| 1. |
- de Ghellinck, Alexis, et al.
(författare)
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Lipid polyunsaturation determines the extent of membrane structural changes induced by Amphotericin B in Pichia pastoris yeast
- 2015
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736. ; 1848:10, s. 2317-2325
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Tidskriftsartikel (refereegranskat)abstract
- The activity of the potent but highly toxic antifungal drug Amphotericin B (AmB), used intravenously to treat systemic fungal and parasitic infections, is widely accepted to result from its specific interaction with the fungal sterol ergosterol. While the effect of sterols on AmB activity has been intensely investigated, the role of membrane phospholipid composition has largely been ignored, and structural studies of native membranes have been hampered by their complex and disordered nature. We show for the first time that the structure of fungal membranes derived from Pichia pastoris yeast depends on the degree of lipid polyunsaturation, which has an impact on the structural consequences of AmB activity. AmB inserts in yeast membranes even in the absence of ergosterol, and forms an extra-membraneous layer whose thickness is resolved to be 4-5 nm. In ergosterol-containing membranes, AmB insertion is accompanied by ergosterol extraction into this layer. The AmB-sponge mediated depletion of ergosterol from P. pastoris membranes gives rise to a significant membrane thinning effect that depends on the degree of lipid polyunsaturation. The resulting hydrophobic mismatch is likely to interfere with a much broader range of membrane protein functions than those directly involving ergosterol, and suggests that polyunsaturated lipids could boost the efficiency of AmB. Furthermore, a low degree of lipid polyunsaturation leads to least AmB insertion and may protect host cells against the toxic effects of AmB. These results provide a new framework based on lipid composition and membrane structure through which we can understand its antifungal action and develop better treatments. (C) 2015 Elsevier B.V. All rights reserved.
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| 2. |
- de Ghellinck, Alexis, et al.
(författare)
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Production and Analysis of Perdeuterated Lipids from Pichia pastoris Cells
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.
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| 3. |
- Delhom, Robin, et al.
(författare)
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The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry
- 2020
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Ingår i: Nanomaterials. - : MDPI AG. - 2079-4991. ; 10:12
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Tidskriftsartikel (refereegranskat)abstract
- We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.
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| 4. |
- Grimaldo, Marco, et al.
(författare)
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Protein Short-Time Diffusion in a Naturally Crowded Environment
- 2019
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Ingår i: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 10:8, s. 1709-1715
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Tidskriftsartikel (refereegranskat)abstract
- The interior of living cells is a dense and polydisperse suspension of macromolecules. Such a complex system challenges an understanding in terms of colloidal suspensions. As a fundamental test we employ neutron spectroscopy to measure the diffusion of tracer proteins (immunoglobulins) in a cell-like environment (cell lysate) with explicit control over crowding conditions. In combination with Stokesian dynamics simulation, we address protein diffusion on nanosecond time scales where hydrodynamic interactions dominate over negligible protein collisions. We successfully link the experimental results on these complex, flexible molecules with coarse-grained simulations providing a consistent understanding by colloid theories. Both experiments and simulations show that tracers in polydisperse solutions close to the effective particle radius R eff = R i 3 1/3 diffuse approximately as if the suspension was monodisperse. The simulations further show that macromolecules of sizes R > R eff (R < R eff ) are slowed more (less) effectively even at nanosecond time scales, which is highly relevant for a quantitative understanding of cellular processes.
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| 5. |
- Luchini, Alessandra, et al.
(författare)
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The impact of deuteration on natural and synthetic lipids : A neutron diffraction study
- 2018
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Ingår i: Colloids and Surfaces B: Biointerfaces. - : Elsevier BV. - 0927-7765. ; 168, s. 126-133
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Tidskriftsartikel (refereegranskat)abstract
- The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures.
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| 6. |
- Ramos, Joao, et al.
(författare)
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The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme
- 2021
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 1579-1590
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Tidskriftsartikel (refereegranskat)abstract
- The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97-Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.
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