Localization and Orientation of Xanthophylls in a Lipid Bilayer

Abstract

Xanthophylls (polar carotenoids) play diverse biological roles, among which are modulation of the physical properties of lipid membranes and protection of biomembranes against oxidative damage. Molecular mechanisms underlying these functions are intimately related to the localization and orientation of xanthophyll molecules in lipid membranes. In the present work, we address the problem of localization and orientation of two xanthophylls present in the photosynthetic apparatus of plants and in the retina of the human eye, zeaxanthin and lutein, in a single lipid bilayer membrane formed with dimyristoylphosphatidylcholine. By using fluorescence microscopic analysis and Raman imaging of giant unilamellar vesicles, as well as molecular dynamics simulations, we show that lutein and zeaxanthin adopt a very similar transmembrane orientation within a lipid membrane. In experimental and computational approach, the average tilt angle of xanthophylls relative to the membrane normal is independently found to be ~40 deg, and results from hydrophobic mismatch between the membrane thickness and the distance between the terminal hydroxyl groups of the xanthophylls. Consequences of such a localization and orientation for biological activity of xanthophylls are discussed.

Figure 1

A scheme presenting localization of the molecule of zeaxanthin in the lipid bilayer. Orientation of the axis normal to the plane of the membrane, molecular axis and the transition dipole are marked. The molecular axis has been defined by the linear part of the conjugated C = C double bound system.

Figure 2

The results of microscopic imaging of single lipid vesicles containing xanthophylls incorporated to the lipid phase. Three panels show fluorescence intensity, anisotropy and lifetime. The images represent an equatorial vesicle cross-section in the focal plane of a microscope. The upper panel presents a liposome containing incorporated zeaxanthin, the lower panel presents a liposome containing incorporated lutein. The direction of the electric vector of probing light is shown below the images (collinear with the Y axis).

Figure 3

The results of Raman microscopic imaging of single lipid vesicles containing xanthophylls incorporated to the lipid phase. Three panels show optical images, Raman images and averaged Raman scattering spectra recorded during imaging. The images represent an equatorial vesicle cross-section in the focal plane of a microscope. The upper panel presents a liposome containing incorporated zeaxanthin, the lower panel presents a liposome containing incorporated lutein. The images are based on the integration of the spectra in the range 1500–1550 cm⁻¹. The direction of the electric vector of probing light is shown below the images (collinear with the Y axis).

Figure 4

Results of molecular dynamics simulations of xanthophylls embedded in the lipid bilayer. (A) Free energy profiles (green) and corresponding probability distributions (red) of the angle between the axis of the polyene chromophore of lutein (left) or zeaxanthin (right) and the membrane normal (φ-angle). The vertical dashed lines show the equilibrium averages of φ-angle obtained from the calculated distributions. (B) Probability distributions of the distance between the terminal rings of both xanthophylls projected on the membrane normal (RR-distance). The vertical dashed lines show the thickness of the hydrophobic core of the membrane (D_h). (C) MD simulation snapshot showing a representative arrangement of a lutein molecule embedded in the DMPC membrane.