Biology subjects

Ebenhan, J.

Publications and source records attributed to Ebenhan, J..

3 recordsLinked to original sources

bioRxiv2024

Caveolin assemblies displace one bilayer leaflet to organize and bend membranes

Caveolin is a monotopic integral membrane protein, widely expressed in metazoa and responsible for constructing enigmatic membrane invaginations known as caveolae. Recently, the high-resolution structure of a purified human caveolin assembly, the CAV1-8S complex, revealed a unique organization of 11 protomers arranged in a tightly packed, radially symmetric spiral disc. One face and the outer rim of this disc are hydrophobic, suggesting that the complex incorporates into membranes by displacing hundreds of lipids from one leaflet. The feasibility of this unique molecular architecture and its biophysical and functional consequences are currently unknown. Using Langmuir film balance measurements, we find that CAV1-8S is highly surface active, intercalating into lipid monolayers of various compositions. CAV1-8S can also incorporate into preformed bilayers, but only upon removal of phospholipids from the outer-facing leaflet. Atomistic and coarse-grained simulations of biomimetic bilayers support this leaflet replacement model and also reveal that CAV1-8S accumulates 40-70 cholesterol molecules into a disordered monolayer between the complex and its distal lipid leaflet. We find that CAV1-8S preferentially associates with positively curved membrane surfaces due to its influence on the conformations of distal leaflet lipids, and that these effects laterally sort lipids. Large-scale simulations of multiple caveolin assemblies confirmed their association with large, positively curved membrane morphologies consistent with the shape of caveolae. Further, association with curved membranes regulates the exposure of caveolin residues implicated in protein-protein interactions. Altogether, the unique structure of CAV1-8S imparts unusual modes of membrane interaction with implications for membrane organization, morphology, and physiology. STATEMENT OF SIGNIFICANCECaveolae are membrane invaginations heavily implicated in cellular physiology and disease; however, how their unique shape and function are produced remains enigmatic. Here, following on recent characterization of the unusual structure of the CAV1-8S oligomer, we examine the molecular details of its interactions with its surrounding lipid membrane using simulations and reconstitution experiments. We describe a novel mode of membrane interaction-which we term leaflet replacement-for the CAV1-8S complex that has not previously been observed for any other protein. The biophysical consequences of this unique molecular organization provide mechanistic insights into the functions and organization of caveolae in cells.

biophysics↗

bioRxiv2020

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.

biophysics↗

bioRxiv2017

Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy

Fluorescence Correlation Spectroscopy (FCS) has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners however comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a binding model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants KD. The total protein concentration, but not the fraction of labeled protein needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds major-minor-mix liposomes. A Langmuir isotherm model yields KD = (2.1{+/-}1.1)M as the single site dissociation constant. The dual-color FCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.

biophysics↗