Active Fluctuations and Electrostatic Interactions in Floating Lipid Membranes

Orateur : Soutenance de thèse de Tetiana MUKHINA

Lipid bilayer systems serve as an excellent platform for biological and physical studies and represent a suitable model system to probe protein-membrane and membrane-membrane interactions. Within this thesis work, the structure, function and physical properties of lipid bilayer systems were investigated by combining a large number of experimental techniques. Two different projects were carried out involving double lipid bilayer systems as useful platforms to investigate I) the out-of-equilibrium membrane fluctuations caused by protein pumping activity and II) the interaction between strongly confined highly negatively charged lipid bilayers in presence of only monovalent counterions. Within project I, protocols for detergent-mediated bacteriorhodopsin (BR) reconstitution in the gel and fluid phase single and floating lipid bilayer systems on planar substrates were developed. The protein insertion was monitored, confirmed and quantified by combining laboratory techniques (QCM-D, AFM, fluorescence microscopy, XRR), with neutron and synchrotron radiation reflectometry (SR) experiments. A reversible effect of illumination (light on/light off states) on the structure and fluctuations of the membrane-protein system was studied by means of specular and off-specular SR reflectometry, which demonstrated the magnification of membrane shape fluctuations induced by BR activity. These results will allow to investigate, for the first time, the fluctuation spectrum of a planar membrane-protein system at the nanoscale and to access the physical properties of the system such as bending modulus, surface tension and interaction potential between adjacent membranes. Within project II, the structure of double DPPS bilayers was fully characterized by means of neutron and SR reflectometry experiments focusing on the equilibrium distance between like-charged bilayers. The influence of added salt and temperature on the lipid bilayer structure and interlayer water thicknesses was monitored as well. The existence of an attractive interaction between highly charged bilayers was demonstrated, in good agreement with the theoretical description of like-charge attraction within the SC limit, taking into account the decrease of water dielectric constant induced by the strong confinement. The obtained results provide a deeper understanding of electrostatic interactions in strong confinement beyond the continuous models, where the discrete nature of charges must be taken into account explicitly.

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