The interaction of proteins with membranes: combined computational and neutron studies
Keywords: PH domains
membrane distance tuning
peripheral membrane proteins
neutron refletcometry
free floating membrane
calcium lipid interactions
model membranes
self-assembling monolayer
molecular dynamics

The interaction between proteins and membranes is essential for cell signalling. Planar model membrane sample systems enable structural and functional investigations of protein/membrane interactions via a variety of experimental methods, like neutron reflectometry (NR), quartz crystal microbalance (QCM) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The free floating membrane (FFM) sample system is a promising, novel planar model membrane system, in which a water layer prevents direct contact between the membrane and the adjacent substrate surface, consisting of a carboxylate terminated oligo (ethylene glycol) self assembling monolayer (OEG-SAM). Our molecular dynamics (MD) simulations of the FFM system showed that membrane dynamics like lipid diffusion are not affected by the substrate surface system above a water inter-layer extent of around 25 Å. Therefore, the FFM system provides better membrane mimicking characteristics compared to previous planar model membrane systems, in which the membrane is in close contact with the substrate surface. Furthermore, our NR and QCM measurements demonstrated how changes in the buffer solution cation concentrations within physiological ranges cause an, as yet, unseen range (over 200 Å) of reversible bilayer movements from the surface. MD simulations suggested that cations, especially Ca2+ ions decrease the electrostatic repulsion between the anionic membrane and OEG-SAM by modulating the OEG-SAM surface charge, while structured hydration layers above the OEG-SAM surface prevent contact between OEG-SAM and membrane. Changing the nature and concentration of cations allowed us to modulate the extent of electrostatic repulsion between OEG-SAM and membrane. Different pH values of the buffer solution showed bilayer movements from the surface within the same range as buffer cations did, supporting our findings that the OEG-SAM surface charge modulation is the main cause of the large reversible membrane movements. These results suggest that within a complex interplay of forces acting in the free floating bilayer sample system, electrostatic interactions can be manipulated to provide a direct means of controlling the membrane-to-surface distance. This "cation switchable" sample system offers a new opportunity to mimic the intracellular and extracellular environment using a planar membrane model system. Thus, our goal was to use this novel sample system to investigate the interaction between peripheral membrane proteins and their target membranes. The proteins of interest were the Kindlin-2 and the T-lymphoma invasion and metastasis 2 (TIAM2) proteins, involved in integrin activation and Rac1 GTPase activation, respectively. Both proteins contain a phosphatidylinositol lipid binding pleckstrin homology domain, responsible for membrane localization of the protein. The combination of NR and MD provided structural insights into the membrane binding orientation and position of the proteins. For instance, our computational and experimental results confirmed the previously suggested clustering of Kindlin-2 upon membrane binding. Together, this work provides an in-depth understanding of the processes within the FFM system in order to make it accessible to complex membrane interaction studies. The full understanding of acting forces within the system allowed us to apply the FFM system for protein/membrane interactions. Our investigations pave the way to further protein/membrane studies using the FFM system, as well as to an extension of the usage to integral membrane proteins and native membranes. Furthermore, our studies showed the powerful approach of combining NR with MD.