Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif

2022

Sci Rep

Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a β-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from Escherichia coli (E. coli). Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes with various lipid compositions was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.

“…4 a,b]. This amplitude was similar to that previously reported for OmpG channels in the BLM system 23 . This open-close signal shape is specific to OmpG.…”

Section: Resultssupporting

confidence: 88%

Formation and function of OmpG or OmpA-incorporated liposomes using an in vitro translation system

2022

Sci Rep

“…We obtained an OmpG current amplitude of approximately 150 pA at +100 mV with 1 M KCl [Figures 4 (a) and (b)]. This amplitude was similar to that previously reported for OmpG channels in the BLM system 22 . This openclose signal shape is speci c to OmpG.…”

Section: Electrophysiological Measurement Of Ompg On the Nano-sized Liposomessupporting

confidence: 88%

Formation and function of bacterial outer membrane proteins- incorporated liposomes using an in vitro translation system

2021

Preprint

Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a β-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from E. coli.. Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes, with various lipid compositions, was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.

“…Therefore, Omp nanopores have a constant diameter, and Omp nanopores with different numbers of β-strands have different pore diameters. The functions and structures of nanopores with β-barrel structures have been investigated to understand the biophysical and biochemical aspects of these nanopores. − To enable single-molecule detection of various types of molecules in terms of size, surface charge, and shape using biological nanopores, the structures of biological nanopores with β-barrel structures have been modified by changing the pore diameter, pore selectivity, and pore length. − For instance, the pore size is changed by duplicating or replacing one or several β-hairpins. Using this method, the pore sizes of LamB, FhuA, and OmpX have been changed.…”

Section: Introductionmentioning

confidence: 99%

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

Tosaka

2022

ACS Appl. Nano Mater.

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.