MspA dimers tested in the study.

*<p>- all constructs were expressed in <i>M. smegmatis</i> ML16.</p

Expression of <i>mspA-mspB</i> fusions in <i>M. smegmatis.</i>

<p>Western blot of detergent extracts of the porin mutant <i>M. smegmatis</i> ML16 expressing different MspA constructs. 15 µl of the extracts were loaded onto 10% polyacrylamide gel followed by transfer onto a PVDF membrane and detection with a polyclonal MspA antiserum. Lanes: M, molecular mass marker; 1, pMN016 (wt <i>mspA</i>); 2, empty vector pMS2; 3, pML870 (<i>mspA-mspB₁₇</i>); 4, pML870-10 (<i>mspA-mspB₄₂</i>); 5, pML870-6 (<i>mspA-mspB₆₂</i>); 6, pML871 (<i>mspA-mspB_16LTR</i>); 7, pML872 (<i>mspA-mspB_14TLT</i>). Abbreviations: o, oligomeris form; d, dimeric form.</p

Histogram of the averaged residual ion current of single-stranded DNA homopolymers in M1 and M1-M1₁₉ MspA

<p>Averaged Gaussian of I_res of M1 MspA (A) and M1-M1₁₉ MspA (B) of ssDNA hairpins with homopolymeric poly-dA or poly-dC tails are shown. Data were recorded at 180 mV transmembrane potential. The data represent an average of four independent experiments.</p

Single-channel recordings and analysis of conductance of purified MspA and MspA-MspB₁₇ dimer in lipid bilayer.

<p>Single-channel recordings of purified wt MspA (A) and MspA-MspB₁₇ dimer (C) in a diphytanoyl phosphatidylcholine (DphPC) membrane in the presence of approximately 100 ng/mL protein sample. Protein solutions were added to both sides of the membrane and data were collected from at least five different membranes. −10 mV transmembrane potential was applied and current was measured in 1 M KCl solution, pH 7.0 Analysis of single-channel conductances of wtMspA (B) and MspA-MspB₁₇ dimer (D). To avoid possible contamination of the MspA-MspB₁₇ preparation with MspB, the subunit dimer protein was excised from the gel and electro-eluted. Analysis of the probability P of a conductance step G for single-channel events. The average single-channel conductances were 4.8 nS, and 2.2 nS for wt MspA, and MspA-MspB₁₇ dimer, respectively.</p

Schematic representation of MspA and MspA-MspB subunit dimer

<p>MspA monomer (A) is encoded by a single copy of <i>mspA</i> gene. Eight monomers self assemble in the outer membrane of <i>Mycobacterium smegmatis</i> to form a functional pore (B and C, top and side view, respectively). MspA-MspB dimer (D) is connected by a (GGGGS)₃ linker via C-terminal asparagine of MspA subunit (shown in red) and N-terminal glycine (in green) of MspB subunit. MspA-MspB dimer (E and F, top and side view, respectively) form a channel in the outer membrane of <i>M. smegmatis.</i></p

Uptake of glucose by <i>M. smegmatis</i> ML16.

<p>Accumulation of [¹⁴C]glucose by <i>M. smegmatis</i> ML16 expressing wt MspA, empty vector pMS2, M1 MspA, and M1-M1₁₉ MspA was measured. The experiments were done in triplicates. The data are shown as averages ± standard deviations. The assay was performed at 37°C at a final glucose concentration of 1 µM. The cells were grown to an A₆₀₀ ∼0.6. At indicated time points 200 µL of cells were drawn from a vial, applied on a 0.22 µm cellulose filter, washed several times with LiCl, and counted on a scintillation counter. Dashed lines represent regression analysis of the first three data points for each strain. Uptake rates for ML16 expressing wt MspA, empty vector, M1 MspA, and M1-M1₁₉ MspA were 0.42, 0.01, 0.44, 0.24 nmol/mg cells/min, respectively.</p

Molecular Dynamics Study of MspA Arginine Mutants Predicts Slow DNA Translocations and Ion Current Blockades Indicative of DNA Sequence

The protein nanopore <i>Mycobacteria smegmatis</i> porin A (MspA), can be used to sense individual nucleotides within DNA, potentially enabling a technique known as nanopore sequencing. In this technique, single-stranded DNA electrophoretically moves through the nanopore and results in an ionic current that is nucleotide-specific. However, with a high transport velocity of the DNA within the nanopore, the ionic current cannot be used to distinguish signals within noise. Through extensive (∼100 μs in total) all-atom molecular dynamics simulations, we examine the effect of positively charged residues on DNA translocation rate and the ionic current blockades in MspA. Simulation of several arginine mutations show a ∼10–30 fold reduction of DNA translocation speed without eliminating the nucleotide induced current blockages. Comparison of our results with similar engineering efforts on a different nanopore (α-hemolysin) reveals a nontrivial effect of nanopore geometry on the ionic current blockades in mutant nanopores