Scattering versus fluorescence self-quenching:more than a question of faith for the quantificationof water flux in large unilamellar vesicles?
Sprache des Vortragstitels:
Water homeostasis is fundamental to all forms of life andplays a major role in human health and disease, plant growthand bacterial survival. The permeation of water is driven byosmotic imbalances of solutes and occurs in either unfacilitatedfashion through the cell membrane or in a facilitatedone through membrane spanning protein channels. To quantifythe effect of the lipid or polymer membrane on channelfunction, to expedite the development of specific water flowinhibitors or facilitate the design of artificial water channelsand aquaporins, accurate ways of determining water permeabilities(Pf) are necessary. A commonly used method is toreconstitute membrane channels into large unilamellar vesicles(LUVs) and to subject these vesicles to an osmotic gradientin a stopped-flow device. Fast recordings of either scatteredlight intensity or fluorescence self-quenching signals aretaken as a readout for vesicle volume change, which in turncan be recalculated to Pf values. By means of computationaland experimental data, we extensively discuss advantagesand disadvantages of using scattering and self-quenching experiments.We for the first time thoroughly assess the effectof LUVs size distribution and remaining detergent after proteinreconstitution on Pf values. Furthermore, we demonstratethe impact of channel distribution between proteoliposomes(PLs) on Pf and the activation energy (EA) of waterpermeation, which can be calculated from temperature dependentPf measurements.