G. van Ginkel

The static and dynamic behaviour of fluorescent probe molecules in lipid bilayers

Abstract Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in multibilayers of dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) above their respective phase transitions. The finding that the order parameter 〈P2〉 of the absorption moment is significantly higher than that for the emission moment for each probe is shown to arise from a tilt of the emission moment relative to the molecular symmetry axis. It is further shown that while the order parameter 〈P2〉 is the same for both probes in DMPC bilayers, it is higher for TMA-DPH than for DPH molecules in POPC bilayers. Considerations of the order parameters 〈P4〉, however, show that this difference can be ascribed solely to the higher fraction of DPH molecules lying with their axes parallel to the bilayer surface. Furthermore it is found that TMA-DPH molecules undergo slower reorientational motions than DPH molecules in the same bilayer system. Nevertheless the motion of both probe molecules is faster in DMPC than in POPC bilayers. The results indicate that TMA-DPH is a more useful probe than DPH in the systems investigated.

Study of the orientational ordering of carotenoids in lipid bilayers by resonance-Raman spectroscopy.

The orientational ordering of beta-carotene and crocetin embedded in lamellar model membranes has been investigated by angle-resolved resonance Raman scattering at a temperature well above the phase transition of the lipid chains. It is shown that the ordering of the carotenoids is dependent on the chemical composition of the lipid bilayers. The orientational distribution functions found clearly show that beta-carotene is oriented parallel to the bilayer plane (dioleoyl lecithin) or perpendicular to it (soybean lecithin). For dimyristoyl lecithin at 40 degrees C, egg-lecithin, and digalactosyl diacylglycerol two maxima were found in the orientational distribution: one parallel and one perpendicular to the bilayer surface. Crocetin embedded in soybean lecithin bilayers yields a similar bimodal distribution function. Because of rapid photodegradation no results could be obtained for spirilloxanthin.

THE ORIENTATION OF THE TRANSITION DIPOLE MOMENTS OF CHLOROPHYLL a and PHEOPHYTIN a IN THEIR MOLECULAR FRAME

Abstract— In this paper we describe the determination of the orientation of the absorption and emission transition dipoles of chlorophyll a and pheophytin a in their molecular frame. For this purpose we have embedded the pigments in anhydrous nitrocellulose films with a concentration of 2 × 10‐7 mol/g. We have shown previously that under these conditions the pigments are in a purely monomeric state, are distributed uniformly both before and after stretching and that no intermolecular energy transfer among the molecules takes place.

DPH is localised in two distinct populations in lipid vesicles

Abstract The fluorescence lifetime and anisotropy of DPH molecules in vesicles of lipids is commonly interpreted in terms of intrinsic structural heterogeneities of the bilayer. However, these heterogeneities are not revealed in experiments using other probe molecules. Here, we present an alternative analysis in terms of two distinct populations of DPH molecules in the bilayer structure. The results indicate that the heterogeneities seen by DPH reflect the variations of the packing density of the lipids in a direction perpendicular to the bilayer surface. The two populations are created by a competition between the interactions between the DPH molecules with the lipid chains and their tendency to access the regions of free volume in the middle of the bilayer.

CHLOROPHYLLS IN POLYMERS. I. STATE OF CHLOROPHYLL a IN UNSTRETCHED POLYMER SYSTEMS

Abstract— Model systems for the study of energy transfer processes are useful for the elucidation of the various factors governing the mechanism of energy transfer in photosynthetic systems. Here we describe the characterization of two systems, consisting of chlorophyll a incorporated in anhydrous nitrocellulose and polyvinylalcohol films. First, optical spectroscopy and time‐resolved fluorescence techniques are used to characterize the state of the chlorophyll molecules in the films. We find that in nitrocellulose films the state of chlorophyll a depends strongly on the ratio of nitrocellulose to dimeth‐ylsulfoxide in the solutions from which the films are cast. The state of chlorophyll a in polyvinylalcohol films does not depend on the amount of polymer originally dissolved in dimethylsulfoxide. In these films the pigment is monomeric at low concentrations of chlorophyll a, but aggregates are formed at much lower concentrations than in nitrocellulose. The latter fact is explained by the existence of pockets in polyvinylalcohol, leading to high local concentrations.

Determination of transition moment directions in molecules of low symmetry using polarized fluorescence. II. Applications to pyranine, perylene, and DPH

The application of angle‐resolved fluorescence depolarization techniques to the simultaneous determination of the directions of the transition moments and orientational order of dye molecules incorporated into uniaxially stretched polymer films is discussed and illustrated. The directions of the transition moments of pyranine molecules are determined in a general way, on exciting the molecules in two distinct electronic transitions and monitoring the fluorescence emission under the same conditions. This assumes that the fluorescence emission occurs via the same transition moment for every excited absorption band. Experiments on perylene molecules show that the directions of the transition moments can also be determined from measurements utilizing a single electronic absorption band. In this case the orientational distribution of the molecules is assumed to have the form given by the maximum entropy method. Finally, it is shown the directions of the transition moments of DPH molecules in unstretched polyme...

The fidelity of response by 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene in time-resolved fluorescence anisotropy measurements on lipid vesicles

Time-resolved fluorescence anisotropy measurements on 10-[4-(tri-methylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules in lipid vesicles of palmitoyloleoylphosphatidylcholine (POPC), PC extracted from egg yolk (EggPC), dioleoyl-PC (DOPC), dilinoleoyl-PC (DLPC), phosphatidylglycerol extracted from egg yolk (EggPG), dioleoyl-PG (DOPG), sulfoquinovosyldiacylglycerol (SQDG) and digalactosyl-DG (DGDG) with and without cholesterol are presented. The observed intensity decay curves are analyzed simultaneously in terms of the Brownian rotational diffusion model. The analysis thus yields the isotropic fluorescence decay, the initial anisotropy r (0), the order parameters 〈P2〉 and 〈P2〉 as well as the diffusion coefficient of the long molecular axis. It is shown that increasing unsaturation in the acyl chains of the PC lipids results in an increase in the rotational diffusion rates of the probes and a decrease in the order parameter 〈P2〉. However, the value of 〈P2〉 remains unchanged. The corresponding orientational distribution function of the probes is bimodal, with fractions lying preferentially parallel and perpendicular to the local vesicle surface. Surprisingly, the fraction of probe molecules lying with their long axes parallel to the bilayer surface increases with increasing unsaturation with a concomitant narrowing in the width of the distribution of the fraction lying perpendicular to it. As expected, cholesterol is found to increase the order parameters in all the systems and to suppress the tendency of the molecules to lie parallel to the bilayer surface. Furthermore, the rotational diffusion coefficients of the probes is found to increase in all the systems except for DLPC. Interestingly, the effects of unsaturation on the reorientational dynamics of TMA-DPH molecules in the vesicle systems are opposite to those found in the corresponding planar multibilayers (Deinum et al. 1988), whereas the same cholesterol effect is observed for the two systems. Nevertheless, the TMA-DPH molecules exhibit higher diffusion coefficients in the vesicle than in the planar multibilayer systems. In addition, a unimodal distribution of the probe molecules is found in the multibilayer systems. The differences between the two systems are ascribed to the differences in the radius of curvature and the hydration of the bilayers. Lastly we rationalize the bimodal distribution of the TMA-DPH molecules in the vesicles in terms of their observed partition between the lipid and aqueous phases.

HEADGROUP HYDRATION IN EGG-LECITHIN MULTIBILAYERS AFFECTS THE BEHAVIOUR OF DPH PROBES

Angle-resolved fluorescence depolarization experiments were carried out on DPH molecules embedded in egg-licithin bilayers of different water contents. It was found that the order parameter ( Pz) of the absorption moment is significantly higher than that of the emission moment and that the difference increases on hydrating the lecithin headgroups. The changes in the order parameter ( Pz) and (P.,) of the DPH molecules induced by hydration are consistent with X-ray diffraction data. Furthermore the reorientational motions of the DPH molecules were found to become faster with increasing hydration.

Fluorescence anisotropy of chlorophyll a and chlorophyll b in castor oil

A detailed study of the fluorescence anisotropy of chlorophyll a and chlorophyll b in castor oil is presented. Scanningacross the wavelength of emission the value of the fluorescence anisotropy shows a considerable variation. This indicates that differently polarized emission moments are present. When excitation spectra of the fluorescence anisotropy are recorded at two distinct emission wavelengths, two separate curves are foundthat cross each other several times. The difference between the two spectra yields information about the extent to which absorption transitions overlap. It is found that over the whole spectrum of visible light, absorption transition bands overlap, so that the valueof the fluorescence anisotropy cannot be interpreted in terms of a single anglebetween absorption and emission moment. It rather yields an average direction of the overlapping transitions. Castor oil at room temperature is not sufficiently viscous to quench the rotational depolarization of the chlorophyll fluorescence. A limiting valueof the fluorescence anisotropy is obtained using time-resolved measurements.

Orientational properties of biological pigments in ordered systems studied with polarized light: photosynthetic pigment-protein complexes in membranes.

A discussion is presented of the problems involved in the interpretation of linear dichroism and fluorescence depolarization experiments on macroscopically ordered membrane systems. Particular attention has been paid to ordered membranes containing photosynthetic pigment-protein complexes, but the mathematical treatment can equally well be applied to other systems. The information about the orientational properties of the pigments is obtained by the application of the theories developed for the characterization of the molecular orientational order in liquid-crystalline materials. It is shown that while linear dichroism only yields the order parameter S mu of the absorption transition moment, fluorescence depolarization experiments yield in addition the order parameter Sv of the emission transition moment as well as three orientational correlation functions of the two transition moments. It is argued that in general the latter information can only be obtained on utilizing a number of experimental scattering geometries. In particular, the merits of angle-resolved experiments are illustrated.