Modified Outer Membrane Protein-G Nanopores with Expanded and Truncated β-Hairpins for Recognition of Double-Stranded DNA

Tosaka, Toshiyuki; Kamiya, Kanichi

doi:10.1021/acsanm.1c04417

Cited by 5 publications

(13 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To investigate K + transport properties of a single bpy-AmB channel in the presence of transition metal ions (Cu 2+ , Co 2+ , or Ni 2+ ions), we recorded a single current signal of bpy-AmB in the planar lipid bilayer using an on-chip lipid bilayer system. , First, to confirm channel formation of bpy-AmB, the single current signal of bpy-AmB on a planar DPhPC bilayer containing 5 mol % ergosterol (5 mol % Ergo/DPhPC) was observed in 3 M KCl containing 2.5 mM HEPES and 1 mM CaCl 2 (pH 9.0) at +150 mV. Figure S5a shows an open-close signal of bpy-AmB.…”

Section: Resultsmentioning

confidence: 99%

Cu(II)-Triggered Ion Channel Properties of a 2,2′-Bipyridine-Modified Amphotericin B

Komaki

Kasuya

Toda

et al. 2023

ACS Appl. Bio Mater.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Cu(II)-Triggered Ion Channel Properties of a 2,2′-Bipyridine-Modified Amphotericin B

Komaki

Kasuya

Toda

et al. 2023

ACS Appl. Bio Mater.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, the conversion of the pore diameter has been reported by duplicating or replacing one or several β-hairpins of β−barrel nanopores, such as LamB, FhuA, and OmpX [ 139 , 140 , 141 ]. OmpG mutants with different numbers of β-hairpins showed that the pore diameter and ion current amplitude depend on the number of β-hairpins and the balance of charged amino acids on the inner surface of the OmpG nanopore [ 142 ]. Additionally, the R. Kawano group designed de novo nanopores formed by β−barrels and investigated the formation mechanism and ion transport properties of the nanopore using BLM systems and MD simulation [ 143 ].…”

Section: Single-channel Recording Using Planar Bilayer Lipid Membrane...mentioning

confidence: 99%

“…In 2016, the application of this technique was commercialized as a DNA sequencer by Oxford Nanopore Technologies. Single-molecule detection techniques, such as DNA, RNA, and small organic molecules, have been reported using aerolysin (AeL) [ 145 , 146 , 147 ], fragaceatoxin C (FraC) [ 148 , 149 ], OmpG [ 142 , 150 ], and FhuA [ 151 , 152 ] ( Figure 6 a–d). Recently, the development of peptide sequencing techniques using protein nanopores, such as AeL, Mycobacterium smegmatis porin A (MspA), and proteasome-nanopore, has been reported [ 153 , 154 , 155 ].…”

Section: Single-channel Recording Using Planar Bilayer Lipid Membrane...mentioning

confidence: 99%

“…( b ) Peptides are pre-hydrolyzed by a specific protease and the resulting peptides are measured as they translocate the FraC nanopore [ 148 ] *. ( c ) Detection of various structure of DNA depending on the nanopore size using OmpG WT or mutated OmpG with different number of β−hairpins [ 142 ] *. ( d ) Detection of the protein analyze outside the FhuA nanopore by means of tethered protein bait [ 151 ] *.…”

Section: Figurementioning

confidence: 99%

See 1 more Smart Citation

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes

Tosaka

Kamiya

2023

IJMS

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Proteo-GUVs prepared using our method contain buffer and lipid components that are suitable for the functions of each membrane protein. In this study, we sought to evaluate molecular transport on GUVs and a bilayer lipid membrane (BLM) integrated with a β-barrel porin and outer membrane protein G (OmpG), which is applied to biological sensors for the detection of small molecules, such as peptides and adenosine triphosphate. − To the best of our knowledge, the formation of OmpG-GUV has not been reported. The reconstitution of Omps into GUV is difficult because the folding of Omps in the cell membrane is catalyzed by the β-barrel assembly machinery complex in Gram-negative bacteria. , Our reconstitution method can be one of the proteo-GUV formation methods in Omps that are difficult to reconstitute into GUV by other methods.…”

Section: Introductionmentioning

confidence: 99%

Formation of Cell-Sized Liposomes Incorporating a β-Barrel-Structured Porin through Rehydration of a Phospholipid-Membrane Protein Dried Film

Tosaka,

Kamiya

2024

ACS Omega

Self Cite

View full text Add to dashboard Cite

Giant unilamellar vesicles (GUVs) integrated with membrane proteins (proteo-GUVs) are attractive tools for visualizing membrane protein functions such as enzyme reactions and molecular transportation. In the dehydration−rehydration method, one of the methods used to form proteo-GUVs, they are formed by using a dried film containing phospholipids and membrane proteins through rehydration with an alternating current electric field and a supporting gel. However, these methods make it difficult to form proteo-GUVs under physiological salt concentration and charged phospholipid conditions or carry the risk of gel contamination of lipid membranes. Therefore, proteo-GUVs formed by these rehydration methods may be harmful to membrane proteins. Here, we propose a method for the formation of proteo-GUVs containing physiological salt concentrations and negatively charged phospholipids that do not require an electric field and a supporting gel. To investigate the molecular transport of modified outer membrane protein G (OmpG), OmpG-giant unilamellar vesicles (GUVs) and OmpG-large unilamellar vesicles (LUVs) were formed. The structure and function of different mutants reconstituted into LUVs were evaluated by using circular dichroism spectroscopy and electrophysiological measurements. In addition, the molecular transport of OmpG in GUVs was evaluated by monitoring the Ca 2+ influx into GUVs and fluorescent molecule leakage from GUVs through OmpG nanopores. We found that the amount of Ca 2+ influx into GUVs through the OmpG nanopores depended on the pore size of OmpG. Our method for forming proteo-GUVs can be applied for the functional evaluation of β-barrel porin and in biological sensors using β-barrel porin.

show abstract

Modified Outer Membrane Protein-G Nanopores with Expanded and Truncated β-Hairpins for Recognition of Double-Stranded DNA

Cited by 5 publications

References 50 publications

Cu(II)-Triggered Ion Channel Properties of a 2,2′-Bipyridine-Modified Amphotericin B

Cu(II)-Triggered Ion Channel Properties of a 2,2′-Bipyridine-Modified Amphotericin B

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes

Formation of Cell-Sized Liposomes Incorporating a β-Barrel-Structured Porin through Rehydration of a Phospholipid-Membrane Protein Dried Film

Contact Info

Product

Resources

About