Fluorescence Correlation Spectroscopy Reveals Additional Information on Protein Insertions into Phospholipid Monolayers

Abstract

The generation of curvature in phospholipid membranes is a crucial step in vesicle and tube formation. A variety of proteins in eukaryotic cells are able to induce such curved membrane structures and produce coated transport vesicles or tubular carriers. Membrane curvature can be generated in different ways.Sar1, which is a small GTPase of the RAS superfamily and a part of the coat protein complex II (COPII), and the N-BAR dimer of amphiphysin II, which is involved in clathrin-coated vesicle formation, both have amphipathic α-helices, which insert only into the proximal leaflet of a phospholipid membrane. Phospholipid monolayers are therefore convenient model systems for analyzing the protein-lipid interactions.While a typical Langmuir film balance setup allows to observe the interaction of proteins with phospholipid monolayers (by changing the surface pressure or the surface area),the determination of protein concentrations and diffusion properties is not possible.To overcome these limitations, we combine a Langmuir film balance with a confocal fluorescence correlation spectroscopy setup for optical detection. This setup, which was described for the first time by Gudmand et.al (Biophys. J., 2009, 96, 4598-4609), allows FCS measurements in a monolayer system.Here we show some basic interaction studies of Sar1 and N-BAR with phospholipid monolayers at different surface pressures. In all cases, FCS measurements were carried out to obtain additional information on the protein.