Theory of fluorescence polarization of oriented pigment molecules in spherical arrays

Summary Theoretical results are presented which are appropriate for the analysis of the static polarized fluorescence experiment with oriented pigment molecules in spherical arrays (vesicles). Though the global orientation mediated over the whole sphere is isotropic, the fluorescent molecules may have preferred local orientation with respect to the local plane. As in a former paper, concerning fluorescence polarization in planar arrays, three basic (local) orientation distributions of the electronic transition moments are investigated, which may be expected to describe a wide class of real cases with sufficient accuracy. Analytic expressions for the degree of polarization are derived. One important result is that the degree of polarization may be extremely dependent on the local orientation of transition moments. Hence the usual method of determination of microviscosities from experiments with vesicles with the use of the theory of fluorescence polarization for macromolecules in solutions should be regarded with great caution.I wish to thank prof. P. LÄuger and Dr. G. Pohl for interesting discussions. This work has been financially supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 138).     

A theory for polarized fluorescence microscopy of chromosome structures

A theory for the determination of DNA arrangements in DNA-containing specimens, using planar aromatic dye molecules as probes for plane polarization of fluorescence, has been described. At low dye-to-DNA concentrations, the dye molecules are sandwiched between the stacked bases of DNA; hence, the fluorescence from the dye bound to a local region of DNA helix is plane-polarized with the polarization direction perpendicular to the local axis of DNA. The degree of such polarization from an aligned DNA-specimen complexed with dye is determined both by the DNA orientation and the conformational state (e.g., base tilt) of DNA into that specimen. Analysis has been made of the relationship between the degree of polarization and the orientation of the emitting dipoles of dye. The dye complexes may be aligned in a mechanical shear or electric field. However, any change in the orientation distribution of the emitting dipoles due to force fields should be taken into account. With some assumptions and approximations, the magnitude and the direction of maximum polarization can be related to different orders of DNA coiling and to their various combinations. Since the measured polarization is averaged over all DNA regions of the specimen, if the magnitude of polarization is appreciable and the polarization occurs in the specific direction of the specimen, the theory helps to eliminate several probable arrangements of DNA. The predominant molecular features of the actual DNA arrangement can be determined through this process of elimination, as explained in two subsequent papers with T-even bacteriophage and chromosome systems.     

Fluorescence polarization in a planar array of pigment molecules: theoretical treatment and application to flavins incorporated into artificial membranes.

A quantitative fluorescence polarization theory of molecules bound to two-dimensional plane layers has been developed when the electronic transition moments of absorption and emission are parallel within the fluorescent molecules. The transition moments are assumed to be in preferred orientation with respect to the normal to the plane and to be randomly oriented within the plane (rotational symmetry with the normal as axis of symmetry). Three basic model distributions of transition moments are investigated quantitatively. These model distributions represent a simplification but in most cases may be expected to describe reality with sufficient accuracy. For all distributions, two cases of different mobility of molecules are treated: (a) the lifetime of fluorescence is small compared with the characteristic relaxation time of the distribution, and (b) the lifetime of fluorescence is long, so that a complete reorientation of transition moments during the excited state can take place. From the quantitative calculations four characteristic quantities are derived, which are appropriate for the analysis of experimental data. Experiments are carried out with phosphatidylcholine bilayer membranes which contain three differently substituted amphiphilic flavins. All three flavins yield similar data. Their analyses predict free and fast mobility of the flavin chromophore.     

Anisotropy of photosynthetic membranes and the degree of fluorescence polarization

The degree of fluoresence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400–680 nm) and emission wavelengths (675–750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, P_IN and P_AN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization P_IN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Q_y (≈ 660−680 nm) absorption band of chlorophyll in vivo. With 670–680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts, P_IN is shown to be +5±1%. The excitation wavelength dependence studies of P_IN indicate that the carotenoid and chlorophyll Q_y transition moments tend to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).     

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.     

Polarized fluorescence of aggregated bacteriochlorophyll c and baseplate bacteriochlorophyll a in single chlorosomes isolated from Chloroflexus aurantiacus

The polarization anisotropy of fluorescence from single chlorosomes isolated from a green filamentous bacterium, Chloroflexus aurantiacus, was measured using a confocal laser microscope at 13 K. Each single chlorosome that is floating in a frozen solvent exhibited strong polarization anisotropy of fluorescence. We calculated the degrees of fluorescence polarization for 51 floating single chlorosomes. The value ranged from 0.1 to 0.76 for the BChl-c aggregate in the core chlorosomes and from 0 to 0.4 for the energy acceptor BChl-a in the baseplate protein in the outer membrane. The shifts in polarization angles between the two emission bands were distributed over all the possible values with a sharp peak around 90 degrees , suggesting the perpendicular orientation between the transition dipoles of the fluorescence emission from the BChl-c aggregate and that from BChl-a. A simulation assuming a random orientation of chlorosomes reproduced the experimental results exactly. The analysis further indicated the appreciable contribution of the transition dipole of BChl-c that has an orientation perpendicular to the major polarization axis in each chlorosome. Small values of the degrees of polarization implied the BChl-a transition dipole to be somewhat tilted with respect to the normal of the cytoplasmic membrane to which chlorosomes are attached. These conclusions can be obtained only by observing the fluorescence of single chlorosomes.     

Quantitative method for studying orientation of transition dipoles in membrane vesicles of spherical symmetry

Pigmented vesicular membranes embedded in polyacrylamide gel exhibit linear dichroism when the gel sample is squeezed [Abdourakhmanov, I.A., Ganago, A.O., Erokhin, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183-186]. The orientation technique of gel-squeezing was modified to enhance polarization effects in membrane vesicles of spherical symmetry. Model calculations were carried out to provide a tool for the quantitative evaluation of the dichroism of squeezed gel samples. The orientation angles of the dipoles can be calculated with reasonable precision by measuring two quantities: (i) the macroscopic deformation parameter of the gel sample, and (ii) a parameter (e.g. the polarization ratio of the fluorescence emission) characterizing the orientation of the transition dipoles in the membranes embedded in the squeezed gel. The validity of the model was confirmed through a series of polarization measurements relating to the fluorescence of chlorophyll a in membranes of osmotically shocked chloroplasts, 'blebs'.     

Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phophoslipid artificial membranes.

The fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles is a function of the physical state of the lipid. Below the phase transition, the polarization approaches the theoretical maximum for total immobilization while above the phase transition the fluorescence becomes nearly completely depolarized. The discontinuity in the temperature dependence of polarization occurs within a temperature range under 5 degrees C in the case of pure phospholipids, but for mixed phospholipids occurs over a temperature range greater than 20 degrees C. From these data, phase diagrams describing the gel-sol equilibrium can be constructed; the phase diagrams correspond well with those described in the literature which were constructed using spin-label probes or from x-ray diffraction patterns. The marked change in polarization at the phase transition may be related to the packing of the probe molecule into the lipid bilayer: fluorescence measurements on oriented bilayers indicate that below the phase transition the long axis of the probe is oriented perpendicular to the plane of the membrane while above the transition the probe is oriented randomly relative to the plane of the membrane.     

Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi

We have investigated the process of intermolecular excitation energy transfer and the relative orientation of the chlorophyll molecules in the unicellular green alga Chlamydomonas reinhardi. The principal experiments involved in vivo measurements of the fluorescence polarization as a function of the exciting-light wavelength in the presence and in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We found that as the fluorescence lifetime increases upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea that the degree of fluorescence polarization decreases over the excitation region from 600 to 660 nm. This result, we argue, implies that a Förster mechanism of excitation energy transfer is involved for Photosystem II chlorophyll molecules absorbing primarily below 660 nm. We must add that our results do not exclude the possibility of a delocalized transfer process from being involved as well. Fluorescence polarization measurements using chloroplast fragments are also discussed in terms of a Förster transfer mechanism. As the excitation wavelength approaches 670 nm the fluorescence polarization is nearly constant upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.Experiments performed using either vertically or horizontally polarized exciting light show that the fluorescence polarization increases as the exciting light wavelength increases from 650 to 673 nm. This suggests the possibility that chlorophyll molecules absorbing at longer wavelengths have a higher degree of relative order. Furthermore, these studies imply that chlorophyll molecules exist in discrete groups that are characterized by different absorption maxima and by different degrees of the fluorescence polarization. In view of these results we discuss different models for the Photosystem II antenna system and energy transfer between different groups of optically distinguishable chlorophyll molecules.     

Effects of local anesthetics on membrane properties. I. Changes in the fluidity of phospholipid bilayers.

The effect of the local anesthetic dibucaine on the solid to liquid-crystalline phase transition in phospholipid vesicles was studied by calorimetry and fluorescence polarization. The partition coefficient (greater than 3000) of dibucaine in the membranes of vesicles prepared from acidic phospholipids was more than 20 times higher than in neutral phospholipid membranes under the same conditions. Calorimetric measurements on vesicles prepared form acidic phospholipids (bovine brain phosphatidylserine; dipalmitoylphosphatidylglycerol) showed that dibucaine (1 with 10(-4) M) produced a significant reduction in the gel-liquid crystalline transition temperature (Tc). This fluidizing effect of dibucaine on acidic phospholipid membranes was even more marked in the presence of Ca2+. In contrast, dibucaine at the same concentration did not alter the Tc of neutral phospholipids (dipalmitoylphosphatidylcholine). Significant increase in the fluidity of neutral phospholipid membranes occurred only at higher dibucaine concentrations (2 with 10(-3) M). Measurements of the fluorescence polarization and lifetime of the probe, 1,6-diphenylhexatriene, in acidic phospholipid vesicles revealed that dibucaine (1 with 10(-4) M) caused an increase in the probe rotation rate indicating an increase in the fluidity of the phospholipid membranes. A good correlation was obtained between fluorescence polarization data on dibucaine-induced changes in membrane fluidity and calorimetric measurements on vesicles of the same type.     

Fluorescence of squid axon membrane labelled with hydrophobic probes

Summary Extrinsic fluorescence changes in squid giant axons were examined under a variety of experimental conditions using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and other fluorescent probes. Measurements of the degree of polarization of the fluorescent light (with the axis of the polarizer parallel to the longitudinal axis of the axon) indicated that the class of the TNS molecules in the axon membrane which participate in production of fluorescence signals have a definite orientation with their absorption and emission oscillators directed parallel to the long axis of the axon. Rectangular depolarizing voltage pulses produced a transient decrease in the fluorescent intensity, of which the early component is correlated tentatively with the rise in the membrane conductance. In response to hyperpolarizing pulses, there was an increase in fluorescence intensity which may be explained in terms of increased incorporation of TNS into the ordered structure in the membrane. Hyperpolarizing responses in KCl depolarized axons were accompanied by a change in fluorescent intensity. Tetrodotoxin appeared to suppress the initial component of the fluorescence signal produced by depolarizing clamping pulses. The technique for detecting these fluorescence changes and the physico-chemical properties of TNS are described in some detail.     

Phase transitions in phospholipid vesicles Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol

We have studied the solid to liquid-crystalline phase transition of sonicated vesicles of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine. The transition was studied by both fluorescence polarization of perylene embedded in the vesicles, and by the efflux rate of trapped ²²Na⁺.Fluorescence polarization generally decreases with temperature, showing an inflection in the region 32–42°C with a mid-point of approximately 37.5 °C. On the other hand, the perylene fluorescence intensity increases abruptly in this region. To explain this result, we have proposed that, for $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ where $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ is the transition temperature, perylene is excluded from the hydrocarbon interior of the membranes, whereas, $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ this probe may be accommodated in the membrane interior to a large extent.The self-diffusion rates of ²²Na⁺ through dipalmitoylphosphatidylglycerol vesicles exhibit a complex dependence on temperature. There is an initial large increase in diffusion rates (approximately 100-fold) between 30 and 38 °C, followed by a decrease (approximately 4-fold) between 38 and 48 °C. A monotonic increase is then observed at temperatures higher than 48 °C. The local maximum of ²²Na⁺ self-diffusion rates at approximately 38 °C coincides with the mid-point of phase transition as detected by changes in fluorescence polarization of perylene with the same vesicles. Vesicles composed of dipalmitoylphosphatidylcholine show the same general behavior in terms of ²²Na⁺ self-diffusion rates at different temperatures, except that the local maximum occurs at approximately 42 °C.The temperature dependence of the permeability and the appearance of a local maximum at the phase transition region could be explained in terms of a domain structure within the plane of the membranes. This explanation is based on the possibility that boundary regions between liquid and solid domains would exhibit relatively high permeability to ²²Na⁺.Mixed vesicles composed of equimolar amounts of dipalmitoyl phospholipids and cholesterol show no abrupt changes in the temperature dependence of either perylene fluorescence polarization or ²²Na⁺ diffusion rate measurements. This is taken to indicate the absence of agross phase transition in the presence of cholesterol.     

Aliphatic chain transitions of phospholipid vesicles and phospholipid dispersions determined by polarization of fluoroscence

The polarization of fluorescence of dansyl phosphatidylethanolamine and 9-methylanthracene shows that these compounds are reliable indicators of the order-disorder transitions of the phospholipid aliphatic chains in bilayer systems. The transition is better defined in phospholipid dispersion than in vesicles. It is concluded that the two models are not identical as far as the structure near the melting temperature is concerned. Experiments in turbid solutions were performed with horizontal slits either in the incident or emitted beams, which eliminate the effect of light scattering. This improvement in experimental technique may facilitate the fluorescence polarization study of membrane suspensions.     

Determination of the orientation distribution of adsorbed fluorophores using TIRF. I. Theory.

The spectroscopic technique total internal reflection fluorescence can be used for determination of the orientation of adsorbed fluorescent molecules. The underlying theory is presented in general terms and elaborated in detail for the case that the fluorescent group is a porphyrin ring. It is shown that order parameters of the orientation distribution can be obtained if both the fluorescence intensity and its polarization are measured as functions of the polarization of the incident laser beam. From these order parameters an approximation of the orientation distribution can be derived by the maximum-entropy method.     

K+ conduction phenomena applicable to the low frequency impedance of squid axon

Summary The incorporation of four amphiphilic flavins (amphiflavins) as fluorescence markers bearing C₁₈-hydrocarbon chains at various positions of the chromophore into artificial membrane vesicles has been investigated. The vesicles utilized were made from three different saturated phospholipids. The stability of the flavin-charged vesicles was found to be good over several days, depending somewhat on the temperature, the pH, and their concentration. A marked increase of the fluorescence quantum yield near the vesicle phase transition (crystalline liquid crystalline) was found which was taken to indicate that the flavin nuclei are imbedded more deeply into the hydrophobic portion of the membranes. This is further supported by a hypsochromic shift of the near flavin UV-peak and the increase of absorbance at 450 nm upon melting. Rotational relaxation times of the various amphiflavins bound to the different vesicles are obtained from measurements of the fluorescence polarizations as a function of temperature. From these data, the microviscosities in the region of the chromophors are calculated. Measurements of the fluorescence polarization as a function of the solvent viscosity and vesicle phase (crystalline-liquid crystalline) indicate that below the phase transition the flavin nucleus is protected from the suspension medium by a lipid-water interphase, which softens above phase transition. The dependence of the flavin orientation and microenvironment on the position of the substitution of the aliphatic chain is reflected in the differences of the fluorescence yields and the shape of the emission spectra.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

Cooperative partition model of nystatin interaction with phospholipid vesicles

Nystatin is a membrane-active polyene antibiotic that is thought to kill fungal cells by forming ion-permeable channels. In this report we have investigated nystatin interaction with phosphatidylcholine liposomes of different sizes (large and small unilamellar vesicles) by time-resolved fluorescence measurements. Our data show that the fluorescence emission decay kinetics of the antibiotic interacting with gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles is controlled by the mean number of membrane-bound antibiotic molecules per liposome, . The transition from a monomeric to an oligomeric state of the antibiotic, which is associated with a sharp increase in nystatin mean fluorescence lifetime from approximately 7-10 to 35 ns, begins to occur at a critical concentration of 10 nystatin molecules per lipid vesicle. To gain further information about the transverse location (degree of penetration) of the membrane-bound antibiotic molecules, the spin-labeled fatty acids (5- and 16-doxyl stearic acids) were used in depth-dependent fluorescence quenching experiments. The results obtained show that monomeric nystatin is anchored at the phospholipid/water interface and suggest that nystatin oligomerization is accompanied by its insertion into the membrane. Globally, the experimental data was quantitatively described by a cooperative partition model which assumes that monomeric nystatin molecules partition into the lipid bilayer surface and reversibly assemble into aggregates of 6 +/- 2 antibiotic molecules.     

Force spectroscopy and fluorescence microscopy of dsDNA–YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region

When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye–DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0°± 3.2°). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting.     

Global analysis of steady-state polarized fluorescence spectra using trilinear curve resolution.

Global analysis using trilinear curve resolution is described and shown to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separable function of polarization orientation, excitation wavelength, and emission wavelength. This methodology is applicable to mixtures the components of which have linearly independent excitation and emission spectra and distinct anisotropies. Normalized excitation and emission spectra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and without the uncertainties inherent in bilinear techniques such as principal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum of the multicomponent mixture to compute absolute absorption and emission spectra. The precision of this methodology is evaluated as a function of noise, overlap, relative intensity, and anisotropy difference between components using simulated mixtures of the DNA bases. The ability of this method to extract individual spectra from steady-state fluorescence data arrays is illustrated for mixtures containing two and three components.     

Comparison of the interaction,positioning, structure induction and membrane perturbation of cell-penetrating peptides and non-translocating variants with phospholipid vesicles

Cell-penetrating peptides (CPPs) are able to translocate and carry cargo molecules across cell membranes. Using fluorescence techniques (polarization and quenching) and CD spectroscopy we studied the interaction, conformation and topology of two such peptides, transportan and 'penetratin' (pAntp), and two variants of differing translocating abilities, with small phospholipid vesicles of varying charge density. The induced structure of transportan is always helical independent of vesicle surface charge. pAntp and its two variants interact significantly only with negatively charged vesicles. The induced secondary structure depends on membrane charge and lipid/peptide ratio. The degree of membrane perturbation, evidenced by fluorescence polarization, of pAntp and its variants is related to their secondary structure. In the helical state, the peptides have little effect on the membrane. Under conditions where pAntp and its variants are converted into beta-structures, they cause membrane perturbation. Oriented CD suggests that the two CPPs (pAntp and transportan) in their helical state lie along the vesicle surface, while the two pAntp variants appear to penetrate deeper into the membrane.