Methods for the Formation of Single Bimolecular Lipid Membranes in Aqueous Solution

Mueller, Paul S.; Rudin, Donald; Tien, H. Ti; Westcott, W. Wynn

doi:10.1021/j100796a046

Cited by 194 publications

(177 citation statements)

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“…Both samples were mixed (1 mg of protein/20 mg of lipid) and dialyzed against 2 liters of 10 mM KCl, 10 mM MOPS/Tris, pH 7.0, for 2 h at 25°C and subsequently overnight at 4°C. Planar lipid bilayers were produced using the painting technique (29). A solution of 75 mg/ml L-␣-phosphatidylcholine in n-decan was applied to a hole in a Teflon septum, separating the two 3-ml chambers.…”

Section: Methodsmentioning

confidence: 99%

The Evolutionarily Related β-Barrel Polypeptide Transporters from Pisum sativum and Nostoc PCC7120 Contain Two Distinct Functional Domains

Ertel

Mirus

Bredemeier

et al. 2005

Journal of Biological Chemistry

118

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Several ␤-barrel-type channels are involved in the translocation or assembly of outer membrane proteins of bacteria or endosymbiotically derived organelles. Here we analyzed the functional units of the ␤-barrel polypeptide transporter Toc75 (translocon in outer envelope of chloroplasts) of the outer envelope of chloroplasts and of a protein, alr2269, from Nostoc PCC7120 with homology to Toc75, both proteins having a similar domain organization. We demonstrated that the N-terminal region functions as a recognition and complex assembly unit, whereas the C terminus forms the ␤-barrel-type pore. The pore region is, in turn, modulated by the N terminus of the proteins. The protein from Nostoc PCC7120, which shares a common ancestor with Toc75, is able to recognize precursor proteins destined for chloroplasts. In contrast, the recognition of peripheral translocon subunits by Toc75 is a novel feature acquired through evolution.␤-barrel-type channels are involved in the translocation of polypeptides (1), the assembly of proteins in the outer membrane of endosymbiotic organelles (2-4), or in the assembly of proteins in the outer membrane of bacteria (5, 6). These proteins belong to one class, which can be termed polypeptide-transporting ␤-barrel channels (2, 4, 7). Four proteins are in the focus of recent investigation, namely the bacterial outer membrane proteins Omp85 and ShlB, the mitochondrial outer membrane protein Tob55/Sam50, and the chloroplast outer envelope protein Toc75.ShlB is an outer membrane protein involved in the secretion of hemolysins or adhesins in various Gram-negative pathogens (8, 9). Omp85 is an essential component for outer membrane biogenesis in Neisseria meningitidis that might have two functions: the assembly of outer membrane proteins (5) and the translocation of lipids (10). Recently, it was discussed that the effect on lipid transfer by Omp85 depletion might be indirect and explained by an assembly defect of the required outer membrane protein, suggesting a function of Omp85 in outer membrane protein assembly only (11). As for ShlB, a ␤-barrel transmembrane structure was suggested for Omp85 (5). Recently, a new polypeptide-transporting protein was identified in the outer membrane of mitochondria and termed Sam50 (3), Tob55 (2), or mitochondrial Omp85 homologue (4). This protein facilitates the assembly of proteins into the outer membrane of mitochondria. Tob55/Sam50 is found in a larger complex with Mas37 (3, 12) and Tob38/Sam35 (13-15).The fourth investigated ␤-barrel-type polypeptide transporter is the 75-kDa subunit of the translocon of the outer envelope of chloroplasts, Toc75. Toc75 forms a complex with Toc34, Toc64, and Toc159 (16). In contrast to the other identified polypeptide transporters, such as Omp85, the translocation of proteins through Toc75 requires the action of assisting proteins, such as Toc159 (17), but still Toc75 seems to contain a preprotein-binding site as determined by electrophysiological measurements (1). Topological modeling of Toc75 from Pisum sativum (18) or T...

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Section: Methodsmentioning

confidence: 99%

The Evolutionarily Related β-Barrel Polypeptide Transporters from Pisum sativum and Nostoc PCC7120 Contain Two Distinct Functional Domains

Ertel

Mirus

Bredemeier

et al. 2005

Journal of Biological Chemistry

118

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“…Bilayers were formed at room temperature by using the brush technique of Mueller et al (18) across a 200-m-diameter hole in a Delrin cup separating 1-ml cis and trans compartments. Symmetric solutions of 100 mM KCl, 25 mM succinic acid, and 1 mM EDTA (pH 5.5) were used.…”

mentioning

confidence: 99%

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

Zhang

Finkelstein

Collier

2004

Proc. Natl. Acad. Sci. U.S.A.

119

149

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“…Planar Lipid Bilayers-Planar lipid bilayers were produced by the painting technique (26). A solution of 80 mg/ml purified azolectin (type IV-S, Sigma) in n-decan (analytical grade; Merck) was applied to a hole (50 -100 m diameter) in a Teflon septum, separating two bath chambers (volume 3 ml each).…”

Section: Methodsmentioning

confidence: 99%

OEP37 Is a New Member of the Chloroplast Outer Membrane Ion Channels

Goetze

Philippar²,

Ilkavets³

et al. 2006

Journal of Biological Chemistry

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The chloroplast outer envelope protein OEP37 is a member of the growing ␤-barrel protein family of the outer chloroplast membrane. The reconstituted recombinant protein OEP37 from pea forms a rectifying high conductance channel with a main conductance () of ⌳ ‫؍‬ 500 picosiemens (symmetrical 250 mM KCl). The OEP37 channel is cation-selective (P K ؉/P Cl ؊ ‫؍‬ 14:1) with a voltage-dependent open probability maximal at V mem ‫؍‬ 0 mV. The channel pore reveals an hourglass-shaped form with different diameters for the vestibule and restriction zone. The diameters of the vestibule at the high conductance side were estimated by d ‫؍‬ 3.0 nm and the restriction zone by d ‫؍‬ 1.5 nm. The OEP37 channel displayed a nanomolar affinity for the precursor of the chloroplast inner membrane protein Tic32, which is imported into the chloroplast through a yet unknown pathway. Pre-proteins imported through the usual Toc pathway and synthetic control peptides, however, did not show a comparable block of the OEP37 channel. In addition to the electrophysiological characterization, we studied the gene expression of OEP37 in the model plant Arabidopsis thaliana. Here, transcripts of AtOEP37 are ubiquitously expressed throughout plant development and accumulate in early germinating seedlings as well as in late embryogenesis. The plastid intrinsic protein could be detected in isolated chloroplasts of cotyledons and rosette leaves. However, the knock-out mutant oep37-1 shows that the proper function of this single copy gene is not essential for development of the mature plant. Moreover, import of Tic32 into chloroplasts of oep37-1 was not impaired when compared with wild type. Thus, OEP37 may constitute a novel peptide-sensitive ion channel in the outer envelope of plastids with function during embryogenesis and germination.The plastid organelle family conducts vital biosynthetic functions in every plant cell. Chloroplasts carry out photosynthesis, which converts atmospheric carbon dioxide to carbohydrates. Furthermore, plastids are the site for fatty acid, amino acid, and porphyrin biosynthesis as well as sulfate and nitrate reduction. Thus, all biosynthetic pathway products are steadily exchanged with the surrounding cell by the assistance of specific transport proteins, localized in the plastid envelope membranes. Although the inner envelope carriers, e.g. the triose phosphatephosphate translocator, the dicarboxylic acid translocator, or the hexose phosphate carrier, show a distinct substrate specificity, until now it remained elusive to what extent transport through outer membrane channels was regulated (for review of plastid envelope transporters compare with Ref. 1).The outer envelope membrane has long been assumed to be freely permeable for small molecular mass solutes of up to 10 kDa (2). Correspondingly, it is believed that the osmotic barrier against the cytosol is formed exclusively by the inner envelope. In Gram-negative bacteria, however, several different types of high conductance channels exist in the outer membrane (3) as ...

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Methods for the Formation of Single Bimolecular Lipid Membranes in Aqueous Solution

Cited by 194 publications

References 0 publications

The Evolutionarily Related β-Barrel Polypeptide Transporters from Pisum sativum and Nostoc PCC7120 Contain Two Distinct Functional Domains

The Evolutionarily Related β-Barrel Polypeptide Transporters from Pisum sativum and Nostoc PCC7120 Contain Two Distinct Functional Domains

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

OEP37 Is a New Member of the Chloroplast Outer Membrane Ion Channels

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