| 1. |
- Al-Jubair, Tamim, et al.
(author)
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Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)
- 2022
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In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
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Journal article (peer-reviewed)abstract
- Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST). MST has the advantage of low sample consumption and high detergent compatibility enabling AQP protein-protein interaction investigation with a high level of control of components and environment. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Roche et al. (2017).
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| 2. |
- Al-Jubair, Tamim, et al.
(author)
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High-yield overproduction and purification of human aquaporins from Pichia pastoris
- 2022
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In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
-
Journal article (peer-reviewed)abstract
- Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1–5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).
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| 3. |
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| 4. |
- Andersson, Magnus, et al.
(author)
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Transport Pathway in Cu+ P-Type ATPases
- 2014
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 106:2, s. 427A-427A
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Journal article (other academic/artistic)
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| 5. |
- Becares, Eva Ramos, et al.
(author)
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Overproduction of human Zip (SLC39) zinc transporters in saccharomyces cerevisiae for biophysical characterization
- 2021
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In: Cells. - : MDPI AG. - 2073-4409. ; 10:2, s. 1-21
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Journal article (peer-reviewed)abstract
- Zinc constitutes the second most abundant transition metal in the human body, and it is implicated in numerous cellular processes, including cell division, DNA and protein synthesis as well as for the catalytic activity of many enzymes. Two major membrane protein families facilitate zinc homeostasis in the animal kingdom, i.e., Zrt/Irt‐like proteins (ZIPs aka solute carrier 39, SLC39, family) and Zn transporters (ZnTs), essentially conducting zinc flux in the opposite directions. Human ZIPs (hZIPs) regulate import of extracellular zinc to the cytosol, being critical in preventing overaccumulation of this potentially toxic metal, and crucial for diverse physiological and pathological processes, including development of neurodegenerative disorders and several cancers. To date, our understanding of structure–function relationships governing hZIP‐mediated zinc transport mechanism is scarce, mainly due to the notorious difficulty in overproduction of these proteins for biophysical characterization. Here we describe employment of a Saccharomyces cerevisiae‐based platform for heterologous expression of hZIPs. We demonstrate that yeast is able to produce four full‐length hZIP members belonging to three different subfamilies. One target (hZIP1) is purified in the high quantity and homogeneity required for the downstream biochemical analysis. Our work demonstrates the potential of the described production system for future structural and functional studies of hZIP transporters.
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| 6. |
- Bjørkskov, Frederik Bühring, et al.
(author)
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Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization
- 2017
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Journal article (peer-reviewed)abstract
- The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization.
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| 7. |
- Couston, Julie, et al.
(author)
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Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs
- 2023
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In: Current Research in Structural Biology. - 2665-928X. ; 6
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Journal article (peer-reviewed)abstract
- Mycobacteria have an atypical thick and waxy cell wall. One of the major building blocks of such mycomembrane is trehalose monomycolate (TMM). TMM is a mycolic acid ester of trehalose that possesses long acyl chains with up to 90 carbon atoms. TMM represents an essential component of mycobacteria and is synthesized in the cytoplasm, and then flipped over the plasma membrane by a specific transporter known as MmpL3. Over the last decade, MmpL3 has emerged as an attractive drug target to combat mycobacterial infections. Recent three-dimensional structures of MmpL3 determined by X-ray crystallography and cryo-EM have increased our understanding of the TMM transport, and the mode of action of inhibiting compounds. These structures were obtained in the presence of detergent and/or in a lipidic environment. In this study, we demonstrate the possibility of obtaining a high-quality cryo-EM structure of MmpL3 without any presence of detergent through the reconstitution of the protein into peptidiscs. The structure was determined at an overall resolution of 3.2 Å and demonstrates that the overall structure of MmpL3 is preserved as compared to previous structures. Further, the study identified a new structural arrangement of the linker that fuses the two subdomains of the transmembrane domain, suggesting the feature may serve a role in the transport process.
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| 8. |
- Georgiou, P., et al.
(author)
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Picosecond calorimetry: Time-resolved x-ray diffraction studies of liquid CH2Cl2
- 2006
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In: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 124:23, s. 234507-
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Journal article (peer-reviewed)abstract
- Liquid phase time-resolved x-ray diffraction with 100 ps resolution has recently emerged as a powerful technique for probing the structural dynamics of transient photochemical species in solution. It is intrinsic to the method, however, that a structural signal is observed not only from the photochemical of interest but also from the embedding solvent matrix. To experimentally characterize the x-ray diffraction signal deriving from the solvent alone we performed time-resolved diffraction studies of a pure liquid sample over a time domain from -250 ps to 2.5 mu s. Multiphoton excitation was used to rapidly heat liquid CH2Cl2 using UV pulses of 100 fs duration. A significant x-ray diffraction signal is visible prior to the onset of thermal expansion, which characterizes a highly compressed superheated liquid. Liquid CH2Cl2 then expands as a shock wave propagates through the sample and the temporal dependence of this phenomenon is in good agreement with theory. An unexpectedly slow initial release of energy into the liquid as heat is observed from multiphoton excited CH2Cl2, revealing the presence of a metastable state of multiphoton excited CH2Cl2.
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| 9. |
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| 10. |
- Górecki, Kamil, et al.
(author)
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Microfluidic-Derived Detection of Protein-Facilitated Copper Flux Across Lipid Membranes
- 2022
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:34, s. 11831-11837
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Journal article (peer-reviewed)abstract
- Measurement of protein-facilitated copper flux across biological membranes is a considerable challenge. Here, we demonstrate a straightforward microfluidic-derived approach for visualization and measurement of membranous Cu flux. Giant unilamellar vesicles, reconstituted with the membrane protein of interest, are prepared, surface-immobilized, and assessed using a novel quencher-sensor reporter system for detection of copper. With the aid of a syringe pump, the external buffer is exchanged, enabling consistent and precise exchange of solutes, without causing vesicle rupture or uneven local metal concentrations brought about by rapid mixing. This approach bypasses common issues encountered when studying heavy metal-ion flux, thereby providing a new platform for in vitro studies of metal homeostasis aspects that are critical for all cells, health, and disease.
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