Gijs van Ginkel

Effects of steroid molecules on the dynamical structure of dioleoylphosphatidylcholine and digalactosyldiacylglycerol bilayers

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, beta-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21-55 degrees C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35 degrees C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter mean value of P2, without affecting the diffusion coefficients. Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D perpendicular of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225-233, and 982, 196-204).

The correlation between molecular orientational order and reorientational dynamics of probe molecules in lipid multibilayers

The behaviour of probe molecules in lipid systems can be characterized in terms of molecular order parameters and rates of reorientational motion. The correlation between these parameters has been investigated in oriented lipid multibilayer systems on changing the degree of unsaturation of the chains and the cholesterol content.Angle-resolved fluorescence depolarization experiments were carried out as a function of two angles: (1) the angle of incidence of the excitation beam and (2) the angle between the directions of incidence and observation. The e.s.r. spectra from nitroxide spin labels were simulated numerically with a general stochastic Liouville equation formalism. The probe molecules were assumed to undergo stochastic small-step reorientational diffusion subject to an orienting potential.Both techniques indicate that the introduction of unsaturation into the lipid chains lowers both the order parameters and the diffusion coefficients of the probe molecules. The incorporation of cholesterol reverses this trend. These findings are at odds with current ideas about the fluidity of membrane systems.

Slow-motion ESR of cholestane spin labels in planar multibilayers of diacyldigalactosyldiglycerides

The orientation and restricted motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) incorporated into planar multibilayers of diacyldigalactosyldiglycerides extracted from the thylakoid membranes of chloroplasts from different plant leaves has been studied. The experimental ESR spectra were simulated in terms of the slow-tumbling ESR formalism of Freed and co-workers (Polnaszek, C.F., Bruno, G.V. and Freed, J.H. (1973) J. Chem. Phys. 58, 3185–3199). The analysis shows that the degree of orientational order is low. The spin label molecules undergo a faster reorientational motion about their long molecular axes than perpendicular to them. At room temperature the reorientational rate around the long molecular axis falls within the fast-motional limit, while the reorientation rate of the long axis itself corresponds to the slow-tumbling regime. The results indicate that the motion of the labels in bilayers of diacyldigalactosyldiglycerides is considerably slower than that of the same label incorporated into bilayers of saturated phosphatidylcholines above the main phase transition. Differences between bilayers of diacyldigalactosyldiglycerides extracted from different plant membranes have been observed.

A Comparison of the Molecular Dynamics of Fluorescent Probes in Curved Lipid Vesicles and Planar Multibilayers

Abstract The dynamic behaviour of the probe molecules DPH and TMA-DPH embedded in small unilamellar vesicles and planar multibilayers of POPC has been studied by time-resolved fluorescence depolarization techniques. The molecular dynamics of the probe molecules was analysed in terms of the rotational diffusion model. It is found that analysis of the time-dependent fluorescence anisotropy from the vesicle system yields two distinct, though statistically equivalent solutions. On the other hand the measurements on planar multibilayers can be interpreted unequivocally. It is shown that the order parameters of the probe molecules are higher in the multibilayers than in the vesicles. A reconstruction of the orientational distribution function reveals that the TMA-DPH molecules have a more pronounced tendency to lie with their long axes parallel to the bilayer surface in the curved vesicles than in the planar multibilayers. An intriguing finding is that the reorientational motion of the probes is considerably sl...

Angle-Resolved Techniques in Studies of Organic Molecules in Ordered Systems Using Polarized Light

Angle-resolved linear dichroism and fluorescence depolarization experiments on organic and biological molecules aligned in stretched polymer films, liquid crystalline materials and biological membranes are described. The theory underlying the experiments is set out in detail and the experimental procedures and sources of error are discussed and illustrated. The mathematical treatment makes use of the Wigner rotation matrix formalism so as to provide a unified description of the static orientational order and reorientational dynamics of the guest molecules in their host medium. It is shown that angle-resolved fluorescence depolarization measurements afford the simultaneous determination of the directions of the transition moments of the guest molecules as well as their orientational order in the medium. The application of the technique to the study of the reorientational motions of the molecules is also discussed. It is argued that angleresolved measurements on ordered systems under conditions of continuous illumination provide a valuable approach to the characterization of the dynamic behaviour of the molecules.

An investigation of various wavelength-shifting compounds for improving counting efficiency when 32P-cerenkov radiation is measured in aqueous samples

Abstract Various water-soluble wavelength-shifting compounds were investigated to assess their suitability for the improvement of counting efficiency when Cerenkov radiation from phosphorous-32 is measured in a liquid scintillation counter. Of these compounds esculin, β-methyl-umbelliferon and sodium salicylate led to the greatest improvement in counting efficiency. Especially esculin and β-methyl-umbelliferon are fairly stable under a variety of experimental conditions and improve counting efficiencies by a factor of about 1.3 and 1.2, respectively. The use of ethanol as a water-miscible solvent combined with wavelength shifters soluble in both solvents does not improve counting efficiency.

On the model-independent determination of 〈P4〉 from time- and angle-resolved fluorescence depolarization on macroscopically oriented lipid multibilayer systems

Abstract Analysis of angle- and time-resolved fluorescence depolarization experiments on DPH and TMA-DPH embedded in macroscopically ordered lipid multibilayer systems shows that the order parameters 〈 P 2 〉 and 〈 P 4 〉 and the angle between the transition dipoles of the probe molecule can be obtained without making use of a particular reorientational model. The values found for 〈 P 4 〉 correspond to the higher values obtained from model-dependent analysis of anisotropy experiments on vesicle systems. The angles between the transition dipoles obtained here are in excellent agreement with those obtained from steady-state experiments.

ANGLE RESOLVED FLUORESCENCE DEPOLARISATION OF 1,6-DIPHENYL-1,3,5 HEXATRIENE (DPH) IN UNSATURATED DIGALACTOSYL DIACYLGLYCEROL (DGDG) LIPID BILAYERS

The orientational ordering and dynamics of DPH molecules embedded in macroscopically ordered bilayers of the unsaturated galactolipid digalactosyl diacylglycerol (DGDG) were studied by angle-resolved fluorescence depolarisation at room temperature. The depolarisation ratios exhibited a weaker angular dependence than those observed for DPH incorporated in multi-bilayers of saturated lecithins above their phase transition. The results reflect the differences in the modes of reorientational motion of the DPH molecules in these systems while a similar orientational order is found. It is suggested that DPH molecules in bilayers of DGDG cannot be considered to possess an effective cylindrical symmetry in contrast to DPH in bilayers of saturated lecithins.

Molecular Dynamics in Liquid-Crystalline Systems Studied by Fluorescence Depolarisation Techniques

Fluorescence depolarisation experiments provide detailed information about molecular orientational dynamics on the nanosecond time scale. However, as in general, liquid-crystalline systems do not exhibit an intrinsic fluorescence emission, the technique can only be exploited through the introduction of fluorescent probes into the structure. In this way, the dynamics of the mesophase is studied indirectly by monitoring the behaviour of the probes. In this Chapter we present a summary of the principles of fluorescence spectroscopy and discuss the instrumentation necessary for time resolved and steady state experiments. In particular, we review the theory of experiments applied to oriented and macroscopically isotropic samples and consider the problems arising in the analysis of the experimental data. It is argued that angle resolved experiments on ordered systems under conditions of continuous illumination provide a valuable approach to the characterisation of the molecular dynamics in liquid-crystalline mesophases.

On the interpretation of fluorescence anisotropy decays from probe molecules in lipid vesicle systems

Measurements of fluorescence depolarization decays are widely used to obtain information about the molecular order and rotational dynamics of fluorescent probe molecules in membrane systems. This information is obtained by least-squares fits of the experimental data to the predictions of physical models for motion. Here we present a critical review of the ways and means of the data analysis and address the question how and why totally different models such as Brownian rotational diffusion and “wobble-in-cone” provide such convincing fits to the fluorescence anistropy decay curves. We show that while these models are useful for investigating the general trends in the behavior of the probe molecules, they fail to describe the underlying motional processes. We propose to remedy this situation with a model in which the probe molecules undergo fast, though restricted local motions within a slowly rotating cage in the lipid bilayer structure. The cage may be envisaged as a free volume cavity between the lipid molecules, so that its position and orientation change with the internal conformational motions of the lipid chains. This approach may be considered to be a synthesis of the wobble-in-cone and Brownian rotational diffusion models. Importantly, this compound motion model appears to provide a consistent picture of fluorescent probe behavior in both oriented lipid bilayers and lipid vesicle systems.