Molecular dynamics simulations and Kelvin probe force microscopy to study of cholesterol-induced electrostatic nanodomains in complex lipid mixtures

Abstract

The molecular arrangement of lipids and proteins within biomembranes and monolayers gives rise to complex film morphologies as well as regions of distinct electrical surface potential, topographical and electrostatic nanoscale domains. To probe these nanodomains in soft matter is a challenging task both experimentally and theoretically. This work addresses the effects of cholesterol, lipid composition, lipid charge, and lipid phase on the monolayer structure and the electrical surface potential distribution. Atomic force microscopy (AFM) was used to resolve topographical nanodomains and Kelvin probe force microscopy (KPFM) to resolve electrical surface potential of these nanodomains in lipid monolayers. Model monolayers composed of dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (DOPG), and cholesterol were studied. It is shown that cholesterol changes nanoscale domain formation, affecting both topography and electrical surface potential. The molecular basis for differences in electrical surface potential was addressed with atomistic molecular dynamics (MD). MD simulations are compared the experimental results, with 100 s of mV difference in electrostatic potential between liquid-disordered bilayer (L_d, less cholesterol and lower chain order) and a liquid-ordered bilayer (L_o, more cholesterol and higher chain order). Importantly, the difference in electrostatic properties between L_o and L_d phases suggests a new mechanism by which membrane composition couples to membrane function.

Fig. 1

Schematic of monolayer arrangement in a two-component lipid system. The hydrophilic head groups of the lipids interact with the hydrophilic surface of the mica substrate, resulting in the tail groups facing upwards into the air. The difference in height (Δh) is extracted from AFM topography and difference in electrical surface potential (ΔV, equivalent to V_topo(high) — V_topo(low)) is determined using KPFM.

Fig. 2

AFM (in gold) and corresponding FM-KPFM (in blue) illustrating the effect of cholesterol on both the topography and electrical surface potential of a lipid mixture of DPPC and DOPC. AFM images (A) and FM-KPFM images (B) of a DPPC-DOPC mixture (at a ratio of 607:393) are compared to topography captured by AFM and electrical surface potential captured by KPFM of a DPPC-DOPC-cholesterol sample (at a ratio of 560 : 346: 94, images C and D respectively). AFM and KPFM images were scanned in air in ambient conditions. These images are 2 μm by 2 μm; brighter regions correspond to higher topography/electrical surface potential, with darker regions corresponding to regions of lower topography/electrical surface potential, as shown in the labelled scale bars to the right.

Fig. 3

Snapshots of the different simulated systems. cholesterol: yellow, water: cyan, lipid tail: grey, phosphorous: red, nitrogen: blue, oxygen on PG head group: orange, and Na⁺: green L_o and L_d denote liquid-ordered and liquid-disordered, respectively.

Fig. 4

Density profiles and electrostatic potentials across one leaflet of the gel DPPC (pure) bilayer and the liquid DOPC (pure) bilayer. (A) Density profiles of one leaflet of a fluid phase DOPC bilayer (black) and DPPC gel phase bilayer (red). Total density and contributions of water and lipids are shown separately. (B) Contributions to the electrostatic potential from the lipids and water for both systems (see Methods). (C) Total electrostatic potential in both systems. The electrostatic potentials were shifted to zero in bulk water for comparison.

Fig. 5

Density profiles and electrostatic potentials across one leaflet of the ternary mixtures of DPPC/DOPC/Chol bilayers. (A) Density profiles of one leaflet of the L_d bilayer (black) and L_o bilayer (red). Total density as well as contributions of water and lipids are shown separately. (B) Contributions to the electrostatic potential from the lipids and water for both systems (see Methods). (C) Total electrostatic potential in both systems. The electrostatic potentials were shifted to zero in bulk water for comparison.

Fig. 6

AFM and corresponding FM-KPFM illustrating the effect of cholesterol on both the topography and electrical surface potential of a lipid mixture of DPPC and negatively charged dopg. AFM images (A) and FM-KPFM images (B) of a DPPC-DOPG mixture (50 : 50 w/w) are compared to topography captured by AFM and electrical surface potential captured by FM-KPFM of a DPPC-DOPG-Chol (40:40 :20 w/w) sample (images C and D respectively). AFM and FM-KPFM images were scanned in air in ambient conditions. these images are 2 μm by 2 μm.

Fig. 7

Density profiles and electrostatic potentials across one leaflet of the ternary mixtures of DPPG/DOPG/Chol bilayers. (A) Density profiles of one leaflet of the L_d bilayer (black) and L_o bilayer (red). Total density as well as contributions of water and lipids are shown separately. (B) Contributions to the electrostatic potential from the lipids and water for both systems (see Methods). (C) Total electrostatic potential in both systems. tHe electrostatic potentials were shifted to zero in bulk water for comparison.