Intrinsic gramicidin fluorescence lifetimes in bilayer lipid membranes and in vesicles

Abstract

Intrinsic Gramicidin A′ tryptophan steady-state fluorescence anisotropies and fluorescence lifetimes have been determined in bilayer lipid membranes (BLMs) prepared from glyceryl monooleate (GMO). In GMO BLMs, fluorescence anisotropy, the r value, was found to be 0.05 ± 0.02. Decays of Gramicidin A′ fluorescence intensities were fitted to the sum of three exponentials (τ1, τ2, and τ3) and appropriate pre-exponentials (A1, A2, and A3). These values allowed for the assessment of average fluorescence lifetimes, [Formula: see text]. These values related to those determined in vesicles prepared from dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and diphytanoylphosphatidylcholine (DPhPC). In BLMs, [Formula: see text], 3.6 ns, and 2.3 ns were determined for vertically, horizontally, and unpolarized average fluorescence lifetimes, respectively. Increasing the applied potential across the BLM from 0 to 80 mV increased [Formula: see text] from 2.2 ns to 4.9 ns and τ1 from 0.43 ± 0.05 to 0.73 ± 0.06 ns, as well as the contributions and lifetimes of the longer lived fluorescence (A2 and A3, τ2 and τ3). The emission maximum of Gramicidin A′ (334 nm in DPPC) and the absence of quenching by iodide ions indicated complete incorporation of the polypeptide into vesicles. The r values were of the order of 0.10 in vesicles prepared from DPPC and DSPC, both in the absence and in the presence of added 1.2 × 10−4 M CsCl. In vesicles prepared from DOPC and DPhPC, r values increased to 0.13 and 0.14 in water and to 0.15 and 0.20 in 1.2 × 10−4 M CsCl, respectively. At 25.0 °C, the temperature of the measurements, DPPC and DSPC are in their "solid" states, but DOPC and DPhPC are in their "liquid" states, [Formula: see text] values for Gramicidin A′ in vesicles prepared from DPPC, DOPC, and DSPC were all in the 3.0 ± 0.3 ns range. In DPhPC vesicles, [Formula: see text] was determined. Time-dependent anisotropics became observable in DOPC and DPhPC vesicles, particularly in the presence of 1.2 × 10−4 M CsCl. Keywords: gramicidin, fluorescence lifetimes, vesicles, bilayer lipid membranes, time-dependent anisotropics.