Pressure Effects on Submicrosecond Phospholipid Dynamics Using a Long-Lived Fluorescence Probe

Abstract

The effects of applied external hydrostatic pressure on submicrosecond lipid motions in DPPC⁴ bilayers have been examined using coronene (a long-lived planar fluorescent molecule) and DPH. Steady-state fluorescence emission anisotropy (EA) values (<r>) obtained for probe-labeled DPPC SUVs measured at different fixed temperatures above T _c as a function of increasing hydrostatic pressure reveal pressure-induced lipid phase transition profiles. For coronene-labeled samples, the observed lipid “melt” profiles are broad and shifted to higher midpoint EA pressure values (P _1/2) compared with corresponding DPH-labeled SUVs at the same temperature. The data suggest lipid motions occurring on the submicrosecond time scale, detected only by using a long-lived fluorescence probe, which occur well above the normally reported “fluid–gel” lipid phase transition. Slopes of the pressure-to-temperature equivalence plots (dP _1/2/dT = 39 bar/K) obtained for DPH-or coronene-labeled DPPC SUVs are identical within experimental error and reflect probe independence. For DPH, the slope of the P _1/2(T) plot provides the expected phase transition phospholipid volume change. However, intercept values (at P _1/2 = 1 bar) or apparent phase transition temperatures obtained from the equivalence plots for the two probes are not equal. Differences appear to arise due to the very disparate fluorescence lifetime values of the two probes, which result in rotational sensitivity of coronene to gel lipid volume fluctuations occurring during the extended time window provided by coronene fluorescence.