Outer membrane protein G: engineering a quiet pore for biosensing
Outer membrane protein G: engineering a quiet pore for biosensing
Bacterial outer membrane porins have a robust ?-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 ?12 and ?13. Second, the interstrand hydrogen bonding between strands ?11 and ?12 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.
nanopore sequencing technology, constriction loop, ompg, gating, noncovalent molecular adapter, md simulation, spontaneous gating activity, ion-channel, ompc porin, beta-cyclodextrin, stochastic sensor, monomeric porin, escherichia-coli porin, simulations
6272-6277
Chen, Min
8ba2b581-ac04-4528-bbfe-7daf7f18bd75
Khalid, Syma
90fbd954-7248-4f47-9525-4d6af9636394
Sansom, Mark S.P.
ed30b4fc-bc73-4ad7-8c56-f51a67136e4e
Bayley, Hagan
929363fb-8539-43de-9d86-153ceb26e03e
29 April 2008
Chen, Min
8ba2b581-ac04-4528-bbfe-7daf7f18bd75
Khalid, Syma
90fbd954-7248-4f47-9525-4d6af9636394
Sansom, Mark S.P.
ed30b4fc-bc73-4ad7-8c56-f51a67136e4e
Bayley, Hagan
929363fb-8539-43de-9d86-153ceb26e03e
Chen, Min, Khalid, Syma, Sansom, Mark S.P. and Bayley, Hagan
(2008)
Outer membrane protein G: engineering a quiet pore for biosensing.
Proceedings of the National Academy of Sciences of the United States of America, 105 (17), .
(doi:10.1073/pnas.0711561105).
Abstract
Bacterial outer membrane porins have a robust ?-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 ?12 and ?13. Second, the interstrand hydrogen bonding between strands ?11 and ?12 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.
Text
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More information
Published date: 29 April 2008
Keywords:
nanopore sequencing technology, constriction loop, ompg, gating, noncovalent molecular adapter, md simulation, spontaneous gating activity, ion-channel, ompc porin, beta-cyclodextrin, stochastic sensor, monomeric porin, escherichia-coli porin, simulations
Organisations:
Chemistry, Computational Systems Chemistry
Identifiers
Local EPrints ID: 54435
URI: http://eprints.soton.ac.uk/id/eprint/54435
ISSN: 0027-8424
PURE UUID: 95c3b5d6-1059-43f7-81c7-0a9126b1ca2e
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Date deposited: 05 Aug 2008
Last modified: 16 Mar 2024 03:56
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Contributors
Author:
Min Chen
Author:
Syma Khalid
Author:
Mark S.P. Sansom
Author:
Hagan Bayley
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