Proteo-lipobeads to encapsulate cytochrome c oxidase from Paracoccus denitrificans.
Proteo-lipobeads (PLBs) are investigated as cell-free model systems to encapsulate membrane proteins such as ion channels and transporters. PLBs are based on nickel nitrile tri-acetic acid (Ni-NTA)-functionalized agarose beads, onto which membrane proteins (MP) are bound via histidine(his)-tag. Composite beads thus obtained (subsequently called proteobeads) are dialyzed in the presence of lipid micelles to form PLBs. As an example we employed cytochrome c oxidase from P. denitrificans with a his-tag fused to the C-terminus of subunitI. In this orientation the P side of CcO faces the outside of the PLB and hence protons are released to the outer aqueous phase, when electron transfer is initiated by light excitation of Ru complexes. Proton release kinetics was probed by fluorescence microscopy using the pH-sensitive sensor molecule fluorescein DHPE inserted into the lipid layer. In order to monitor the generation of membrane potentials we performed a FLIPR assay on the CcO embedded in PLBs using the FRET pair CC2-DMPE/DiSBAC2(3). The combined results show that PLBs can be used as a model system designed to quantify the kinetic parameters of membrane proteins. In addition, the FLIPR assay demonstrates the feasibility of PLBs for high throughput screening applications.
['Geiss|Andreas F|AF|', 'Bliem|Christina|C|', 'Frank|Pinar|P|', 'Reiner-Rozman|Ciril|C|', 'Kewney|Justin|J|', 'Boersch|Michael|M|', 'Naumann|Renate L C|RLC|']
D004337:Drug Carriers D065546:Drug Liberation D004579:Electron Transport D003576:Electron Transport Complex IV D005456:Fluorescent Dyes D057166:High-Throughput Screening Assays D007700:Kinetics D008051:Lipid Bilayers D008564:Membrane Potentials D008863:Microspheres D010231:Paracoccus denitrificans D011510:Proteolipids D012685:Sepharose D013499:Surface Properties
Proteo-lipobead (PLB) Fluorescence microscopy Confocal laser-scanning microscopy Cytochrome c oxidase (CcO) Fluorescence imaging plate reader assay (FLIPR) Protein-tethered bilayer lipid membrane (ptBLM) Proteoliposome