Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 17, Pages 6272-6277Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0711561105
Keywords
gating; MD simulation; OmpG; stochastic sensor
Categories
Funding
- MRC [G0300122] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [B19456, BEP17032, BBS/B/16011] Funding Source: researchfish
- Medical Research Council [G0300122] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BBS/B/16011, BEP17032, B19456] Funding Source: Medline
- Medical Research Council [G0300122] Funding Source: Medline
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Bacterial outer membrane porins have a robust P-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 beta 12 and beta 13. Second, the interstrand hydrogen bonding between strands beta 11 and beta 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.
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