Figure 1.
Schematic representation of MspA and MspA-MspB subunit dimer
MspA monomer (A) is encoded by a single copy of mspA gene. Eight monomers self assemble in the outer membrane of Mycobacterium smegmatis 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 M. smegmatis.
Table 1.
MspA dimers tested in the study.
Figure 2.
Expression of mspA-mspB fusions in M. smegmatis.
Western blot of detergent extracts of the porin mutant M. smegmatis 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 mspA); 2, empty vector pMS2; 3, pML870 (mspA-mspB17); 4, pML870-10 (mspA-mspB42); 5, pML870-6 (mspA-mspB62); 6, pML871 (mspA-mspB16LTR); 7, pML872 (mspA-mspB14TLT). Abbreviations: o, oligomeris form; d, dimeric form.
Figure 3.
Single-channel recordings and analysis of conductance of purified MspA and MspA-MspB17 dimer in lipid bilayer.
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.
Figure 4.
Expression of M1-M119 MspA on the surface of M. smegmatis and purification by gel extraction.
(A) Western blot of the samples after selective extraction of MspA proteins at 100°C from M. smegmatis ML16 cells. 15 µl of the extracts have been loaded onto 10% polyacrylamide gel followed by transfer onto the PVDF membrane, and detected with polyclonal MspA antiserum. Lanes: 1, raw extract of MspA; 2, empty vector pMS2; 3, M1 MspA; 4, M1-M1 MspA. Molecular mass marker is shown next to the lane 1. (B) Analysis of M1-M119 MspA gel extraction procedure. After selective extraction of M1-M119 MspA from M. smegmatis ML16 cells the sample was concentrated and loaded onto SDS-PAGE. The band corresponding to oligomeric form of the protein was cut from the gel and electoreluted followed by analysis for channel forming properties. After bilayer experiments the sample was loaded on the gel to monitor its stability. Lanes: 1, raw extract of wt MspA; 2, M1-M119 MspA before gel extraction and bilayer analysis; 3, M1-M119 after gel extraction and bilayer analysis. Molecular mass marker is shown next to lane 1. SDS-PAGE gel was stained with Simple Blue Safe Stain (Invitrogen). Abbreviations: o, oligomeris form; d, dimeric form; m, monomeric form.
Figure 5.
Characterization of M1 MspA and M1-M119 MspA dimer in lipid bilayer experiments.
Single-channel recordings of purified M1 MspA (A) and gel extracted M1-M119 MspA (B) in a diphytanoyl phosphatidylcholine membrane in the presence of approximately 100 ng/mL protein. 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 M1 MspA and M1-M119 MspA dimer (C and D, respectively). Analysis of the probability P of a conductance step G for single-channel events. The peak single-channel conductance for both M1 MspA and gel extracted MspA made from M1-M119 subunit dimer was 1.6 nS.
Figure 6.
Uptake of glucose by M. smegmatis ML16.
Accumulation of [14C]glucose by M. smegmatis 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.
Figure 7.
Histogram of the averaged residual ion current of single-stranded DNA homopolymers in M1 and M1-M119 MspA
Averaged Gaussian of Ires 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.