Staphylococcal α-hemolysin (αHL) forms a heptameric pore that features a 14-stranded transmembrane β-barrel. We attempted to force the αHL pore to adopt novel stoichiometries by oligomerizing subunit dimers generated by in vitro transcription and translation of a tandem gene. However, in vitro transcription and translation also produced truncated proteins, monomers, that were preferentially incorporated into oligomers. These oligomers were shown to be functional heptamers by single-channel recording and had a similar mobility to wild-type heptamers in SDS-polyacrylamide gels. Purified full-length subunit dimers were then prepared by using His-tagged protein. Again, single-channel recording showed that oligomers made from these dimers are functional heptamers, implying that one or more subunits are excluded from the central pore. Therefore, the αHL pore resists all structures except those that possess seven subunits immediately surrounding the central axis. Although we were not able to change the stoichiometry of the central pore of αHL by the concatenation of subunits, we extended our findings to prepare pores containing one subunit dimer and five monomers and purified them by SDS-PAGE. Two half-chelating ligands were then installed at adjacent sites, one on each subunit of the dimer. Single-channel recording showed that pores formed from this construct formed complexes with divalent metal ions in a similar fashion to pores containing two half-chelating ligands on the same subunit, confirming that the oligomers had assembled with seven subunits around the central lumen. The ability to incorporate subunit dimers into αHL pores increases the range of structures that can be obtained from engineered protein nanopores.
"Our strategy is based on designing distinguishable translocation time statistics for any given sequence by engineering the polymer-pore interactions and combining readouts from multiple pores for rapid convergence. The desired patterns in surface interaction could be achieved by using biological nanopores with appropriate modification   or those with known hydrophobic-hydrophilic pattern structure , as well as solid-state nanopores with tailormade surface interactions     . The proposed approach could potentially improve the overall speed of sequence detection by orders of magnitude, and could be integrated in high throughput microfluidic devices. "
[Show abstract][Hide abstract] ABSTRACT: The effect of the microscopic structure of a pore on polymer translocation is
studied using Langevin dynamics simulation, and the consequence of introducing
patterned stickiness inside the pore is investigated. It is found that the
translocation process is extremely sensitive to the detailed structure of such
patterns with faster than exponential dependence of translocation times on the
stickiness of the pore. The stochastic nature of the translocation process
leads to discernable differences between how polymers with different sequences
go through specifically patterned pores. This notion is utilized to propose a
stochastic sensing protocol for polynucleotides, and it is demonstrated that
the method, which would be significantly faster than the existing methods,
could be made arbitrarily robust.
Physical Review X 01/2012; 2(2). DOI:10.1103/PhysRevX.2.021002 · 9.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA.
PLoS ONE 06/2012; 7(6):e38726. DOI:10.1371/journal.pone.0038726 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A study was conducted to report about the biophysical investigations of the channel-forming bacterial toxins that provided information about the mechanisms of their toxicity and proposed new approaches to block their virulent action. Imitating nature, which created the channel-inhibiting toxins, made it possible to design the potent antitoxins specifically blocking the conductive pathways of the channel-forming toxins with an ultimate goal to defend from the cytotoxic action of these agents. All bacterial toxins were divided into two functionally different groups, such as endotoxins and exotoxins. The endotoxins were components of the outer membrane of Gram-negative bacteria. The exotoxins were proteins secreted by a number of Gram-positive and Gram-negative bacteria, which acted on eukaryotic cells far off from the host bacterium.
Chemical Reviews 10/2012; 112(12). DOI:10.1021/cr300141q · 46.57 Impact Factor
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