MspA Nanopores from Subunit Dimers

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.

BROMOC suite: Monte Carlo/Brownian dynamics suite for studies of ion permeation and DNA transport in biological and artificial pores with effective potentials

“…K + is the main current carrier with ∼10:1 ratio to Cl − . The result is in agreement with recently reported experimental and computational work . The developed method shows a clear separation of the blocked current between A and C with poly(dA), allowing for considerably higher residual currents, which are in excellent accord with experimental data reported for the low‐conductance state of MspA.…”

“…This may raise some concerns regarding the usefulness of the crystal structure for simulations of ss‐DNA translocations. Experimentally most of the residual currents for ssDNA sequencing are reported for the selected states with G ∼ 1.6 to 2.2 nS …”

Properties and Phylogeny of 76 Families of Bacterial and Eukaryotic Organellar Outer Membrane Pore-Forming Proteins

“…Acid fast Gram-positive Actinobacteria have OMPPs of two types, β-barrel and α-helical, in their outer membranes (see Table 1 and TCDB [ 120 , 121 ]). Two of these OMPP families, the Mycobacterial OMPP (MBP or MspA) Family (1.B.24) and the Nocardial Heterooligomeric Cell Wall Channel (NfpA/B) Family (1.B.58) are believed to consist of β-barrels and comprise Superfamily II [ 122 , 123 ]. These two families include proteins, most of which are of 200–290 aas with a single N-terminal α-helical TMS followed by a proposed β-barrel OMPP-type structure.…”

Single molecule observation of hard–soft-acid–base (HSAB) interaction in engineeredMycobacterium smegmatisporin A (MspA) nanopores

“…Alternatively, all eight subunits of MspA may be genetically chained into a single unit. 41 However, difficulties in pore oligomerization or an extremely low expression yield may be encountered. The monomeric OmpG nanopore 42,43 may be promising, despite the fact that its pore geometry may or may not be sensitive enough to report a single ion binding event.…”