MspA dimers tested in the study.

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

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

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

Investigating asymmetric salt profiles for nanopore DNA sequencing with biological porin MspA

<div><p>Nanopore DNA sequencing is a promising single-molecule analysis technology. This technique relies on a DNA motor enzyme to control movement of DNA precisely through a nanopore. Specific experimental buffer conditions are required based on the preferred operating conditions of the DNA motor enzyme. While many DNA motor enzymes typically operate in salt concentrations under 100 mM, salt concentration simultaneously affects signal and noise magnitude as well as DNA capture rate in nanopore sequencing, limiting standard experimental conditions to salt concentrations greater than ~100 mM in order to maintain adequate resolution and experimental throughput. We evaluated the signal contribution from ions on both sides of the membrane (<i>cis</i> and <i>trans</i>) by varying <i>cis</i> and <i>trans</i> [KCl] independently during phi29 DNA Polymerase-controlled translocation of DNA through the biological porin MspA. Our studies reveal that during DNA translocation, the negatively charged DNA increases cation selectivity through MspA with the majority of current produced by the flow of K⁺ ions from <i>trans</i> to <i>cis</i>. Varying <i>trans</i> [K⁺] has dramatic effects on the signal magnitude, whereas changing <i>cis</i> [Cl^-] produces only small effects. Good signal-to-noise can be maintained with <i>cis</i> [Cl^-] as small as 20 mM, if the concentration of KCl on the <i>trans</i> side is kept high. These results demonstrate the potential of using salt-sensitive motor enzymes (helicases, polymerases, recombinases) in nanopore systems and offer a guide for selecting buffer conditions in future experiments to simultaneously optimize signal, throughput, and enzyme activity.</p></div

Effect of <i>cis</i> and <i>trans</i> [KCl] on DNA capture rate by MspA.

<p>DNA capture rate, the number of DNA molecules threading through MspA per second, was measured using short hairpin DNA (500 nM) over a range of <i>cis</i> [KCl] at three <i>trans</i> [KCl] with an applied voltage of 180 mV. No phi29 DNAP enzyme was included in this set of experiments. Trend lines are to guide the eye. Errors are S.E.M.</p

Basic nanopore sensing and sequencing schematic.

<p>(A) A single MspA channel is inserted into a lipid bilayer membrane. Positive (blue) K⁺ ions and negative (red) Cl^- ions are contained on either side of the membrane. An applied electric field drives K⁺ ions from the <i>trans</i> chamber to the <i>cis</i> chamber and Cl^- ions from <i>cis</i> to <i>trans</i> through MspA, producing the unblocked pore current. The region shaded in red at the base of the pore marks the constriction zone of MspA. (B) A motor enzyme controls the translocation of single stranded DNA through MspA. In the schematic depicted here, the motor enzyme translocates and unwinds double stranded DNA (dsDNA), allowing the passage of the ssDNA further into the pore with each step. The flow of both K⁺ and Cl^- ions is modulated by the presence of the DNA within the pore.</p