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Self-assembly is a fundamental feature of biological systems, and control of such processes offers fascinating opportunities to regulate function. Fragaceatoxin C (FraC) is a toxin that upon binding to the surface of sphingomyelin-rich cells undergoes a structural metamorphosis, leading to the assembly of nanopores at the cell membrane and causing cell death. In this study we attached photoswitchable azobenzene pendants to various locations near the sphingomyelin binding pocket of FraC with the aim of remote controlling the nanopore assembly using light. We found several constructs in which the affinity of the toxin for biological membranes could be activated or deactivated by irradiation, thus enabling reversible photocontrol of pore formation. Notably, one construct was completely inactive in the thermally adapted state; it however induced full lysis of cultured cancer cells upon light irradiation. Selective irradiation also allowed isolation of individual nanopores in artificial lipid membranes. Photocontrolled FraC might find applications in photopharmacology for cancer therapeutics and has potential to be used for the fabrication of nanopore arrays in nanopore sensing devices, where the reconstitution, with high spatiotemporal precision, of single nanopores must be controlled.

Nanopores have been used to detect molecules, to sequence DNA, or to investigate chemical reactions at the single-molecule level. Because they approach the absolute limit of sensor miniaturization, nanopores are amenable to parallelization and could be used in single-cell measurements. Here we show that single enzymes can be functionally and reversibly trapped inside the confined space of a ClyA nanopore. Remarkably, the binding of ligands to the internalized proteins is mirrored by specific changes to the nanopore conductance. Conveniently, the manipulation of the charge of the protein allowed increasing of the residence time of the protein inside the nanopore. Nanopores with internalized protein adaptors can be used to study proteins in real time or can be incorporated into inexpensive portable devices for the detection of analytes with high selectivity.

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 native substrate-binding domains (SBD) of an ATP-Binding Cassette importer in real-time. Comparison with single-molecule Förster Resonance Energy Transfer (smFRET) 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.

Nanopores have recently emerged as powerful tools in single-molecule investigations. Biological nanopores, however, have drawbacks, including a fixed size and limited stability in lipid bilayers. Inspired by the great success of directed evolution approaches in tailoring enzyme properties, in this work we evolved Cytolysin A from Salmonella typhi (ClyA) to a high level of soluble expression and desired electrical properties in lipid bilayers. Evolved ClyA nanopores remained open up to -150 mV applied potential, which allowed the detailed characterization of folded proteins by ionic current recordings. Remarkably, we also found that ClyA forms several nanopore species; among which we could isolate and characterize three nanopore types most likely corresponding to the 12mer, 13mer, and 14mer oligomeric forms of ClyA. Protein current blockades to the three ClyA nanopores showed that subnanometer variations in the diameter of nanopores greatly affect the recognition of analyte proteins. ispartof: Journal of the American Chemical Society vol:135 issue:36 pages:13456-63 ispartof: location:United States status: published