Dynamic properties of the lipid-water interface of model membranes as revealed by lifetime-resolved fluorescence emission spectra

Abstract

We examined the dynamic properties of the lipid-water interface region of model membranes, on the nanosecond time scale, by using the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). In particular, we examined the steady-state emission spectra of TNS as its average lifetime was decreased by oxygen quenching. Under these quenching conditions the centers of gravity (Vcg) of the emission spectra shift ot shorter wavelengths. The lifetime dependence of these shifts reveals the time dependence of membrane relaxation around the excited-state dipole moment of TNS. The lipids investigated include dioleoyl-, dimyristoyl-, and dipalmitoyl-L-alpha-phosphatidylcholines, bilayers containing cholesterol, and an ether analogue of dipalmitoyl-L-alpha-phosphatidylcholine. For these lipids, the spectral relaxation times and the temperature dependence of the relaxations are similar in magnitude. Most relaxation times fall in the range of 0.6-6 ns, and except for the ether analogue, the inactivation energies for spectral relaxation are 10 plus or minus 2 kcal/mol. The average energy loss during spectral relaxation was 1000 cm(-1). However, for the saturated phosphatidylcholines at temperatures below their transition temperatures, smaller relaxation losses were observed (approximately 600 cm(-1)). We attribute these smaller losses to ordering of the polar head groups around the ground-state dipole moment of TNS. Overall, these results indicate that the dynamic properties of the lipid-water interface region are similar among the phosphatidylcholines and depend only slightly on the chemical composition and phase state of the acyl side chains.

FIGURE 1:

Effects of temperature on fluorescence emission spectra of TNS-labeled vesicles. The vertical solid and dashed lines indicate v_cg⁻¹, in nanometers, at 5 and 55°C, respectively.

FIGURE 2:

Stern-Volmer plot for oxygen quenching of TNS-labeled DMPC/Chol (3: 1) vesicles. The fluorescence intensities were calculated from the integrals of the emission spectra from 340 to 580 nm. The dashed line illustrates the τ₀/τ values expected at 25°C for the dynamic component of the quenching (K_D, Table I).

FIGURE 3:

Effects of oxygen quenching on fluorescence emission spectra of TNS-labeled DMPC/Chol(3:1) vesicles. The vertical solid and dotted lines illustrate v_cg⁻¹, in nanometers, in the absence and presence of oxygen, respectively. The fluorescence lifetimes at each oxygen pressure are shown on the figure.

FIGURE 4:

Fluorescence emission spectra of TNS-labeled vesicles in the absence and presence of oxygen quenching. The vertical solid and dashed lines illustrate the unquenched and quenched value, respectively, of v_cg⁻¹, in nanometers.

FIGURE 5:

Lifetime-resolved centers of gravity for TNS-labeled DOPC and DPPC-E vesicles. The solid and dashed lines are both used to represent the theoretical curves, fit through the data points shown, for the v₀, v_∞, and τ_R values listed in Table II. The dashed lines are used selectively to illustrate those data which could not be fit to the v₀ and v_∞, observed at temperatures above the transition temperature of the vesicles.

FIGURE 6:

Lifetime-resolved centers of gravity for TNS-labeled DMPC and DPPC vesicles.

FIGURE 7:

Lifetime-resolved centers of gravity for TNS-labeled DMPC/cholesterol vesicles at 3:1 and 6:1 molar ratios.

FIGURE 8:

Quenching of DPPC-bound TNS by N-methylnicotinamide. The bimolecular quenching constants (k_q) were obtained from τ₀/τ = 1 + k_qτ₀ [quencher]. 10-MHz phase lifetimes were used. The excitation wavelength was 335 nm, and the emission filters were Corning 7–51 and 0–51.

FIGURE 9:

Arrhenius plots for membrane relaxation times of TNS-labeled DOPC, DMPC, and DPPC-E vesicles.