Localization of Cholesterol within Supported Lipid Bilayers Made of a Natural Extract of Tailor-Deuterated Phosphatidylcholine

Abstract

Cholesterol is an essential component of mammalian membranes and is known to induce a series of physicochemical changes in the lipid bilayer. Such changes include the formation of liquid-ordered phases with an increased thickness and a configurational order as compared to liquid-disordered phases. For saturated lipid membranes, cholesterol molecules localize close to the lipid head group-tail interface. However, the presence of polyunsaturated lipids was recently shown to promote relocation of cholesterol toward the inner interface between the two bilayer leaflets. Here, neutron reflection is used to study the location of cholesterol (both non-deuterated and per-deuterated versions are used) within supported lipid bilayers composed of a natural mixture of phosphatidylcholine (PC). The lipids were produced in a genetically modified strain of Escherichia coli and grown under specific deuterated conditions to give an overall neutron scattering length density (which depends on the level of deuteration) of the lipids matching that of DO. The combination of solvent contrast variation method with specific deuteration shows that cholesterol is located closer to the lipid head group-tail interface in this natural PC extract rather than in the center of the core of the bilayer as seen for very thin or polyunsaturated membranes.

The Production of Matchout-Deuterated Cholesterol and the Study of Bilayer-Cholesterol Interactions

“…The bilayer is broken up into five-layers: an inner head group, three distinct tail regions and an outer head group exposed to the solvent contrast. The three sections of the tail core allow for an asymmetric distribution of cholesterol, as reported previously 26 . The thickness, coverage and roughness of the tail regions were all constrained during the fitting process to be symmetric (each of these parameters were kept equal throughout all three core layers) to limit the number of variables.…”

“…However, the fits were not satisfactory, suggesting an oversimplification of the composition of the SLB, assumed to be symmetrical and uniform throughout the core. A five-layer model was therefore necessary to understand where the cholesterol was located in the core - whether it accumulates in the centre of the bilayer core or positions itself at the edges of the bilayer close to the headgroups as seen previously 11,26 .…”

Assessing molecular simulation for the analysis of lipid monolayer reflectometry

“…Neutron and x-ray reflectometry techniques are popular in the study of layered structures, such as polyelectrolyte-surfactant mixtures [1], lipid bilayer systems [2], electrodeposited films [3], and dye-sensitised solar cell materials [4]. Unlike other surface-sensitive techniques, such as atomic force microscopy (AFM) or scanning electron microscopy (SEM), reflectometry methods can investigate buried interfaces in addition to the material surface.…”

Production of perdeuterated fucose from glyco-engineered bacteria

“…The deuteration of biomolecules by stable isotope labelling is of high interest for nuclear magnetic resonance (NMR) spectroscopy and neutron scattering studies of the structure and dynamics of biological macromolecules and is now widely used (Haertlein et al 2016, Blakeley andPodjarny 2018). Tailor-designed deuteration can be used very effectively in studies of multicomponent systems by small-angle neutron solution scattering (Laux et al 2008, Cuypers et al 2013b, Dunne et al 2017, Josts et al 2018, Maric et al 2019 and neutron reflection (Grage et al 2011, Hellstrand et al 2013, Waldie et al 2018. In Laue neutron crystallography, the use of perdeuterated protein imparts major benefits in terms of data quality and interpretation (Haupt et al 2014, Dajnowicz et al 2017, Yee et al 2019, Kwon et al 2020 and the same is true in the case of monochromatic neutron crystallography (Cuypers et al 2013a, Cuypers et al 2016.…”