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 f...

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Main Authors: Mikhail Pavlenok, Ian M Derrington, Jens H Gundlach, Michael Niederweis
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2012-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC3377714?pdf=render
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spelling doaj-8c9a2a71ca6f4a00bde02dda66b9b44f2020-11-25T01:24:02ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0176e3872610.1371/journal.pone.0038726MspA nanopores from subunit dimers.Mikhail PavlenokIan M DerringtonJens H GundlachMichael NiederweisMycobacterium 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.http://europepmc.org/articles/PMC3377714?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Mikhail Pavlenok
Ian M Derrington
Jens H Gundlach
Michael Niederweis
spellingShingle Mikhail Pavlenok
Ian M Derrington
Jens H Gundlach
Michael Niederweis
MspA nanopores from subunit dimers.
PLoS ONE
author_facet Mikhail Pavlenok
Ian M Derrington
Jens H Gundlach
Michael Niederweis
author_sort Mikhail Pavlenok
title MspA nanopores from subunit dimers.
title_short MspA nanopores from subunit dimers.
title_full MspA nanopores from subunit dimers.
title_fullStr MspA nanopores from subunit dimers.
title_full_unstemmed MspA nanopores from subunit dimers.
title_sort mspa nanopores from subunit dimers.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2012-01-01
description 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.
url http://europepmc.org/articles/PMC3377714?pdf=render
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