Effect of gramicidin addition upon the physicochemical properties of dipalmitoyl phosphatidyl choline large unilamellar vesicles

Abstract

In this work, we report the results bearing on the effect of gramicidin incorporation upon the dynamic and structural properties of dipalmitoyl phosphatidyl choline (DPPC) large unilamellar vesicle (LUVs). Results were obtained at different depths in the bilayer, both in the bulk of the bilayer and the vicinity of the added pentadecapeptide. This analysis was performed employing a series of extrinsic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 6-dodecanoyl-2-dimethyl aminenaphthalene (Laurdan), pyrenedodecanoic acid, pyrene butyric acid, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (Py–C 10–PC) and 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (bis-Py–C 10–PC)). Fluorescence measurements were carried out after direct excitation of the probe or after its excitation by resonant energy transfer (RET) from the intrinsic tryptophan groups of the polypeptide. The last procedure allows an evaluation of the lipidic annulus properties in the vicinity of the incorporated polypeptide. In the gel state, gramicidin incorporation increases the order near the vesicle interface, as sensed by TMA-DPH. This increase is observed, above the phase transition temperature, over all the bilayer. This effect is probably related to a decrease in the lateral diffusion of the lipids, as evidenced by the reduced formation of intermolecular excimers following Py–C 10–PC excitation. Gramicidin incorporation also increases the penetration and/or mobility of water molecules located near the bilayer interface. In the gel phase, these changes are more relevant in the central part of the bilayer. When the LUVs are in the liquid crystalline state, the effect is observed through all the bilayer. In this state, the polypeptide incorporation also increases the diffusion/concentration of oxygen, irrespective of the probe localization. Resonance energy transfer between Trp residues and Laurdan, Py–C 10–PC or bis-Py–C 10–PC dyes was employed to evaluate the properties of the lipidic annulus surrounding the polypeptide. In the gel phase, gramicidin incorporation produces an increase in the penetration and/or dynamics of water molecules, while in the liquid crystalline state it leads to a decrease of the acyl chain mobility in the deepest parts of the bilayer.