Single-molecule fluorescence vistas of how lipids regulate membrane proteins

Abstract

The study of membrane proteins is undergoing a golden era, and we are gaining unprecedented knowledge on how this key group of proteins works. However, we still have only a basic understanding of how the chemical composition and the physical properties of lipid bilayers control the activity of membrane proteins. Single-molecule (SM) fluorescence methods can resolve sample heterogeneity, allowing to discriminate between the different molecular populations that biological systems often adopt. This short review highlights relevant examples of how SM fluorescence methodologies can illuminate the different ways in which lipids regulate the activity of membrane proteins. These studies are not limited to lipid molecules acting as ligands, but also consider how the physical properties of the bilayer can be determining factors on how membrane proteins function.

Keywords: EphA2; PIP2; SMALP; cholesterol; fluorescence resonance energy transfer; hydrophobic mismatch.

Figure 1.. Lipids use different mechanisms to activate membrane proteins.

A, The presence of the lipid ligand PIP₂ (red), which is distributed asymmetrically across the lipid bilayer, can induce a conformational change in a membrane protein (blue) that alters function. B, A change in protein conformation, activity, and/or dynamics can also occur when specific lipids (green) surround the TMD causing changes in the physical properties of the protein solvent (marked as arrows).

Figure 2.. Experimental diagram of TIRF single molecule microscopy.

A, Generalized TIRF microscope schematic. The magnified area shows a membrane protein isolated by detergent solubilization or reconstitution using membrane-scaffolding proteins into nanodiscs (blue cylinders), styrene maleic-acid for SMALPs (green band), and liposomes. B, Example fluorescent traces are shown for the main SM modalities: photobleaching, Förster resonance energy transfer (FRET), and protein induced fluorescence enhancement (PIFE). In FRET experiments, green lines denote the donor fluorescence and red traces correspond to the acceptor.

Figure 3:. KirBac1.1 closure is induced by PIP₂.

A, The red arrow marks the distance between the cysteine residues where the donor and acceptor dyes are located (T120C and A270C, shown as red dots). The graphs show raw fluorescence and FRET (dark blue). Data are shown for control conditions (top), and in the presence of PIP₂ (bottom). B, FRET contour plots show that when the channel is closed by PIP₂, there is an increase in the distance between the TMD and C-terminus domain, revealed as in increase of the channel population with a FRET efficiency of ~0.15. Experiments were performed in liposomes of POPE:POPG (3:1) in the presence of 1% PIP₂. Figure modified from (48), with permission.

Figure 4.. EphA2 signaling is determined by ligand and lipid interactions.

A, Two alternate EphA2 activation mechanisms. B, Transmembrane domain crossing angles of EphA2 in thin (14:1 PC) and thick (22:1 PC) bilayers. C, Single molecule photobleaching of fluorescently conjugated TMJM EphA2 in SMALPs. Quantification of TMD monomers (left) and dimers (middle) indicate a monomer-dimer equilibrium (right). Adapted from (35) with permission.

Figure 5.. Lipid solvation controls the dimerization of the CLC-ec1 transporter.

A, Dimerization of CLC-ec1 is mediated by a small protein interface, showed in green and blue for each monomer. Arrows indicate the pathways for Cl⁻ and H⁺ transport. The hydrophobic core of the membrane is shown in yellow, and the hydrated regions in cyan. B, side view of the interface can be observed when the figure in A is rotated, and a monomer removed. The dashed red lines mark the hydrophobic surface, which show two polar pockets (red arrows) that cause membrane defects. C, Molecular dynamics simulation of CLC-ec1 shows acyl chain deformation around the transporter dimerization interface, resulting in local bilayer thinning. Data are shown for a monomer, with interface helices in yellow. Figure modified from (71), with permission.