|dc.description.abstracteng||Lipid and protein diffusion in membranes is a fundamental requirement for many signaling processes in biological cells. Therefore, measuring protein and lipid mobility in lipid bilayers with high accuracy is essential for understanding biological mechanisms.
In 1975, Philip Saffman and Max Delbrück developed a model to describe protein diffusion in membranes. They predicted a logarithmic dependence of the protein's diffusion coefficient on its hydrodynamic radius. Recently, however, Gambin et al. observed a more Stokes-Einstein-like behavior, where the protein's diffusion coefficient and hydrodynamic radius are inversely proportional. Previous theoretical and experimental studies reflect this discrepancy, illustrating the urgent need for accurate diffusion measurements in lipid bilayers.
To measure diffusion in membranes, Dual-focus Fluorescence Correlation Spectroscopy (2fFCS) was used. For correctly positioning the foci on the bilayer, a new method based on the maximum molecular brightness was developed, which is just as precise but much faster than the previously reported z-scan FCS.
The maximum molecular brightness method was first applied to investigate lipid diffusion in Black Lipid Membranes (BLMs), in particular the influence of mono- and divalent ions on neutral and charged lipid bilayers. It was found that monovalent ions do not influence lipid diffusion in neutral and charged BLMs. In contrast, divalent ions affect lipid diffusion differently depending on the lipid composition. While lipid diffusion in neutral membranes remained unchanged, lipid mobility in charged BLMs was decreased significantly upon addition of calcium ions. The reason for this is, that calcium can link two negatively charged head groups together thus increasing the membrane viscosity.
Secondly, protein diffusion in lipid bilayers was investigated in order to study the applicability of the Saffman-Delbrück model. Therefore, proteins of different sizes spanning one order of magnitude in radii were reconstituted into BLMs. The results revealed that, despite its simplicity, the Saffman-Delbrück model is suitable for describing protein diffusion in membranes. The Stokes-Einstein-like model, however, does not fit the obtained data all. A newer theoretical model developed by Petrov and Schwille was able to reproduce the Saffman-Delbrück results in the size range investigated.
In summary, BLMs in combination with 2fFCS provide a robust tool for investigating diffusion processes in lipid bilayers with high accuracy. For future studies, this system provides a great opportunity for investigating other membrane characteristics, such as membrane tension, which have been challenging to determine previously.||de