Outer membrane protein G: Engineering a quiet pore for biosensing

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.gating ͉ MD simulation ͉ OmpG ͉ stochastic sensor E ngineered protein pores can be used as stochastic sensors for single-molecule detection (1). The ionic current flowing through a pore under an applied potential is altered when an analyte binds within the lumen (1). Measurement of the frequency of occurrence of the binding events allows the determination of the concentration of an analyte, while the nature of the events (e.g., their amplitude or duration) aids in analyte identification. To date, studies of proteinaceous stochastic sensing elements have mostly focused on staphylococcal ␣-hemolysin (␣HL), a ␤-barrel pore-forming toxin (1, 2). However, the stoichiometry and symmetry of the heptameric ␣HL pore generate a large number of combinations and permutations when more than one type of subunit is used (3), which makes it difficult to fine-tune the properties of the pore. Therefore, it would be highly desirable to design a stochastic sensor based on a monomeric ␤-barrel.OmpG is a monomeric porin from the outer membrane of Escherichia coli that presents an attractive alternative to ␣HL for stochastic sensing (4-7). Previous studies have shown that OmpG undergoes pH-dependent, voltage-dependent, and spontaneous gating (4, 5). Specifically, the pore tends to close when the pH decreases to below 7 or when the voltage is higher than Ϯ100 mV (5). At neutral pH and an applied potential lower than Ϯ100 mV, OmpG exhibits a spontaneous gating behavior-i.e., it rapidly switches between open and closed states (Fig. 1A). Such spontaneous gating interferes with the application of a pore as a biosensor. Therefore, eliminating or significantly reducing the intrinsic gating activity of OmpG would be an important step in the development of an al...

Engineering channels: Atomic biology

“…The results of Chen et al (10) are already an important advance in stochastic sensing and will become even more so once ompG and cyclodextrin adapters become widely used in many laboratories. Just as interesting to me are the implications of the clever tools used by Bayley's laboratory in this study, their limitations, and their possible extensions.…”

“…ompG, however, has natural gating that interferes with its use. In this issue of PNAS, Chen et al (10) address that issue with a clever combination of molecular dynamics and protein engineering. They show how mutations that change flexibility of parts of the protein can greatly reduce natural gating.…”

“…Just as importantly, Chen et al (10) show that a cyclodextrin molecular adapter can be used with ompG to allow chemical engineering of its properties as a stochastic sensor. They use a specific cyclodextrin to make ompF respond to ADP, although they do not investigate (in this first article on the molecule) the specificity of that sensing.…”

“…Chen et al (10) use molecular dynamics in a most successful way, following the work of Sansom's group on porins (18) to motivate molecular design. Investigating the flexibility of the ompG molecule with principal component analysis, Chen et al identify promising sites for mutations, make two distinct mutations, combine them, and achieve the desired result.…”

Carbon nanotube-based nano-fluidic devices

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