Measuring Protein Insertion Areas in Lipid Monolayers by Fluorescence Correlation Spectroscopy

The insertion of protein domains into membranes is an important step in many membrane remodeling processes, for example in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. Total area changes in lipid monolayers can be measured on a Langmuir film balance. Finding the area per inserted protein however proves challenging for two reasons: The number of inserted proteins must be determined without disturbing the binding equilibrium and the change in the film area can be very small. Here we address both issues using Fluorescence Correlation Spectroscopy (FCS): Firstly, by labeling a fraction of the protein molecules fluorescently and performing FCS experiments directly on the monolayer, the number of inserted proteins is determined in situ without having to rely on invasive techniques, such as collecting the monolayer by aspiration. Secondly, by using another FCS color channel and adding a small fraction of fluorescent lipids, the reduction in fluorescent lipid density accompanying protein insertion can be monitored to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation, in a lipid monolayer. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm2 was obtained for Sar1 in monolayers from a lipid mixture typically used in reconstitution, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to be applied also to laterally heterogeneous monolayers and bilayers.Statement of SignificanceWe show that two color Fluorescence Correlation Spectroscopy (FCS) measurements can be applied to the binding of a protein to a lipid monolayer on a Langmuir film balance in order to determine the protein insertion area. One labelling color was used to determine the number of bound proteins and the other one to monitor the area expansion of the lipid monolayer upon protein binding. A strategy for the FCS data analysis is provided, which includes focal area calibration by raster image correlation spectroscopy and a framework for applying z-scan FCS and including free protein in the aqueous subphase. This approach allows determining an area occupied by a protein in a quasi-planar model membrane from a local, non-invasive, optical measurement.

Structure–property relations of amphiphilic poly(furfuryl glycidyl ether)-block-poly(ethylene glycol) macromonomers at the air–water interface

Structure–property relations of poly(furfuryl glycidyl ether)-block-poly(ethylene glycol) macromonomers at the air–water interface are studied with a Langmuir film balance.

Influence of the isolation method of the soapstockfatty component on its characteristics

From soapstock, which is a waste product of sunflower oil production, the fatty component was isolated using isopropyl alcohol, toluene, fusel oil and a mixture of common salt and nonionic surfactants. With the help of the Langmuir film balance the molecular areas of isolated fats were found and the degree of hydrophobization in comparison with the original soapstock was evaluated. It was found that the strongest compression of the monolayer is observed when using fusel oil. The surface active properties of emulsifiers, synthesized by alkaline hydrolysis method on the basis of the obtained fat extracts and original soapstock, were studied. These results are consistent with measurements on the Langmuir balance.

LIPOPHILIC-FULLERENE DERIVATIVE MONOLAYERS AT THE AIR–WATER INTERFACE

Monolayers of a lipophilic C 60-derivative (FPTL) mixed in dipalmitoyl-phosphatidyl-choline (DPPC) have been studied by the Langmuir film balance technique and Brewster angle microscopy. Previous X-ray scattering studies showed that the FPTL molecules intercalated into the DPPC monolayers and modified the bending and compression modulus of the host DPPC membranes. Combined study of surface pressure–area isotherms and Brewster angle microscopy measurements clearly established that the liquid-condensed domain structures are strongly influenced by an addition of the fullerene bearing lipid molecules, where it caused smaller liquid-condensed domain structures.