Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quiet Outer Membrane Protein G (OmpG) Nanopore for Biosensing.

ABSTRACT: Interest in nanopore technology has been growing due to nanopores' unique capabilities in small molecule sensing, measurement of protein folding, and low-cost DNA and RNA sequencing. The E. coli ?-barrel outer membrane protein OmpG is an excellent alternative to other protein nanopores because of its single polypeptide chain. However, the flexibility of its extracellular loops ultimately limits applications in traditional biosensing. We deleted several residues in and near loop 6 of OmpG. The dynamic structure of the new construct determined by NMR shows that loops 1, 2, 6, and 7 have reduced flexibilities compared to those of wild-type. Electrophysiological measurements show that the new design virtually eliminates flickering between open and closed states across a wide pH range. Modification of the pore lumen with a copper chelating moiety facilitates detection of small molecules. As proof of concept, we demonstrate concurrent single-molecule biosensing of glutamate and adenosine triphosphate.

SUBMITTER: Sanganna Gari RR

PROVIDER: S-EPMC6596296 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Quiet Outer Membrane Protein G (OmpG) Nanopore for Biosensing.

Sanganna Gari Raghavendar Reddy RR Seelheim Patrick P Liang Binyong B Tamm Lukas K LK

ACS sensors 20190425 5

Interest in nanopore technology has been growing due to nanopores' unique capabilities in small molecule sensing, measurement of protein folding, and low-cost DNA and RNA sequencing. The E. coli β-barrel outer membrane protein OmpG is an excellent alternative to other protein nanopores because of its single polypeptide chain. However, the flexibility of its extracellular loops ultimately limits applications in traditional biosensing. We deleted several residues in and near loop 6 of OmpG. The dy ...[more]

PMID: 30990011

Similar Datasets

Outer membrane protein G: Engineering a quiet pore for biosensing.

Project description:Bacterial outer membrane porins have a robust beta-barrel structure and therefore show potential for use as stochastic sensors based on single-molecule detection. The monomeric porin OmpG is especially attractive compared with multisubunit proteins because appropriate modifications of the pore can be easily achieved by mutagenesis. However, the gating of OmpG causes transient current blockades in single-channel recordings that would interfere with analyte detection. To eliminate this spontaneous gating activity, we used molecular dynamics simulations to identify regions of OmpG implicated in the gating. Based on our findings, two approaches were used to enhance the stability of the open conformation by site-directed mutagenesis. First, the mobility of loop 6 was reduced by introducing a disulfide bond between the extracellular ends of strands beta12 and beta13. Second, the interstrand hydrogen bonding between strands beta11 and beta12 was optimized by deletion of residue D215. The OmpG porin with both stabilizing mutations exhibited a 95% reduction in gating activity. We used this mutant for the detection of adenosine diphosphate at the single-molecule level, after equipping the porin with a cyclodextrin molecular adapter, thereby demonstrating its potential for use in stochastic sensing applications.

| S-EPMC2359795 | biostudies-literature

Membrane protein biosensing with plasmonic nanopore arrays and pore-spanning lipid membranes.

Project description:Integration of solid-state biosensors and lipid bilayer membranes is important for membrane protein research and drug discovery. In these sensors, it is critical that the solid-state sensing material does not have adverse effects on the conformation or functionality of membrane-bound molecules. In this work, pore-spanning lipid membranes are formed over an array of periodic nanopores in free-standing gold films for surface plasmon resonance (SPR) kinetic binding assays. The ability to perform kinetic assays with a transmembrane protein is demonstrated with α-hemolysin (α-HL). The incorporation of α-HL into the membrane followed by specific antibody binding (anti-α-HL) red-shifts the plasmon resonance of the gold nanopore array, which is optically monitored in real time. Subsequent fluorescence imaging reveals that the antibodies primarily bind in nanopore regions, indicating that α-HL incorporation preferentially occurs into areas of pore-spanning lipid membranes.

| S-EPMC3015192 | biostudies-literature

Selective Detection of Protein Homologues in Serum Using an OmpG Nanopore.

Project description:Outer membrane protein G is a monomeric β-barrel porin that has seven flexible loops on its extracellular side. Conformational changes of these labile loops induce gating spikes in current recordings that we exploited as the prime sensing element for protein detection. The gating characteristics, open probability, frequency, and current decrease, provide rich information for analyte identification. Here, we show that two antibiotin antibodies each induced a distinct gating pattern, which allowed them to be readily detected and simultaneously discriminated by a single OmpG nanopore in the presence of fetal bovine serum. Our results demonstrate the feasibility of directly profiling proteins in real-world samples with minimal or no sample pretreatment.

| S-EPMC5065927 | biostudies-literature

Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms.

Project description:The flexible loops decorating the entrance of OmpG nanopore move dynamically during ionic current recording. The gating caused by these flexible loops changes when a target protein is bound. The gating is characterized by parameters including frequency, duration, and open-pore current, and these features combine to reveal the identity of a specific analyte protein. Here, we show that OmpG nanopore equipped with a biotin ligand can distinguish glycosylated and deglycosylated isoforms of avidin by their differences in surface charge. Our studies demonstrate that the direct interaction between the nanopore and analyte surface, induced by the electrostatic attraction between the two molecules, is essential for protein isoform detection. Our technique is remarkably sensitive to the analyte surface, which may provide a useful tool for glycoprotein profiling.

| S-EPMC4958047 | biostudies-literature

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single-Molecule Multiplex Protein Detection.

Project description:Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate single-molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.

| S-EPMC9898208 | biostudies-literature

A pH-independent quiet OmpG pore with enhanced electrostatic repulsion among the extracellular loops.

Project description:Membrane protein pores have emerged as powerful nanopore sensors for single-molecule detection. OmpG, a monomeric nanopore, is comprised of fourteen β-strands connected by seven flexible extracellular loops. The OmpG nanopore exhibits pH-dependent gating as revealed by planar lipid bilayer studies. Current evidence strongly suggests that the dynamic movement of loop 6 is responsible for the gating mechanism. In this work, we have shown that enhancing the electrostatic repulsion forces between extracellular loops suppressed the pH-dependent gating. Our mutant containing additional negative charges in loop 6 and loop 1 exhibited minimal spontaneous gating and reduced sensitivity to pH changes compared to the wild type OmpG. These results provide new evidence to support the mechanism of OmpG gating controlled by the complex electrostatic network around the gating loop 6. The pH-independent quiet OmpG pores could potentially be used as a sensing platform that operates at a broad range of pH conditions.

| S-EPMC7704735 | biostudies-literature

Tuning Protein Discrimination Through Altering the Sampling Interface Formed between the Analyte and the OmpG Nanopore.

Project description:Nanopore sensors capable of distinguishing homologous protein analytes are highly desirable tools for proteomics research and disease diagnostics. Recently, an engineered outer membrane protein G (OmpG) nanopore with a high-affinity ligand attached to a gating loop 6 showed specificity for distinguishing homologous proteins in complex mixtures. Here, we report the development of OmpG nanopores with the other six loops used as the anchoring point to host an affinity ligand for protein sensing. We investigated how the analyte binding to the affinity ligand located at different loops affects the detection sensitivity, selectivity, and specificity. Our results reveal that analytes weakly attracted to the OmpG nanopore surface are only detectable when the ligand is tethered to loop 6. In contrast, protein analytes that form a strong interaction with the OmpG surface via electrostatic attractions are distinguishable by all seven OmpG nanopore constructs. In addition, the same analyte can generate distinct binding signals with different OmpG nanopore constructs. The ability to exploit all seven OmpG loops will aid the design of a new generation of OmpG sensors with increased sensitivity, selectivity, and specificity for biomarker sensing.

| S-EPMC8500544 | biostudies-literature

Outer membrane protein evolution.

Project description:Outer membrane proteins have remarkably homogeneous structure. They are all up down β-barrels. Up down barrels themselves are composed of repeated sets of β-hairpins. The consistency of the usage of the β-hairpin throughout the outer membrane milieu allows for interrogation of the evolution of these repetitive structures. Here we describe recent investigations of outer membrane protein evolution and how evolutionary precepts have been used for novel outer membrane protein design.

| S-EPMC8222147 | biostudies-literature

Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies.

Project description:Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. Whereas solubilized membrane proteins lack the functionally important lipid membrane, reconstitution embeds them into artificial lipid bilayers, which lack characteristic features of cellular membranes including lipid diversity, composition and asymmetry. Here, we utilize outer membrane vesicles (OMVs) released from Escherichia coli to study outer membrane proteins (Omps) in the native membrane environment. Enriched in the native membrane of the OMV we characterize the assembly, folding, and structure of OmpG, FhuA, Tsx, and BamA. Comparing Omps in OMVs to those reconstituted into artificial lipid membranes, we observe different unfolding pathways for some Omps. This observation highlights the importance of the native membrane environment to maintain the native structure and function relationship of Omps. Our fast and easy approach paves the way for functional and structural studies of Omps in the native membrane.

| S-EPMC6123736 | biostudies-literature

Modifying the pH sensitivity of OmpG nanopore for improved detection at acidic pH.

Project description:The outer membrane protein G (OmpG) nanopore is a monomeric β-barrel channel consisting of seven flexible extracellular loops. Its most flexible loop, loop 6, can be used to host high-affinity binding ligands for the capture of protein analytes, which induces characteristic current patterns for protein identification. At acidic pH, the ability of OmpG to detect protein analytes is hampered by its tendency toward the closed state, which renders the nanopore unable to reveal current signal changes induced by bound analytes. In this work, critical residues that control the pH-dependent gating of loop 6 were identified, and an OmpG nanopore that can stay predominantly open at a broad range of pHs was created by mutating these pH-sensitive residues. A short single-stranded DNA was chemically tethered to the pH-insensitive OmpG to demonstrate the utility of the OmpG nanopore for sensing complementary DNA and a DNA binding protein at an acidic pH.

| S-EPMC8943698 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data