Plasma cell membranes have a rich diversity of lipids that vary by cell type. However, a common feature among plasma membranes is the predominance of cholesterol and phosphatidylcholine (PC) lipids with different degrees of chain unsaturation, i.e., number of double bonds in the acyl chains. These double-bond structures cause the acyl chains to be more flexible and result in looser lipid packing. In contrast, cholesterol causes lipids to pack more tightly. Such opposing effects have important consequences on local functional properties in cell membranes, including the ability of certain membrane patches to deform or bend during vesiculation or viral budding events (e.g., coronavirus). This necessitates a better understanding of how cholesterol affects the bending rigidity of membranes locally, depending on the surrounding lipid composition including the degree of PC chain unsaturation. We recently showed that cholesterol-induced stiffening of mono-unsaturated PC membranes is associated with changes in lipid packing. Here, we report results on PC membranes with different unsaturation levels and double-bond locations along and across the two acyl chains. To examine the mesoscale bending rigidity, we use neutron spin-echo spectroscopy, solid-state <sup>2</sup>H NMR relaxometry, and molecular dynamics simulations. Our results demonstrate that membrane stiffening by cholesterol is most pronounced for saturated PC membranes and decreases with increasing chain unsaturation. However, stiffening effects are detectable even for highly unsaturated PC membranes with up to 6 double bonds. Further examination of the results shows that the bending rigidity scales with the area per lipid, consistent with structure-property relations of molecular self-assemblies. Our results also illustrate the importance of the length and time scales over which membrane bending rigidity is measured. These observations of emergent membrane properties pave the way to future studies on more complex cell membrane mimics, including laterally heterogeneous membranes.

Cholesterol stiffening of phosphatidylcholine membranes depends on acyl chain unsaturation

ACS Spring 2022

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