Polarity of lipid bilayers. A fluorescence investigation

Abstract

Through steady-state and time-resolved fluorescence experiments, the polarity of the bilayers of egg phosphatidylcholine vesicles was studied by means of the solvatochromic 2-anthroyl fluorophore which we have recently introduced for investigating the environmental micropolarity of membranes and which was incorporated synthetically in phosphatidylcholine molecules (anthroyl-PC) in the form of 8-(2-anthroyl)octanoic acid. Fluorescence quenching experiments carried out with N,N-dimethylaniline and 12-doxylstearic acid as quenchers showed that the 2-anthroyl chromophore was located in depth in the hydrophobic region of the lipid bilayer corresponding to the C9-C16 segment of the acyl chains. Steady-state fluorescence spectroscopy revealed a nonstructured and red-shifted (lambda em(max) = 464 nm) spectrum for the probe in egg-PC bilayers, which greatly differed from the structured and blue (lambda em(max) = 404 nm) spectrum the fluorophore was shown to display in n-hexane. While the fluorescence decays of the fluorophore in organic solvents were monoexponential, three exponentials were required to account for the fluorescence decays of anthroyl-PC in egg-PC vesicles, with average characteristic times of 1.5 ns, 5.5 ns, and 20 ns. These lifetime values were independent of the emission wavelength used. Addition of cholesterol to the lipid did not alter these tau values. One just observed an increase in the fractional population of the 1.5-ns short-living species detrimental to the population of the 20-ns long-living ones. These observations enabled time-resolved fluorescence spectroscopy measurements to be achieved in the case of the 1/1 (mol/mol) egg-PC/cholesterol mixture. Three distinct decay associated spectra (DAS) were recorded, with maximum emission wavelengths, respectively, of 410 nm, 440 nm, and 477 nm for the 1.5-ns, 6-ns, and 20-ns lifetimes found in this system. On account of the properties and the polarity scale previously established for the 2-anthroyl chromophore in organic solvents, these data strongly suggest the occurrence of three distinct excited states for anthroyl-PC in egg-PC bilayers, corresponding to three environments for the 2-anthroyl chromophore, differing in polarity. The lifetime of 1.5 ns and the corresponding structured and blue (lambda em(max) = 410 nm) DAS account for a hydrophobic environment, with an apparent dielectric constant of 2, which is that expected for the hydrophobic core of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)

Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

“…The precise orientation and location of the NBD group of this molecule in the membrane is known London, 1987, 1988;Mitra and Hammes, 1990;Wolf et al, 1992;Abrams and London, 1993). This group has been found to be localized at the membrane interface which has unique motional and dielectric characteristics distinct from both the bulk aqueous phase and the hydrocarbon-like interior of the membrane (Seelig, 1977;Ashcroft et al, 1981;Stubbs et al, 1985;Perochon et al, 1992;Slater et al, 1993;Venable et al, 1993;White and Wimley, 1994;Gawrisch et al, 1995) thus making it an ideal probe for monitoring red edge effects. Furthermore, previous electrophoretic measurements have shown that the NBD group in NBD-PE is uncharged at neutral pH in the membrane (Chattopadhyay and London, 1988).…”

“…This not only results in the anisotropic behavior of the constituent lipid molecules, but more importantly, the environment of a probe molecule becomes very much dependent on its precise localization in the membrane. While the center of the bilayer is nearly isotropic, the upper portion, only a few angstroms away toward the membrane surface, is highly ordered (Seelig, 1977;Ashcroft et al, 1981;Stubbs et al, 1985;Perochon et al, 1992;Slater et al, 1993;Venable et al, 1993;White and Wimley, 1994;Gawrisch et al, 1995). As a result, properties such as polarity, fluidity, segmental motion, ability to form hydrogen bonds and extent of solvent (water) penetration would vary in a depth-dependent manner in the membrane (Fig.…”

“…In addition, it is becoming increasingly evident that tryptophan residues in integral membrane proteins and peptides are not uniformly distributed and that they tend to be localized toward the membrane interface, possibly because they are involved in hydrogen bonding (Ippolito et al, 1990) with the lipid carbonyl groups or interfacial water molecules. As mentioned earlier, the interfacial region in membranes is characterized by unique motional and dielectric characteristics distinct from both the bulk aqueous phase and the hydrocarbon-like interior of the membrane (Seelig, 1977;Ashcroft et al, 1981;Stubbs et al, 1985;Perochon et al, 1992;Slater et al, 1993;Venable et al, 1993;White and Wimley, 1994;Gawrisch et al, 1995). The tryptophan residue has a large indole side chain that consists of two fused aromatic rings.…”

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

“…While the center of the bilayer is nearly isotropic, the upper portion, only a few angstroms away toward the membrane surface, is highly ordered. 38,[101][102][103][104][105] Properties such as polarity, fluidity, segmental motion, ability to form hydrogen bonds and extent of solvent penetration vary in a depth-dependent manner in the membrane. The interfacial region in membranes is the most important region so far as the dynamics and function of the membrane is concerned.…”

“…The membrane interface is characterized by unique motional and dielectric characteristics distinct from both the bulk aqueous phase and the more isotropic hydrocarbon-like interior of the membrane. [101][102][103][104]106 It is a chemically heterogeneous region composed of lipid headgroup, water and portions of the acyl chain. 107 Overall, the interfacial region of the membrane accounts for 50% of the thermal thickness of the bilayer.…”