MspA Nanopores from Subunit Dimers

Mikhail Pavlenok,Jens H Gundlach,Ian M Derrington,Michael Niederweis,Jerome Mathe

doi:10.1371/journal.pone.0038726

Mikhail Pavlenok, Jens H Gundlach + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0038726

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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.

Highlights

Pore proteins have important transport functions in biological cells [1,2] and are versatile tools in nanotechnology which have been utilized as sensors of small molecules [3], proteins [4] and nucleic acids [5]
We present evidence that these single-chain dimers assemble to channels in the outer membrane of M. smegmatis with very similar properties compared to wt Mycobacterium smegmatis porin A (MspA) pores indicating that the overall number of eight MspA subunits was not altered in the engineered MspA
Membrane topology experiments indicated that both N- and C-termini of adjacent MspA monomers are buried in the outer membrane of M. smegmatis [17]

Summary

Introduction

Pore proteins have important transport functions in biological cells [1,2] and are versatile tools in nanotechnology which have been utilized as sensors of small molecules [3], proteins [4] and nucleic acids [5]. Most of these technological applications have been pioneered with a-hemolysin from Staphylococcus aureus. The membrane-spanning b-barrel has a length of 5 nm It has a rather broad constriction zone consisting of 3 amino acids with a diameter of

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