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Cholesterol is a major constituent of biological membranes in mammalian cells. Experiments have shown that cholesterol influences the physical properties of the plasma membrane, such as lateral diffusion and phase equilibrium. In addition to controlling the 2-dimensional phase behaviour and mobility of lipids in membranes, cholesterol has also been implicated in the transbilayer diffusion of lipids across the bilayer. Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip–flop for 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the presence of cholesterol using planar supported lipid bilayer (PSLB) model membranes. Asymmetric PSLBs were prepared using the Langmuir–Blodgett (LB) method by placing a perdeuterated lipid analogue in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of DSPC within the membrane by measuring the decay in the CH3vs intensity at 2875 cm−1 with time and as a function of temperature. A complete kinetic analysis of DSPC flip–flop and the effect of cholesterol on the DSPC dynamics are presented. An analysis of the kinetic data in the framework of Eyring theory provides important insight into the transition state enthalpy (ΔH‡), entropy (ΔS‡) and free energy (ΔG‡) for this important biological process. In addition, the transmembrane migration of cholesterol molecules was also explored by SFVS. These combined studies are aimed at providing new insight in to the transbilayer migration of phospholipids and cholesterol in biological membranes and the effects cholesterol plays in membrane dynamics.
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