactions 25 can be observed by specific changes to the nanopore conductance.…”