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BIOLOGICAL SCIENCES / BIOPHYSICS
Outer membrane protein G: Engineering a quiet pore for biosensing
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*Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom; and Department of Biochemistry and Oxford Centre for Integrative Systems Biology, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
Edited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved February 20, 2008 (received for review December 7, 2007)
Bacterial outer membrane porins have a robust β-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 β12 and β13. Second, the interstrand hydrogen bonding between strands β11 and β12 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.
gating | MD simulation | OmpG | stochastic sensor
Present address: School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, United Kingdom.
The authors declare no conflict of interest.
See Commentary on page 6211.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0711561105/DCSupplemental.
To whom correspondence may be addressed. E-mail: mark.sansom{at}bioch.ox.ac.uk or hagan.bayley{at}chem.ox.ac.uk
© 2008 by The National Academy of Sciences of the USA
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  B. Eisenberg Engineering channels: Atomic biology PNAS, April 29, 2008; 105(17): 6211 - 6212. [Full Text] [PDF]
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