Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy

Abstract

Flash photolysis studies have shown that the membrane lipid environment strongly influences the ability of rhodopsin to form the key metarhodopsin II intermediate. Here we have used plasmon-waveguide resonance (PWR) spectroscopy, an optical method sensitive to both mass and conformation, to probe the effects of lipid composition on conformational changes of rhodopsin induced by light and due to binding and activation of transducin (G(t)). Octylglucoside-solubilized rhodopsin was incorporated by detergent dilution into solid-supported bilayers composed either of egg phosphatidylcholine or various mixtures of a nonlamellar-forming lipid (dioleoylphosphatidylethanolamine; DOPE) together with a lamellar-forming lipid (dioleoylphosphatidylcholine; DOPC). Light-induced proteolipid conformational changes as a function of pH correlated well with previous flash photolysis studies, indicating that the PWR spectral shifts monitored metarhodopsin II formation. The magnitude of these effects, and hence the extent of the conformational transition, was found to be proportional to the DOPE content. Our data are consistent with previous suggestions that lipids having a negative spontaneous curvature favor elongation of rhodopsin during the activation process. In addition, measurements of the G(t)/rhodopsin interaction in a DOPC/DOPE (25:75) bilayer at pH 5 demonstrated that light activation increased the affinity for G(t) from 64 nM to 0.7 nM, whereas G(t) affinity for dark-adapted rhodopsin was unchanged. By contrast, in DOPC bilayers the affinity of G(t) for light-activated rhodopsin was only 18 nM at pH 5. Moreover exchange of GDP for GTP gamma S was also monitored by PWR spectroscopy. Only the light-activated receptor was able to induce this exchange which was unaffected by DOPE incorporation. These findings demonstrate that nonbilayer-forming lipids can alter functionally linked conformational changes of G-protein-coupled receptors in membranes, as well as their interactions with downstream effector proteins.

FIGURE 1

PWR spectra obtained for egg PC lipid bilayer formation, rhodopsin incorporation in the dark, and light activation, in 10 mM phosphate buffer at pH 5 using p-polarized (A) and s-polarized (B) light excitation. All spectra were measured with 632.8 nm exciting light. In both A and B, curve 1 represents the buffer spectra before bilayer formation, curve 2 shows PWR spectra obtained after the formation of an egg PC bilayer on the resonator surface, curve 3 shows PWR spectra obtained after addition of an octylglucoside-containing buffer solution of rhodopsin to the aqueous compartment of the PWR cell in the dark (the final rhodopsin concentration in the cell sample compartment was ≈1 μM), and curve 4 shows PWR spectra obtained upon saturating yellow light (λ > 500 nm) activation of rhodopsin.

FIGURE 2

PWR spectra obtained upon light activation of rhodopsin incorporated into an egg PC lipid bilayer in 10 mM phosphate buffer, at pH 7.5, using p-polarized (A) and s-polarized (B) light excitation. All spectra were measured with 632.8 nm exciting light. (•) PWR spectra obtained after addition in the dark of an octylglucoside-containing buffer solution of rhodopsin to the aqueous compartment of the sample cell containing a preformed bilayer (final rhodopsin concentration in the cell sample compartment was ≈1 μM); (○) PWR spectra obtained upon saturating yellow light (λ > 500 nm) activation of rhodopsin. Solid lines represent the data; symbols are used to distinguish the two spectral curves. Note that the two sets of spectra superimpose on each other within the spectral resolution (1 mdeg) indicating that no rhodopsin photoactivation occurred.

FIGURE 3

Dependence on pH of the changes in the PWR resonance angular position observed in recombinant rhodopsin/egg PC films in 10 mM phosphate buffer upon saturating yellow light irradiation. Squares represent the results obtained with p-polarized light and triangles with s-polarized light. The solid curve through the data points for both polarizations represents the best fit to the Henderson-Hasselbalch equation with an apparent pK_a value of 6.4 ± 0.05.

FIGURE 4

PWR spectra obtained for a rhodopsin/DOPC recombinant film and changes obtained upon yellow light activation at pH 5 using p-polarized (A) and s-polarized (B) light excitation. All spectra were measured with 632.8 nm exciting light. For both panels, curve 1 represents the PWR spectra obtained after the formation of a DOPC lipid bilayer on the resonator surface, curve 2 shows PWR spectra obtained after addition of an octylglucoside-containing buffer solution of rhodopsin to the aqueous sample compartment in the dark (final rhodopsin concentration in the cell sample compartment was ≈1 μM), and curve 3 shows PWR spectra obtained upon saturating yellow light (λ > 500 nm) activation of rhodopsin.

FIGURE 5

PWR spectra obtained for rhodopsin in DOPC/DOPE (25:75 mol %) recombinant films and changes obtained upon yellow light activation at pH 5 using p-polarized (A) and s-polarized (B) light excitation. All spectra were measured with 632.8 nm exciting light. For both panels, curve 1 represents the PWR spectra obtained after the formation of a DOPC/DOPE lipid bilayer on the resonator surface, curve 2 shows PWR spectra obtained after addition of an octylglucoside-containing buffer solution of rhodopsin to the aqueous sample compartment in the dark (final rhodopsin concentration in the cell sample compartment was ≈1 μM), and curve 3 shows PWR spectra obtained upon saturating yellow light (λ > 500 nm) activation of rhodopsin.

FIGURE 6

Dependence on pH of the changes in the PWR resonance angular position observed for rhodopsin in DOPC/DOPE (25:75 mol %) recombinant films upon saturating yellow light irradiation. Squares represent the results obtained with p-polarized light and triangles those obtained using s-polarized light. The solid curve through the data points for both polarizations represents the best fit to the Henderson-Hasselbalch equation with an apparent pK_a value of 7.3 ± 0.13.

FIGURE 7

Dependence on the mol %/DOPE of the changes in the PWR resonance angle position observed for rhodopsin incorporated into DOPC/DOPE recombinant films at pH 5 upon saturating yellow light irradiation. Conditions as in Figs. 5 and 6. Squares represent the results obtained with p-polarized light and triangles those obtained using s-polarized light. The solid line through the data points represents the best linear least-squares fit.

FIGURE 8

Interaction of transducin (G_t) with a control DOPC/DOPE (25:75 mol %) lipid bilayer in the absence of rhodopsin using p- (A) and s-polarized (B) light excitation. All spectra were measured with 632.8 nm exciting light. In both panels, curve 1 represents the PWR spectra obtained for the DOPC/DOPE lipid bilayer and curve 2 shows PWR spectra after G_t addition to the sample compartment (1 μM was the final G_t concentration in the sample cell).

FIGURE 9

Interaction of transducin (G_t) with dark-adapted rhodopsin in DOPC/DOPE (25:75 mol %) recombinant films at pH 5, and PWR spectral changes obtained upon light activation and GTPγS addition using p- (A) and s-polarized (B) light excitation. All spectra were measured with 632.8 nm exciting light. In both panels, curve 1 represents the PWR spectra obtained after rhodopsin incorporation into the lipid bilayer (DOPC/DOPE; 25:75 mol %) in the dark, curve 2 shows PWR spectra after G_t addition to the sample compartment in the dark (10 nM is the final G_t concentration in the sample cell), curve 3 shows PWR spectra obtained upon saturating yellow light activation of the rhodopsin-G_t complex, and curve 4 shows the PWR spectra obtained after GTPγS addition to the sample compartment in the dark (final GTPγS concentration in the sample cell was 3 μM).

FIGURE 10

Binding curves for the interaction of G_t with dark-adapted (A) and light-activated (B) rhodopsin in a DOPC/DOPE (25:75 mol %) lipid bilayer at pH 5. Isotherms were obtained by plotting the shifts in the resonance angular minimum of PWR spectra measured after several incremental additions of G_t for p- (▪) and s-polarized (▴) light. Note the differences in the concentration ranges, and thus the differences in affinity observed in the two panels. The data are fit to a hyperbolic function (solid curves). Dissociation constant values are given in the figure as well as in Table 1.

FIGURE 11

Binding curves for the interaction of GTPγS with the light-activated rhodopsin-G_t complex in DOPC/DOPE (25:75 mol %) films. Isotherms plot the shifts in the resonance angle minimum of the PWR spectra obtained after addition of aliquots of GTPγS for p- (▪) and s-polarized (▴) light. Solid curves correspond to hyperbolic fits to the data; dissociation constant values are given in Table 1.