Preparation of Cytolysin A (ClyA) Nanopores.

The ionic currents passing through nanopores can be used to sequence DNA and identify molecules at the single-molecule level. Recently, researchers have started using nanopores for the detection and analysis of proteins, providing a new platform for single-molecule enzymology studies and more efficient biomolecular sensing applications. For this approach, the homo-oligomeric Cytolysin A (ClyA) nanopore has been demonstrated as a powerful tool. Here, we describe a simple protocol allowing the production of ClyA nanopores. Monomers of ClyA are expressed in Escherichia coli and oligomerized in the presence of detergent. Subsequently, different oligomer variants are electrophoretically resolved and stored in a gel matrix for long-term use.

Real-Time Conformational Changes and Controlled Orientation of Native Proteins Inside a Protein Nanoreactor.

Protein conformations play crucial roles in most, if not all, biological processes. Here we show that the current carried through a nanopore by ions allows monitoring conformational changes of single and native substrate-binding domains (SBD) of an ATP-Binding Cassette importer in real-time. Comparison with single-molecule Förster Resonance Energy Transfer and ensemble measurements revealed that proteins trapped inside the nanopore have bulk-like properties. Two ligand-free and two ligand-bound conformations of SBD proteins were inferred and their kinetic constants were determined. Remarkably, internalized proteins aligned with the applied voltage bias, and their orientation could be controlled by the addition of a single charge to the protein surface. Nanopores can thus be used to immobilize proteins on a surface with a specific orientation, and will be employed as nanoreactors for single-molecule studies of native proteins. Moreover, nanopores with internal protein adaptors might find further practical applications in multianalyte sensing devices.

Single-molecule nanopore enzymology.

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'.

Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore.

The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.

Engineering a nanopore with co-chaperonin function.

The emergence of an enzymatic function can reveal functional insights and allows the engineering of biological systems with enhanced properties. We engineered an alpha hemolysin nanopore to function as GroES, a protein that, in complex with GroEL, forms a two-stroke protein-folding nanomachine. The transmembrane co-chaperonin was prepared by recombination of GroES functional elements with the nanopore, suggesting that emergent functions in molecular machines can be added bottom-up by incorporating modular elements into preexisting protein scaffolds. The binding of a single-ring version of GroEL to individual GroES nanopores prompted large changes to the unitary nanopore current, most likely reflecting the allosteric transitions of the chaperonin apical domains. One of the GroEL-induced current levels showed fast fluctuations (<1 ms), a characteristic that might be instrumental for efficient substrate encapsulation or folding. In the presence of unfolded proteins, the pattern of current transitions changed, suggesting a possible mechanism in which the free energy of adenosine triphosphate binding and hydrolysis is expended only when substrate proteins are occupied.

Detection of two isomeric binding configurations in a protein-aptamer complex with a biological nanopore.

Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we showed that folded proteins can be internalized inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalization of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores can be used to probe conformational heterogeneity in protein:DNA interactions.