The detection of single molecules such as singlestranded DNA (ssDNA) and other small molecules through biological nanopores is a powerful approach for analyzing DNA sequences and DNA shapes, as well as for disease diagnostics. The fixed diameter of biological nanopores restricts the size of biomolecules translocated through them. Although some nanopores such as ClyA, FraC, Phi29p, and γ-hemolysin have been shown to translocate double-stranded DNA (dsDNA), identifying the difference between dsDNA and three-way junction DNA is difficult using these native biological nanopores. OmpG, a major outer membrane protein, forms a nanosized pore with 14 β-strands.Here, we create a modified OmpG that expands and truncates βhairpins, allowing the generation of small or large nanopores compared to that of wild-type (WT) OmpG nanopores. To determine the pore diameters of modified OmpGs, the change in the current amplitude of the various modified OmpGs was measured in the presence or absence of poly(ethylene glycol) at different molecular weights. Finally, we demonstrated the detection of various structures of DNA (branched DNA) depending on the nanopore size using OmpG WT or mutated OmpG nanopores. Insights into the changes in pore diameters will be crucial to form precise pore diameters for the detection of various types of single biomolecules and for sequencing DNA, peptides, and proteins.

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

The development of stimuli−responsive synthetic channels that open and close in response to physical and chemical changes in the surrounding environment has attracted attention because of their potential bioapplications such as sensing, drug release, antibiotics, and molecular manipulation tools to control membrane transport in cells. Metal coordination is ideal as a stimulus for stimuli−responsive channels because it allows for reversible gating behavior through the addition and removal of metal ions and fine-tuning of channel structure through coordination geometry defined by the type of the metal ion and ligand. We have previously reported on transition metal-ion dependent ion permeability control of Amphotericin B (AmB) modified with a metal coordination site, 2,2′-bipyridine ligand (bpy-AmB). AmB is one of the polyene macrolide antibiotics, and it is known that the interaction between AmB and ergosterol molecules is required for AmB channel formation. In contrast, the Cu 2+ coordination to the bpy moiety of bpy-AmB induces formation of Ca 2+ ion-permeable channels in the ergosterol-free POPC membrane. However, the details of bpy-AmB properties such as channel stability, ion selectivity, pore size, and the effect of ergosterol on channel formation remain unclear. Here, we investigate bpy-AmB channels triggered by transition metal coordination in POPC or ergosterol-containing POPC liposomes using an HPTS assay, electrophysiological measurements, and time-resolved UV−vis spectral measurements. These analyses reveal that bpy-AmB channels triggered by Cu 2+ ions are more stable and have larger pore sizes than the original AmB channels and enable efficient permeation of various cations. We believe that our channel design will lead to the construction of metal coordination-triggered synthetic ion channels.