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- Ausili, A., et al.
(författare)
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Membrane docking mode of the C2 domain of PKCε: An infrared spectroscopy and FRET study
- 2013
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736. ; 1828:2, s. 552-560
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Tidskriftsartikel (refereegranskat)abstract
- The C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the β-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the β-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45 to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of β-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the β-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49). © 2012 Elsevier B.V. All rights reserved.
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| 2. |
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| 3. |
- Ausili, A., et al.
(författare)
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Quartz crystal microbalance with dissipation monitoring and the real-time study of biological systems and macromolecules at interfaces
- 2012
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Ingår i: Biomedical Spectroscopy and Imaging. - 2212-8794 .- 2212-8808. ; 1:4, s. 325-338
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Tidskriftsartikel (refereegranskat)abstract
- QCM-D technique is based on the physical phenomenon that generates an acoustic shear wave with an oscillating resonance in quartz resulting in an evanescent wave that arises at the interface of the quartz and the solution. The amplitude of the acoustic wave is influenced by the deposition of material onto the quartz surface and from the subsequent decrease of the frequency the bound mass can be calculated. The dissipation shift which arises inform about viscoelasticity and flexibility of the adsorbed material. QCM-D can be applied for real-time studies of several biological systems since it is a simple, fast, low-cost and sensitive technique without having to label any sample. Common applications in biological field include measurements on adsorption of lipids, proteins, DNA and cells directly onto the surface of the sensor, which generally are chemically modified by self-assembled monolayer (SAM) technique or by spin-coated polymers. QCM-D can also be used to study molecular interactions between macromolecules and adsorbed materials. Three examples of the use of this technique are presented, namely the docking orientation of the C2 domain of PKCε on phospholipid membranes, the conformational changes of fibrinogen adsorbed to model acrylic polymers and the attachment of endothelial cells to carboxylated polymers of different configuration.
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