Outer membrane protein G: Engineering a quiet pore for biosensing.
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.
Chen
Min
M
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom.
Khalid
Syma
S
Sansom
Mark S P
MS
Bayley
Hagan
H
D000244:Adenosine Diphosphate; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D015374:Biosensing Techniques; D003198:Computer Simulation; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D015640:Ion Channel Gating; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D018272:Porins / Q000737:chemistry*; D025341:Principal Component Analysis; D011487:Protein Conformation; D015202:Protein Engineering; D013269:Stochastic Processes; D047392:beta-Cyclodextrins / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; C114347:OmpG protein, E coli; D018272:Porins; D047392:beta-Cyclodextrins; D000244:Adenosine Diphosphate; C031215:betadex