Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape.

The chemical nature and precise position of posttranslational modifications (PTMs) in proteins or peptides are crucial for various severe diseases, such as cancer. State-of-the-art PTM diagnosis is based on elaborate and costly mass-spectrometry or immunoassay-based approaches, which are limited in selectivity and specificity. Here, we demonstrate the use of a protein nanopore to differentiate peptides─derived from human histone H4 protein─of identical mass according to the positions of acetylated and methylated lysine residues. Unlike sequencing by stepwise threading, our method detects PTMs and their positions by sensing the shape of a fully entrapped peptide, thus eliminating the need for controlled translocation. Molecular dynamics simulations show that the sensitivity to molecular shape derives from a highly nonuniform electric field along the pore. This molecular shape-sensing principle offers a path to versatile, label-free, and high-throughput characterizations of protein isoforms.

A chip-based array for high-resolution fluorescence characterization of free-standing horizontal lipid membranes under voltage clamp.

Optical techniques, such as fluorescence microscopy, are of great value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine high-resolution optical measurements with high-resolution voltage clamp electrical recordings providing direct information on e.g. single ion channel gating and/or membrane capacitance. Here, we report on a novel chip-based array device which facilitates optical access with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes while controlling membrane voltage and recording currents using individual micropatterned Ag/AgCl-electrodes. Wide-field and confocal imaging, as well as time-resolved single photon counting on free-standing membranes spanning sub-nanoliter cavities are demonstrated while electrical signals, including single channel activity, are simultaneously acquired. This optically addressable microelectrode cavity array will allow combined electrical-optical studies of membranes and membrane proteins to be performed as a routine experiment.

Pore-forming toxins as tools for polymer analytics: From sizing to sequencing.

We report here on the nanopore resistive pulse sensing (Np-RPS) method, involving pore-forming toxins as tools for polymer analytics at single molecule level. Np-RPS is an electrical method for the label-free detection of single molecules. A molecule interacting with the pore causes a change of the electrical resistance of the pore, called a resistive pulse, associated with a measurable transient current blockade. The features of the blockades, in particular their depth and duration, contain information on the molecular properties of the analyte. We first revisit the history of Np-RPS, then we discuss the effect of the configuration of the molecule/nanopore interaction on the molecular information that can be extracted from the signal, illustrated in two different regimes that either favor molecular sequencing or molecular sizing. Specifically, we focus on the sizing regime and on the use of two different pore-forming toxins, staphylococcal α-hemolysin (αHL) and aerolysin (AeL) nanopores, for the characterization of water-soluble polymers (poly-(ethylene glycol), (PEG)), homopeptides, and heteropeptides. We discuss how nanopore sizing of polymers could be envisioned as a new approach for peptide/protein sequencing.

Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore.

Efforts to sequence single protein molecules in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.