Membrane curvature: Bending begets binding

Lipidomics Gateway (23 September 2009) [doi:10.1038/lipidmaps.2009.24]

Curved membranes intrinsically recruit large densities of alkylated or amphipathic proteins independent of affinity, with no requirement for specific recognition motifs.

Bending a membrane introduces packing defects that become adsorption sites for amphiphilic molecules. For full figure and legend see Nat. Chem. Bio. 10.1038/nchembio.213

The tightly-regulated curve of a biological membrane is determined by both the lipid composition and the action of membrane-bending proteins. Membrane curvature (MC) in turn regulates the localization of proteins that contain specific recognition motifs. Amphipathic α-helices are important MC-sensors for a range of proteins, and are thought to have greater affinity for positively curved membranes through recognition of curve-induced defects in lipid packing. MC-dependent measurements, classically made in vitro, are averaged across liposomes of unavoidably variant diameter. This reduces accuracy and makes calculating the affinity difficult.

Dimitrios Stamou and colleagues eliminated this problem by using fluorescence microscopy to measure MC-selective binding on individual liposomes. Their study, published in Nature Chemical Biology, reveals that curved membranes themselves intrinsically accommodate a higher density of amphiphilic molecules. Thus sensing of membrane curvature emerges as a generic property of lipid membranes, rather than a specific protein attribute, and may control the distribution of proteins anchored by any hydrophobic motif.

To make their single liposome binding measurements, Stamou and colleagues used biotinylated liposomes of varied sizes, labeled with a chromophore, and immobilized them on streptavidin. The fluorescence intensity, measured by confocal microscopy, was used to calculate the individual liposomal diameters. Many liposomes were monitored in parallel in one frame of view. A second chromophore was used to tag the molecule of interest, and the ratio of the two fluorescent signals revealed the density of binding of labeled molecules to each individual liposome.

The authors first assayed binding of amphipathic α-helical (AH) peptides to the immobilized liposomes, and plotted the bound AH density against liposomal diameter. The peptide density was constant for liposomes measuring 200nm and above, but below that size it increased sharply, such that 50nm liposomes had an AH density of 50:1 over larger liposomes. This was reflected in a 50-fold greater maximum density at saturating AH concentrations for the smaller liposomes. The equilibrium disassociation constant, K _d, was found to have only a minor contribution to curvature-selective binding, and only at AH concentrations below K _d. These data indicate that curvature sensing by AH is not mediated by varied affinity for different curvatures.

To explain their results, the authors proposed a geometrical model in which highly-curved membranes have a greater density of lipid packing defects, which provide sites for amphiphilic molecules to insert themselves. The density of defects, and thus the density of binding, depends on the increased area of the outer leaflet of a membrane as it is bent. Although specific affinity-based interactions of some proteins might mask this effect at concentrations below K _d, at higher concentrations these interactions are saturated and the defect density effect will predominate. Peptide density will thus be proportional to defect density, and the data for the two AH peptides tested fitted well with the model.

The model is not restricted to amphipathic helices, but rather predicts that insertion of any amphiphilic molecules will be curvature selective. To test this, the authors used simple alkyl chains, including the palmitoyl motif that is the most common membrane anchor. All alkyl chains measured were sensors of MC, and the sensor potency of the palmitoyl group was comparable to that of the AHs. The head group of the alkyl chains had no influence, whereas alterations to the chain itself only fine-tuned the curvature dependency. Furthermore, adding proteins onto the chains did not alter their curvature-sensing properties.

This study proposes that membrane curvature is a generic mechanism for influencing the localization of all amphiphilic molecules. Influenced by the concentration of fusogenic lipids in the membrane, or by the action of proteins, MC may thus be of great importance for protein trafficking and cell signaling.

Emma Leah