Red Edge Excitation Shift of a Deeply Embedded Membrane Probe:  Implications in Water Penetration in the Bilayer

Abstract

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 NBDcholesterol. 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.

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

“…After bath sonication for 10 min and vortexing for 5 min, the concentration of the solution was checked spectrophotometrically at 484 nm, using an extinction coefficient of 22,000 M −1 cm −1 [31]. Several dilutions with running buffer were then made and subjected immediately to SPR analysis.…”

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

“…This offers the possibility of using wavelength-selective fluorescence as a novel approach to investigate the depth of membrane penetration of a reporter fluorophore i.e., as a membrane dipstick. This was tested by demonstrating that chemically identical fluorophores, varying solely in terms of their localization at different depths in the membrane, experience very different local environments, as judged by wavelength-selective fluorescence parameters (Chattopadhyay and Mukherjee, 1999b). Two anthroyloxy stearic acid derivatives where the anthroyloxy group has previously been found to be either shallow (2-AS) or deep (12-AS) were used.…”

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

“…38,112 It is therefore the membrane interface which is most likely to display REES. 105,[113][114][115] The application of REES to study the organization and dynamics of membranes has been reviewed recently. …”

“…Such extrinsic fluorescent probes are widely used to study the dynamics of proteins and membranes. 74,105,113,114,176 The advantage of this approach is that one has a choice of the fluorescent label to be used and, therefore, specific probes with appropriate characteristics can be designed for specific applications. Many of these probes display high sensitivity to the polarity of the local environment.…”

“…35,38 The NBD group therefore has been used extensively to explore the organization and dynamics in membranes and membrane-mimetic media utilizing the REES approach. 113,114,[191][192][193][194][195][196][197] Apolipoprotein C-II (apoC-II) is an exchangeable 79-residue apolipoprotein found in blood plasma and is essential for the activation of lipoprotein lipase and therefore in the hydrolysis of triacylglycerols and the prevention of hypertriglyceridaemia. 198 By covalently labeling the peptide fragment of apolipoprotein C-II (apoC-II ) with NBD, it was found that NBD-apoC-II exhibits an increased REES of 25 nm when bound to egg yolk phosphatidylcholine membranes when compared to the corresponding REES in aqueous solution (9 nm).…”