| 1. |
- 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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| 2. |
- Wadsten, Pia, 1969, et al.
(author)
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Lipidic sponge phase crystallization of membrane proteins.
- 2006
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In: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 364:1, s. 44-53
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Journal article (peer-reviewed)abstract
- Bicontinuous lipidic cubic phases can be used as a host for growing crystals of membrane proteins. Since the cubic phase is stiff, handling is difficult and time-consuming. Moreover, the conventional cubic phase may interfere with the hydrophilic domains of membrane proteins due to the limited size of the aqueous pores. Here, we introduce a new crystallization method that makes use of a liquid analogue of the cubic phase, the sponge phase. This phase facilitates a considerable increase in the allowed size of aqueous domains of membrane proteins, and is easily generalised to a conventional vapour diffusion crystallisation experiment, including the use of nanoliter drop crystallization robots. The appearance of the sponge phase was confirmed by visual inspection, small-angle X-ray scattering and NMR spectroscopy. Crystals of the reaction centre from Rhodobacter sphaeroides were obtained by a conventional hanging-drop experiment, were harvested directly without the addition of lipase or cryoprotectant, and the structure was refined to 2.2 Angstroms resolution. In contrast to our earlier lipidic cubic phase reaction centre structure, the mobile ubiquinone could be built and refined. The practical advantages of the sponge phase make it a potent tool for crystallization of membrane proteins.
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| 3. |
- Engström, Sven, 1951, et al.
(author)
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Cubic, sponge, and lamellar phases in the glyceryl monooleyl ether-propylene glycol-water system
- 2007
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In: Langmuir. - 1520-5827 .- 0743-7463. ; 23:20, s. 10020-10025
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Journal article (peer-reviewed)abstract
- The phase behavior of 1-glyceryl monooleyl ether (GME) in mixtures of propylene glycol (PG) and water was investigated by visual inspection, polarization microscopy, small-angle X-ray diffraction, and conductance measurements. A phase diagram, based on over 200 samples of the ternary system GME-PG-water, was constructed at 20 degrees C. Without PG, GME forms a reverse micellar phase with up to 10 wt % water and a reverse hexagonal liquid-crystalline phase between 10 and 25 wt % water, a phase that can coexist with excess water. If PG is added in amounts exceeding about 10 wt %, then cubic and lamellar liquid-crystalline phases start to form. A cubic phase, belonging to space group Pn3m, can coexist with excess PG-water mixtures. If even more PG is added, then the cubic phase is transformed into a sponge phase. A lamellar phase forms at water contents between 10 and 15 wt % and with widely differing PG/GME weight ratios. We postulate that the phase behavior is caused by the fact that PG makes the interfacial region between self-assembled GME and PG-water less negatively curved, which in turn allows for the formation of the new phases. The phase behavior obtained for the GME system shows a striking similarity with the phase behavior of the corresponding system in which the GME has been replaced by the ester, 1-glycerol monooleate (GMO), differing only in one extra carbonyl oxygen. The major difference is the lower amount of water present in the GME phases, an effect that is mainly due to the more hydrophobic character of GME compared to that of GMO.
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| 4. |
- Hindrichsen, Pia Jeanette, 1969, et al.
(author)
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Aqueous self-assembly of phytantriol in ternary systems: effect of monoolein, distearoylphosphatidylglycerol and three water-miscible solvents
- 2007
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In: Journal of Colloid and Interface Science. - : Elsevier BV. - 1095-7103 .- 0021-9797. ; 315:2, s. 710-713
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Journal article (peer-reviewed)abstract
- The aqueous phase behavior of phytantriol (PT) in mixtures of monoolein (MO), distearoylphosphatidylglycerol (DSPG), propylene glycol (PG), polyethylene glycol 400 (PEG 400) and 2-methyl-2,4-pentanediol (MPD) was investigated by visual inspection, polarized light microscopy and small angle X-ray diffraction at room temperature. The phase diagrams of PT and MO in water are qualitatively very similar and PT/MO mixtures in excess water form one cubic phase of space group Pn3m irrespective of mixing ratio. The addition of the charged membrane lipid DSPG to the PT system gives rise to a considerable water swelling of the cubic phases as well as the occurrence of a cubic phase of space group Im3m. Whereas all three solvents studied give rise to a sponge (L3) phase in the MO-water system, this phase was only found when MPD was added to the PT-water system. The results are discussed with respect to the chemical differences between PT and MO.
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| 5. |
- Hindrichsen, Pia Jeanette, 1969
(author)
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On the crystallization of membrane proteins in lipidic sponge and cubic phases
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Membrane proteins are involved in many important biological processes and in order to understand their mechanism, their three-dimensional structures need to be elucidated to high resolution by for example X-ray crystallography. However, there is only limited structural knowledge for membrane proteins which is partly explained by the difficulties in obtaining well-diffracting crystals. To facilitate production of such crystals, cubic phases of monoolein (MO) can be used, but the mechanism behind this process is still unclear and requires further exploration. In this thesis, an anionic lipid, distearoyl phosphatidyl glycerol (DSPG) was used to enlarge the water pores in the cubic phase since there were indications that the water pore diameter is crucial for inclusion and diffusion of membrane proteins. The phase behaviour was also investigated in the MO-DSPG-water system with small amounts of the membrane protein, bacteriorhodopsin (bR), and detergent, octyl glycoside (OG). It was shown that both bR and OG stabilized the lamellar phase in favour for the cubic phase. The phase behaviour was characterized by visual inspection and small-angle X-ray scattering (SAXS). It was also demonstrated that bR and another membrane protein, reaction centre from Rhodopseudomonas viridis (RCvir), could be crystallized from the swelled MO-DSPG cubic phase. A new crystallization method that makes use of a liquid analogue of the cubic phase, the sponge phase, is also presented in this thesis. The sponge phase facilitates a considerable increase in the allowed size of membrane proteins aqueous domains and is easily setup using a conventional vapour diffusion crystallization experiment. Crystals of the reaction centre from Rhodobacter sphaeroides (RCsph) and RCvir were obtained by this method and the appearance of the sponge phase was confirmed by visual inspection, SAXS and NMR spectroscopy. MO is susceptible to (ester) hydrolysis and the cubic phase of MO proved to be unstable at high pH. Therefore, the possibility to use ester-free lipids, phytantriol (PT) and selachyl alcohol was investigated. Detailed phase diagrams were made with solvents known to create sponge phases in the MO-water system and it was shown that PT and selachyl alcohol could be good alternatives for MO in applications were extreme pH is of importance.
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