The influence of cholesterol on the bending energy of lipid membranes is critical for various cellular processes. Despite contradicting reports, our recent work revealed that cholesterol impacts the bending moduli of phosphatidylcholine (PC) bilayers along with cholesterol-induced condensation, regardless of acyl chain unsaturation 1 . Here, we show that similar results were self-consistently observed using solid-state 2 H NMR studies of lipid bilayers with varying acyl chain unsaturation and cholesterol concentrations. For 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), we used a tracer-lipid approach utilizing chain-perdeuterated POPC- d 31 , which facilitates investigation of unsaturated systems. Using 2 H NMR, we measure both structural and dynamic membrane properties through quadrupolar-echo and spin-lattice relaxation experiments. Utilizing the mean-torque model, our 2 H NMR approach determines the area per lipid, while combined analysis of spin-lattice relaxation and squared order parameters measures the bilayer elasticity. 2 We found that saturated 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) membranes exhibit the greatest response to increasing cholesterol concentration, with the highest bending rigidity and order parameter values. A similar increase, but to a lesser extent, was observed in monounsaturated POPC and di-monounsaturated DOPC bilayers depending on the degree of chain unsaturation. These results are commensurate with cholesterol’s effects on molecular packing, evidenced by decreasing area per lipid. Notably, the bending rigidity exhibits a unified exponential relationship with area per lipid regardless of acyl chain unsaturation or cholesterol content 1 Our findings illustrate that cholesterol exerts control over mechanical membrane properties through molecular packing densities determined by acyl chain unsaturation. Understanding cholesterol interactions across saturated and unsaturated lipid membranes elucidates how lipid composition regulates membrane material properties following established structure-property relations with important biomedical and therapeutic applications. 1 Kumarage, T. et al. (2025). Nat. Comm. 16, 7024. 2 Brown, M. F. et al. (2002). J. Am. Chem. Soc. 124, 8471.
Arruda et al. (Sun,) studied this question.