One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a β-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore. More importantly, this nanostructure maintained its stability under many experimental circumstances, some of which, including low ion concentration and highly acidic aqueous phase, are normally employed to gate, destabilize or unfold β-barrel membrane proteins. To demonstrate these advantageous traits, we show that the engineered FhuA-based protein nanopore functioned as a sensing element for examining the proteolytic activity of an enzyme at highly acidic pH and for determining the kinetics of protein-DNA aptamer interactions at physiological salt concentration.

The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust ␤-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (

5-bisphosphate carboxylase/oxygenase (RuBisCO). The challenge associated with structure and functional investigation of Rca can be attributed to its exceptionally low thermo-stability, high degree of size polydispersity and tendency toward subunit aggregation. In this work we have successfully employed fluorescence fluctuation methods to study the nucleotide-dependent stoichiometry of fluorescently tagged Rca for a wide range of concentrations. Our results show a stepwise assembly pathway of Rca under different assay conditions. In presence of Mgþ2 -ADP, the oligomerization state of Rca is largely dominated by monomers at concentrations below 0.5 mM. The state of oligomerization gradually changes in steps of two subunits. The most probable model for this assembly supports the dissociation coefficients of~4, 1, 1 mM for the monomer-dimer, dimer-tetramer and tetramer-hexamer equlibria respectively. Continued assembly at even higher concentrations suggests self association through the formation of spiral arrangements that grow along the helical axis. 2706-Pos Board B725Inferring Subunit Stoichiometry from Single Molecule Photobleaching Keegan E. Hines. University of Texas at Austin, Austin, TX, USA. Single molecule photobleaching is a powerful tool for determining the stoichiometry of protein complexes. By attaching fluorophores to proteins of interest, the number of associated subunits in a complex can be deduced by imaging single molecules and counting fluorophore photobleaching steps. Since some bleaching steps might be unobserved, the ensemble of bleaching steps will be binomially distributed and it has been commonly assumed that the highest number of observed bleaching steps is indicative of the stoichiometry of the complex. However, we point out that inferring the true composition of a complex from such data is non-trivial because binomial processes are ill-posed. As a result, there may be a significant probability that the true complex is larger than the data indicate simply due to finite sample size and the variance of binomial processes. Because of this possibility, calculating likelihoods to establish parameter confidence can be misleading. What is needed is a reliable method to quantify one's conclusions about stoichiometry. We present a Monte Carlo method which does not rely on likelihood calculation and provides a reliable estimate of confidence. The formalization and methods presented here provide a rigorous analytical basis to this pervasive experimental tool. Nanopores are fast becoming a major scientific tool in molecular analysis and detection due to their ability to detect polynucleotides, proteins, and small molecules. Previous work has included the manipulation of a-hemolysin into a DNA sequencing pore however further development is needed for the use of a simple, monomeric pore for this purpose. Biomimetic modelling of nanopores allows for a specific function to be built into the model based on the replication and analysis of existing selectivity present in proteins. An initial analysis of know...

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