Characterization of Nanostructured Surfaces Generated by Reconstitution of the Porin MspA from <i>Mycobacterium smegmatis</i>

Wörner, Michael; Lioubashevski, Oleg; Basel, Matthew T.; Niebler, Sandra; Gogritchiani, Eliso; Egner, Nicole; Heinz, Christian; Hoferer, Jürgen; Cipolloni, Michela; Janik, Katharine; Katz, Evgeny; Braun, André M.; Willner, Itamar; Niederweis, Michael; Bossmann, Stefan H.

doi:10.1002/smll.200600559

Cited by 21 publications

(22 citation statements)

References 66 publications

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“…The structure has proven to be of immense value not only as a paradigm for a new class of proteins, but also for understanding the function of MspA [50], for elucidating its membrane topology [12], and for applications in nanotechnology [51-53]. The porin has an octameric goblet-like conformation with a single central channel 10 nm in length (Figure 3).…”

Section: Structure Of Mycobacterial Outer Membrane Proteinsmentioning

confidence: 99%

Mycobacterial outer membranes: in search of proteins

Niederweis

Danilchanka

Huff

et al. 2010

Trends in Microbiology

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SummaryThe cell wall is a major virulence factor of Mycobacterium tuberculosis and contributes to its intrinsic drug resistance. Recently, cryo-electron microscopy showed that the mycobacterial cell wall lipids form an unusual outer membrane. Identification of the components of the uptake and secretion machinery across this membrane is critical for understanding the physiology and pathogenicity of M. tuberculosis and for the development of better anti-tuberculosis drugs. Although the genome of M. tuberculosis appears to encode over 100 putative outer membrane proteins, only a few have been identified and characterized. Here, we summarize the current knowledge on the structure of the mycobacterial outer membrane and its known proteins. Through comparison to transport processes in Gram-negative bacteria, we highlight several hypothetical outer membrane proteins of M. tuberculosis awaiting discovery. Mycobacteria have a complex cell envelopeScientific interest in mycobacteria has been sparked by the medical importance of Mycobacterium tuberculosis and by properties that distinguish them from other microorganisms. In particular, mycobacteria possess a remarkably complex cell envelope consisting of a cytoplasmic membrane and a cell wall, which constitutes an efficient permeability barrier and plays a crucial role in the intrinsic drug resistance and in survival under harsh conditions [1].These microbes produce a fascinating diversity of lipids [1,2] such as the mycolic acids, exceptionally long fatty acids that account for 30% to 40% of the cell envelope mass [3,4]. Mycolic acids are covalently linked to peptidoglycan via an arabinogalactan polymer, a polysaccharide composed of arabinose and galactose subunits. In a typical arrangement, the peptidoglycan network is substituted by linear galactan molecules, which bear several branched arabinose chains [1,2]. These branches end in four arabinose dimers, each forming the head group for two mycolic acid molecules.The mycolic acid-arabinogalactan-peptidoglycan polymer is arranged to form a hydrophobic layer with other lipids and the cytoplasmic membrane [5,6]. Recently, a model describing the

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Section: Structure Of Mycobacterial Outer Membrane Proteinsmentioning

confidence: 99%

Mycobacterial outer membranes: in search of proteins

Niederweis

Danilchanka

Huff

et al. 2010

Trends in Microbiology

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205

137

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“…The role of the periplasmic loop L6 in substrate translocation is not surprising given its large size and proximity to the constriction zone. MspA is the prototype of a new family of porins with more than 30 members identified solely in mycolic acid-containing bacteria (38) and has excellent perspectives in nanotechnological applications because of its extraordinary biophysical and biochemical properties (39,40). In addition to the periplasmic loop L6, the constriction zone represents another structural novelty of MspA.…”

Section: Discussionmentioning

confidence: 99%

Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis

Huff¹,

Pavlenok

Sukumaran³

et al. 2009

Journal of Biological Chemistry

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MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants ⌬7, ⌬9, and ⌬11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin.Mycobacteria have a unique outer membrane composed of covalently attached mycolic acids and a variety of other loosely associated lipids. Visualized by cryo-electron tomography (1), the outer membrane is an efficient permeability barrier that renders mycobacteria intrinsically resistant to many drugs and toxic compounds. As is the case for Gramnegative bacteria, this permeability barrier must be functionalized by outer membrane proteins. Although over 60 outer membrane proteins are known to exist in Escherichia coli (41), very few have been described in mycobacteria to date.The major protein of Mycobacterium smegmatis is MspA (42), which is the primary member of the nearly identical Msp (M. smegmatis porin) proteins. Msp proteins account for ϳ80% of detergent-extractable proteins at 100°C (2) in wt 4 M. smegmatis mc 2 155. MspA represents the main pathway for small and hydrophilic solutes and nutrients across the outer membrane (3). One hallmark of MspA is its extremely high stability. Functional pores are detectable at temperatures up to 100°C, a pH range from 0 -14, and resist the action of denaturing agents such as 2% SDS (4), thus providing the utility of MspA for nanotechnological applications. However, it is unknown how M. smegmatis utilizes Msp porins to functionalize the outer membrane in varying conditions or what parts of the channel determine substrate translocation. It is our goal to understand the translocation determinants of the MspA channel.The cry...

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“…Consequently, MspA, which has a length of approx. 10 nm, is towering over the SAM and can be easily detected by AFM 12. Here, MspA extends only slightly (< 2nm) beyond the copolymer layer.…”

Section: Spincasting Experimentsmentioning

confidence: 93%

“…The use of self-assembled monolayers (SAMs) for protein adsorption and protection is prevalent 10,11. Recently, several procedures for the preparation of nanolayers of oligopeptides that were able to stabilize proteins12 and protein complexes13 have been reported. Polymer layers, such as Poly-N-isopropylacrylamide on HOPG surfaces were used as well to provide protein-stabilization 7…”

Section: Introductionmentioning

confidence: 99%

Poly-N-Isopropyl-acrylamide/Acrylic Acid Copolymers for the Generation of Nanostructures on Mica Surfaces and as Hydrophobic Host Systems for the Porin MspA from Mycobacterium smegmatis

et al. 2009

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The work presented here aims at utilizing poly-N-isopropyl-acrylamide/acrylic acid copolymers to create nanostructured layers on mica surfaces by a simple spin-casting procedure. The average composition of the copolymers determined by elemental analysis correlates excellently with the feed composition indicating that the radical polymerization process is statistical. The resulting surfaces were characterized by Atomic Force Microscopy (magnetic AC-mode) at the copolymer/air interface. Postpolymerization modification of the acrylic acid functions with perfluoro-octyl-iodide decreased the tendency towards spontaneous formation of nanopores. Crosslinking of individual polymer chains permitted the generation of ultraflat layers, which hosted the mycobacterial channel protein MspA, without compromising its channel function. The comparison of copolymers of very similar chemical composition that have been prepared by living radical polymerization and classic radical polymerization indicated that differences in polydispersity played only a minor role when poly-N-isopropyl-acrylamide/acrylic acid copolymers were spincast, but a major role when copolymers featuring the strongly hydrophobic perfluoro-octyl-labels were used. The mean pore diameters were 23.8±4.4 nm for P[(NIPAM)95.5-co-(AA)4.5] (PDI (polydispersity index)=1.55) and 21.8±4.2 nm for P[(NIPAM)95.3-co-(AA)4.7] (PDI=1.25). The depth of the nanopores was approx. 4 nm. When depositing P[(NIPAM)95-co-(AA)2.8-AAC8F17 2.2] (PDI=1.29) on Mica, the resulting mean pore diameter was 35.8±7.1 nm, with a depth of only 2 nm.

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Characterization of Nanostructured Surfaces Generated by Reconstitution of the Porin MspA from Mycobacterium smegmatis

Cited by 21 publications

References 66 publications

Mycobacterial outer membranes: in search of proteins

Mycobacterial outer membranes: in search of proteins

Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis

Poly-N-Isopropyl-acrylamide/Acrylic Acid Copolymers for the Generation of Nanostructures on Mica Surfaces and as Hydrophobic Host Systems for the Porin MspA from Mycobacterium smegmatis

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