Single-molecule nanopore enzymology

Abstract

One contribution of 17 to a discussion meeting issue 'Membrane pores: from structure and assembly, to medicine and technology'. Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

“…A single biological membrane protein-based single-biomolecule interface provides a single-molecule nanopore platform for sensitive detection of DNA/RNA [17–22], peptides [23–25], proteins [26–29], enzymes [30, 31], and host-guest molecules [32, 33]. The confined nanopore interface effectively captures a single molecule from the bulk solution at an applied potential, resulting in the typical ionic blockages for each analyte.…”

The Utility of Nanopore Technology for Protein and Peptide Sensing

“…More recently, reports of bionanopore sensors being used to characterize a variety of protein and peptide‐related phenomenon has demonstrated that nanopore‐based sensing and characterization of proteins is entirely possible and provides useful information at the single‐molecule limit. Future efforts in this area will most likely continue to pursue aptamer‐based nanopore protein sensors and the development of optimized sensing geometries (i.e., the discovery of new protein nanopores, improvements to graphene‐based sensors, and controlled synthesis of solid‐state pores via dielectric breakdown, and DNA origami) for development of portable sensing devices and sensors capable of sequencing proteins. The continued growth of nanopore sensors for protein and peptide detection should continue for years to come.…”

“…Although most of the above work relied on solid‐state pores for their geometrical attributes, Maglia and colleagues have developed several different protein–pore systems that allow the detection and characterization of a wide variety of protein analytes . One compelling pore is an engineered version of fragaceatoxin C (Fra C), which allows the detection of a wide range of intact, folded proteins (Figure A) .…”

Snapshotting the transient conformations and tracing the multiple pathways of single peptide folding using a solid-state nanopore

“…The nanopore-based technique is a novel analytical technique that allowed single molecules to be observed over significant lengths of time with high throughput. [29][30][31][32][33][34][35][36][37][38][39] Furthermore, with the high temporal resolution and high sensitivity of the nanopore confined space, transition conformations and individual folding trajectories of protein can be monitored. Here, we developed a new method to achieve the study of the dynamic processes of protein folding by the electrochemically confined effects of the solid-state nanopore.…”