Red edge excitation shift of a deeply embedded membrane probe: Implications in water penetration in the bilayer

The biological membrane is a highly organized anisotropic molecular assembly. While the center of the bilayer is nearly isotropic, the upper portion, only a few angstroms away toward the membrane surface, is highly ordered. How this organization correlates with the degree of water penetration into the bilayer interior is not clear. in general, it is believed that there is not much water in the deeper hydrocarbon regions of: the bilayer. In this study, we have utilized the phenomenon of wavelength-selective fluorescence to address this question. We show here that when the same fluorescent group (i.e., 7-nitrobenz-2-oxa-1,3-diazol-4-yl or NBD) is localized at different depths within the bilayer (viz., near the membrane interface in case of the headgroup-labeled NBD-phosphatidylethanolamine (NBD-PE) and near the center of the bilayer in NBD-cholesterol), the degrees to which their fluorescence properties exhibit solvent-induced effects are markedly different. For example, the headgroup-labeled NBD-PE exhibits a much stronger red edge excitation shift (REES) relative to that of NBD-cholesterol. This indicates lesser restriction to mobility in this region as compared to the polar/hydrocarbon interface. In the gel phase, however, REES of NBD-PE did not show any significant change while NBDcholesterol exhibited no REES. In addition, NBD-cholesterol exhibits a stronger dependence of fluorescence polarization on excitation wavelength in fluid membranes. We attribute these results to the more compact arrangement of the lipid acyl chains in the gel phase which results in lesser water penetration. Since the hydrophobic core of the lipid bilayer is made up of methyl and methylene groups, the only solvent dipoles capable of any interaction with the dipole of the fluorophore giving rise to the REES effect in the fluid phase have to be water molecules that have penetrated deep into the bilayer close to the NBD moiety of NBD-cholesterol. Our results indicate that at least in the fluid phase of the membrane, penetration of water in the deep hydrocarbon region of the bilayer does indeed occur.