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      An engineered dimeric protein pore that spans adjacent lipid bilayers

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          Abstract

          The bottom-up construction of artificial tissues is an underexplored area of synthetic biology. An important challenge is communication between constituent compartments of the engineered tissue and between the engineered tissue and additional compartments, including extracellular fluids, further engineered tissue and living cells. Here we present a dimeric transmembrane pore that can span two adjacent lipid bilayers and thereby allow aqueous compartments to communicate. Two heptameric staphylococcal α-hemolysin (αHL) pores were covalently linked in an aligned cap-to-cap orientation. The structure of the dimer, (α7) 2, was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. We show that one of two β barrels of (α7) 2 can insert into the lipid bilayer of a small unilamellar vesicle, while the other spans a planar lipid bilayer. (α7) 2 pores spanning two bilayers were also observed by TEM.

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          Most cited references 34

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          A vesicle bioreactor as a step toward an artificial cell assembly.

          An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles containing extracts are produced in an oil-extract emulsion. To form a bilayer the vesicles are transferred into a feeding solution that contains ribonucleotides and amino acids. Transcription-translation of plasmid genes is isolated in the vesicles. Whereas in bulk solution expression of enhanced GFP stops after 2 h, inside the vesicle permeability of the membrane to the feeding solution prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the alpha-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein production of 30 muM after 4 days. Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst.
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            Structure of the connexin 26 gap junction channel at 3.5 A resolution.

            Gap junctions consist of arrays of intercellular channels between adjacent cells that permit the exchange of ions and small molecules. Here we report the crystal structure of the gap junction channel formed by human connexin 26 (Cx26, also known as GJB2) at 3.5 A resolution, and discuss structural determinants of solute transport through the channel. The density map showed the two membrane-spanning hemichannels and the arrangement of the four transmembrane helices of the six protomers forming each hemichannel. The hemichannels feature a positively charged cytoplasmic entrance, a funnel, a negatively charged transmembrane pathway, and an extracellular cavity. The pore is narrowed at the funnel, which is formed by the six amino-terminal helices lining the wall of the channel, which thus determines the molecular size restriction at the channel entrance. The structure of the Cx26 gap junction channel also has implications for the gating of the channel by the transjunctional voltage.
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              Stochastic sensors inspired by biology.

              Sensory systems use a variety of membrane-bound receptors, including responsive ion channels, to discriminate between a multitude of stimuli. Here we describe how engineered membrane pores can be used to make rapid and sensitive biosensors with potential applications that range from the detection of biological warfare agents to pharmaceutical screening. Notably, use of the engineered pores in stochastic sensing, a single-molecule detection technology, reveals the identity of an analyte as well as its concentration.
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                Author and article information

                Journal
                101528555
                37539
                Nat Commun
                Nat Commun
                Nature communications
                2041-1723
                24 April 2013
                16 April 2013
                16 October 2013
                : 4
                : 1725
                Affiliations
                [1 ]Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK
                [3 ]Department of Physics, University of Oxford, Oxford OX1 3PU, UK
                Author notes
                [* ]Correspondence and requests for materials should be addressed to H.B. ( hagan.bayley@ 123456chem.ox.ac.uk )
                [2]

                Present address: Alberta Diabetes Institute, Department of Pharmacology, University of Alberta, Edmonton, T6G 2E1, Canada

                Article
                NIHMS454094
                10.1038/ncomms2726
                3644966
                23591892

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Funding
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: R01 HG003709 || HG
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