Chasing a Protein’s Tail: Detection of Polypeptide Translocation through Nanopores

Main Text

First imagined in 1989 (1), nanopore sequencing of DNA is in many respects a compelling scientific success story. During the first decade of this millennium, it sometimes seemed doubtful, at least to the more impatient among us (including this author), that the concept would ever yield a practical sequencing method. Through the contributions of many researchers and despite technical hurdles, nanopore sequencing has been commercialized (2) and seems destined to be the method of choice for some important niches in the field of DNA sequencing (3). However, even when it was arguable whether nanopore-based sequencing could compete with more established sequencing methods, nanopores always held great promise as versatile tools for single-molecule characterization. In fact, some of the earliest experiments that set the groundwork for nanopore sequencing focused on analytes other than nucleic acids, such as polyethylene glycol (4, 5). With nanopore DNA sequencing now a commercial enterprise, academic researchers turn with renewed interest to other applications of nanopore sensing, including peptide and protein characterization (2, 6), which bring a new set of challenges.

A major hurdle in applying nanopore-based sensing to biopolymers other than nucleic acids is the lack of a uniform charge distribution on these polymers to drive them, on average, unidirectionally through the nanopore. The prototypical nanopore apparatus is a thin membrane containing a single small channel. An electrical potential difference is created between the solution volumes on the two sides of the membrane, which has two important effects. First, a measurable current of small ions (usually provided by a simple salt such as KCl) is driven through the pore, providing a sensitive macroscopic readout of the atomic configuration at the constriction (7), which is typically <3 nm in diameter. Second, the electric field funnels appropriately charged molecules into the pore constriction and causes uniformly charged polymers to thread through the pore. Nucleic acids carry a uniform charge of $-e$ per nucleotide at physiological conditions, irrespective of their sequence, which leads to unidirectional threading. This unidirectional motion is superimposed on thermal diffusion, which can cause backward motion of the polymer and failed translocation attempts at probabilities that decrease with the magnitude of the unidirectional driving force (8). In the case of peptides and proteins, capture and threading of the molecules becomes more difficult and failed attempts become more likely.

Similar to DNA, peptides (9) and unfolded proteins (10, 11, 12) can traverse narrow pores with diameters similar to the peptide chain, leading to measurable changes in the current and the possibility of detecting atomic-scale details, e.g., discriminating phosphorylated and nonphosphorylated forms (13). Larger synthetic pores have been used to distinguish different proteins, as well as charge states and conformations of the same protein (14, 15, 16), albeit with presumably less than atomic resolution. None of these works distinguished between failed and completed translocation events using current measurements alone. A nucleic acid tag, together with polymerase chain reaction, has been used to quantify protein translocation independent of the ion current measurements (11), but this strategy seems more cumbersome than real-time tracking of the polypeptide by ion current.

In this issue, Hoogerheide et al. (17) describe experiments in which a polypeptide having regions of different charge densities is translocated through a protein nanopore. As shown in Fig. 1, the 140-residue polypeptide, α-synuclein, possesses an N-terminal region consisting of two long α-helices and a disordered C-terminal region (18). The total charge of the polypeptide is $-9e$; however, most of this negative charge is concentrated in the C-terminal region, whereas residues 1–115 inclusive have a net charge of zero. While threading through the pore, the N-terminal and C-terminal regions cause distinct electrostatic environments in the constriction. The result is unique current signatures corresponding to each region, allowing different stages of the translocation process to be distinguished. Importantly, failed translocation attempts (referred to as “retraction events” by Hoogerheide et al.), wherein the polypeptide partially threads through the pore but then reverses and exits onto the same side of the membrane from which it entered, can be unambiguously identified. Based on this experiment, the authors propose the idea of selectivity tags—regions of the analyte polymer with particular charge distributions that alter the ion selectivity of the pore and allow monitoring of the analyte’s translocation progress by changes in the measured current. This idea appears to be broadly transferable to other polypeptides and even other types of polymer. Hence, these experiments appear to represent a significant advance in high-resolution characterization of peptides and proteins using nanopores.

Figure 1.

Molecular model of the nanopore system described by Hoogerheide et al. (17). A cross section through the lipid bilayer and channel protein is shown, with the polypeptide α-synuclein threaded through the channel. The channel protein (voltage-dependent anion channel, VDAC) is represented by a multicolored surface. Neutral residues of α-synuclein are shown as a yellow tube, and the negatively and positively charged amino acids are highlighted in red and blue, respectively. The C-terminal region of α-synuclein, threaded through the pore, carries a negative charge, whereas the N-terminal helical regions are nearly neutral overall.

Editor: Chris Chipot.