The effect of temperature and pressure on structural and dynamic properties of phospholipid/sterol mixtures - A steady-state and time-resolved fluorescence anisotropy study

Cholesterol and the plant sterol beta-sitosterol were added up to 50 mol% to unilamellar vesicles of the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC). One aim of this study was to investigate the influence of the sterol side chain structure on the acyl chain orientational order of the lipid bilayer by measuring the steady-state fluorescence anisotropy r(ss) of the fluorophore 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Experiments were carried out in the temperature range from 30 to 60 degrees C, i.e. at temperatures below and above the gel to liquid-crystalline phase transition temperature T-m of DPPC bilayers (T-m approximate to 41.5 degrees C). In general, the incorporation of either sterol in the DPPC bilayer enhances the orientational order of TMA-DPH in the liquid-crystalline state of DPPC, whereas the orientational order in the gel state is slightly reduced. It is found that the molecular interactions between DPPC and sterols in the hydrophobic membrane interior distinctly depend on the structure of the side chain of sterols. The presence of the additional ethyl group on the alkyl side chain of beta-sitosterol markedly reduces the effectiveness of the sterol on ordering liquid-crystalline bilayers. In addition to the temperature dependent studies, we measured the pressure dependence of r(ss) at T = 50 degrees C. For this purpose, a pressure vessel was constructed that permits fluorescence polarization measurements up to pressures of 2 kbar. The pressure profiles of r(ss) of the two systems turned out to be essentially mirror images of the temperature profiles of r(ss). To yield also information about the rotational dynamics of the probe TMA-DPH in DPPC/sterol mixtures, time-resolved experiments were carried out by using multifrequency cross-correlation phase fluorometry. The rotational rates of TMA-DPH depolarizing motions are only modestly increased in going from the gel to the liquid-crystalline phase of DPPC. However, a marked increase in the contribution of fast rotational motions of TMA-DPH is found as the temperature is increased through the main transition. No significant differences in rotational rates of TMA-DPH exist between samples with embedded cholesterol or beta-sitosterol at T = 55 degrees C. The rotational rate of TMA-DPH in the gel state of the lipid bilayer seems to be slightly larger in the case of beta-sitosterol. Cholesterol and beta-sitosterol have a similar effect on the contribution of fast rotational dynamics in the ns time regime in the interfacial region of the bilayer. It decreases about 40% upon addition of 50 mol% sterol. The influence of temperature, pressure and sterol concentration on the fluorescence Lifetime of TMA-DPH was also examined and is discussed in the light of water penetration into the bilayer system. The addition of cholesterol results in a marked increase in average fluorescence lifetime in both lipid phases, which is slightly less pronounced for the system DPPC/beta-sitosterol. Embedding sterols in lipid bilayers has a significantly more pronounced effect on the fluorescence lifetime and thus the hydrophibicity of the membrane than an increase of pressure of several hundred to thousand bar. These results clearly demonstrate the ability of sterols to efficiently regulate the structure, motional freedom and hydrophobicity of biomembranes, and their efficiencies seem to depend modestly on the detailed molecular structure of the sterols.