Automated Formation of Lipid Membrane Microarrays for Ionic Single‐Molecule Sensing with Protein Nanopores

Martinez, Juan Miguel Del Rio; Zaitseva, Ekaterina; Petersen, Sönke; Baaken, Gerhard; Behrends, Jan C.

doi:10.1002/smll.201402016

Cited by 58 publications

(66 citation statements)

References 44 publications

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“…In brief, horizontal bilayers of 1,2-diphytanoyl- sn -glycero-3-phosphocholine (Avanti Polar Lipids) were formed over microcavities (of subpicolitre volume) in a 16-channel multielectrode cavity array (MECA) chip (Ionera) 51 . Both the cis and trans cavities above and below the bilayer contained 1.0 M KCl, 25 mM Tris-HCl pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Goyal

Krasteva

Gerven

et al. 2014

Nature

262

306

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Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes)1–3. They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia1,4,5. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF6,7. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The trans-membrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å3 pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.

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Section: Methodsmentioning

confidence: 99%

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Goyal

Krasteva

Gerven

et al. 2014

Nature

262

306

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“…Lipid bilayers were formed by remote liquid spreading of a DOPC/cholesterol (7:3) solution in octane (7 mg/mL) on a MECA-16 microelectrode cavity array chip (49-51) using the Orbit-16 automated parallel bilayer platform (Nanion Technologies, Munich, Germany) as described in detail by del Rio Martinez et al (51). Bilayer formation was controlled with the Orbit-16's internal multichannel patch-clamp amplifier (Triton-16; Tecella, Foothill Ranch, CA) running under TecellaLab (version 0.84b) software.…”

Section: Electrophysiological Recordingsmentioning

confidence: 99%

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

Salnikov

Raya

Zotti

et al. 2016

Biophysical Journal

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Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C 19 F 3 -labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19 F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19 F-19 F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

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“…Thus we prefer to use the term “nanopore-based single-molecule size discrimination” (Np-SMSD) in what follows. While this experiment has been amply reproduced also in higher throughput formats1617181920, an important limitation is that size-dependence of blockade depth is lost when PEG polymers become larger than about 3000 g/mol13.…”

mentioning

confidence: 98%

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore

Piguet¹,

Ouldali²,

Discala³

et al. 2016

Sci Rep

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We explore the effect of temperature on the interaction of polydisperse mixtures of nonionic poly(ethylene glycol) (PEG) polymers of different average molar masses with the biological nanopore α-hemolysin. In contrast with what has been previously observed with various nanopores and analytes, we find that, for PEGs larger than a threshold molar mass (2000 g/mol, PEG 2000), increasing temperature increases the duration of the PEG/nanopore interaction. In the case of PEG 3400 the duration increases by up to a factor of 100 when the temperature increases from 5 °C to 45 °C. Importantly, we find that increasing temperature extends the polymer size range of application of nanopore-based single-molecule mass spectrometry (Np-SMMS)-type size discrimination. Indeed, in the case of PEG 3400, discrimination of individual molecular species of different monomer number is impossible at room temperature but is achieved when the temperature is raised to 45 °C. We interpret our observations as the consequence of a decrease of PEG solubility and a collapse of PEG molecules with higher temperatures. In addition to expanding the range of application of Np-SMMS to larger nonionic polymers, our findings highlight the crucial role of the polymer solubility for the nanopore detection.

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Automated Formation of Lipid Membrane Microarrays for Ionic Single‐Molecule Sensing with Protein Nanopores

Cited by 58 publications

References 44 publications

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore

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