| 1. |
- Backmark, Anna, 1979, et al.
(author)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Journal article (peer-reviewed)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 2. |
- Horsefield, Rob, 1977, et al.
(author)
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High-resolution x-ray structure of human aquaporin 5
- 2008
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In: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:36, s. 13327-13332
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Journal article (peer-reviewed)abstract
- Human aquaporin 5 (HsAQP5)facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-angstrom resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.
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| 3. |
- Törnroth-Horsefield, Susanna, 1973, et al.
(author)
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Aquaporin gating
- 2007
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In: Journal of Biomolecular Structure & Dynamics. ; 24:6, s. 719-721
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Journal article (peer-reviewed)
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| 4. |
- Törnroth-Horsefield, Susanna, 1973, et al.
(author)
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Crystal Structure of AcrB in Complex with a Single Transmembrane Subunit Reveals Another Twist.
- 2007
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In: Structure (London, England : 1993). - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 15:12, s. 1663-73
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Journal article (peer-reviewed)abstract
- Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.
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| 5. |
- Gordon, Euan, 1971, et al.
(author)
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Effective high-throughput overproduction of membrane proteins in Escherichia coli
- 2008
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In: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 62:1, s. 1-8
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Journal article (peer-reviewed)abstract
- Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5 ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-β-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2 mg/l, with 18 targets producing at levels of 5 mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.
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| 6. |
- Wöhri, Annemarie, 1976, et al.
(author)
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A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization
- 2008
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In: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 16:7, s. 1003-1009
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Journal article (peer-reviewed)abstract
- A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.
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