MspA Nanopores from Subunit Dimers

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

Nucleotide Discrimination with DNA Immobilized in the MspA Nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices

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