Biomolecular Detection with Engineered Robust Nanopores

Abstract

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...

Interactions of a Polypeptide with a Protein Nanopore Under Crowding Conditions

“…A normalized single-channel conductance lower than 1 would only occur when PEGs partition into the pore lumen, because they decrease the conductivity of the solution with respect to PEG-free bulk phase. 19,26,[34][35][36][37][38] On the contrary, a normalized unitary conductance greater than 1 would indicate that PEGs do not partition into the pore interior, because of the enhancement in the local salt concentration within the PEG-unoccupied volume. 19,26,39 We determined that PEG-2k did not partition into the pore lumen in 300 mM KCl and at of up to 20% (w/v) ( Fig.…”