Bionanopore Function via In Silico Design: A Biomimetic Approach

The overall aim of this project in to use a combination of in silico (biomolecular simulation) and experimental methods to explore bionanopore function via biomimetic design. This work, which will building on previous and existing collaborations between the Bayley and Sansom research groups, is of considerable importance to rational design of novel bionanopores for use in direct, electrical detection and analysis of single molecules, the latter being the primary aim of Oxford Nanopore Technologies. Previous studies of relevance have included: 1. Simulation studies of model nanopores in lipid bilayers, focussing on protein/lipid lipid interactions and on nanopore clustering (1). 2. Combined computational and experimental study of nanopore mutations and perturbation of transmembrane beta-barrel stability. 3. In silico electrophysiology via direct simulations of ion and water fluxes through alpha-hemolysin pores. 4. Free energy profiles for porins, enabling a quantitative comparison of phosphate and chloride interactions with the OprP pore (2). 5. Design of biomimetic nanopores based on OprP, exploring the importance of sidechain flexibility in smoothing free energy landscapes for permeant ions (Pongprayoon et al., ms. in preparation). Ongoing studies include: 1. Exploration of the mechanism and energetics of porins for hydrophobic/aromatic solutes. Systems being studied include OpdK (selective for vanillate; Pongprayoon et al., ms. in preparation); and TodX (selective for toluene and its derivatives) The current project will exploit and extend these studies in a more synthetic fashion. Specifically, the project will involve: 1. Analysis of existing bionanopores: a semi-quantitative survey of the relationship between structure and function (solute specificity) in bacterial outer membrane proteins (OMPs) using electrostatics and related analytical methods based on lessons learned from the above studies. 2. De novo design of nanopore functionality: using the free energy profile and related methods developed in the studies of biomimetic nanopores based on OprP. 3. Design of existing and novel selectivity functionality into existing templates (e.g. OmpG (3), alpha-hemolysin). 4. Combine our understanding of selectivity for phosphate (OprP) and for aromatics (OpdK; TodX) to design a pore selective for DNA and related polymers. 5. Focus on minimum requirements for single stranded DNA recognition which are consistent with optimal DNA transport rates. 6. Use our understanding of OmpG and of stability of 'featureless' beta-barrels to combine these requirements with a stable nanopore template. 7. Experimental evaluation of design and refinement by combined simulation and experimental studies. References (1) Klingelhoefer, J., Carpenter, T., and Sansom, M. S. P. (2009) Peptide nanopores and lipid bilayers: Interactions by coarse-grained molecular dynamics simulations. Biophys. J. 96, 3519-3528. (2) Pongprayoon, P., Beckstein, O., Wee, C. L., and Sansom, M. S. P. (2009) Simulations of anion transport through OprP reveal the molecular basis for high affinity and selectivity for phosphate. Proc. Natl. Acad. Sci. USA 106, 21614-21618. (3) Chen, M., Khalid, S., Sansom, M. S. P., and Bayley, H. (2008) Outer membrane protein G: engineering a quiet pore for biosensing. Proc. Natl. Acad. Sci. USA 105, 6272-6277.