Viscosity and order in erythrocyte membranes studied with nanosecond fluorometry

The viscosity and the order in the interior of human erythrocyte membranes were investigated by the fluorescence depolarization technique in the nanosecond region with 1,6-diphenyl-1,3,5-hexatriene (DPH). After pulsed excitation with a polarized light, the fluorescence anisotropy ratio of DPH in membranes rapidly decreased and gave a final value (r infinity). The rate of initial decrease and the value of r infinity related to the viscosity in the interior of the membranes and a wobbling angle of DPH which reflects a size of range for the phospholipid motion relating to the order of membrane structure. For normal human erythrocyte membranes the viscosity and the wobbling angle were obtained to be 0.82 poise and 42 degrees, at 37 degrees C. Similar values were obtained for spectrin-free membranes. Hardened membranes by the cross-linking of the cytoskeletal proteins with glutaraldehyde showed a small wobbling angle of 37 degrees, but the viscosity of them was unchanged.     

Dynamic structure of lipid bilayers studied by nanosecond fluorescence techniques

Molecular motions in liposomes of dipalmitoyl-phosphatidylcholine (DPPC) were studied by nanosecond fluorescence techniques. As a fluorescent probe for the hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used. Time courses of fluorescence intensity IT(t) and emission anisotropy r(t) of DPH embedded in DPPC liposomes were measured at various temperatures. The value of the fluorescence lifetime tau obtained froma single exponential decay of IT(t) was somewhat higher than that in liquid paraffin below the transition temperature Tt and decreased above Tt. Higher values of tau below Tt indicate the almost complete hydrophobic environment. The decay curves of r(t) were separated into two phases: an initial fast decreasing phase of the order of one nanosecond and a second almost constant phase. This indicates that the orientational motion of DPH in the hydrocarbon region is described by a wobbling diffusion restricted by a certain anisotropic potential. The results were analyzed on the model that the wobbling diffusion is confined in a cone with a uniform diffusion constant. Though temperature dependence of the cone angle was sigmoidal, that of the wobbling diffusion constant was like the exponential function. The change in the cone angle at Tt was sharper than that in the wobbling diffusion constant at Tt. Estimated values of the viscosity in the cone were an order of magnitude smaller than the values of "microviscosity" which were estimated from the steady-state emission anisotropy without considering the restrictions on the rotational motion.     

A time-resolved fluorescence anisotropy study of bilayer membranes containing alpha-tocopherol

Rotational mobility in fluid phase dipalmitoylphosphatidylcholine unilamellar vesicles containing alpha-tocopherol has been studied by time-resolved anisotropy measurements of fluorescence from a diphenylhexatriene-phosphatidylcholine conjugate. The results are analysed using a simple wobbling-in-cone model. The diphenylhexatriene probe shows an increasing order parameter and more restricted wobbling with increasing alpha-tocopherol content of the membrane. The diffusional rate for wobbling was found not to change significantly.     

Changes in phospholipid constituents in mitochondrial membranes after long lasting exercise in rat heart

Phosphatidylethanolamine, phosphatidylcholine and cholesterol were found to be significantly decreased to 44, 56 and 54% of the control values (p less than 0.001, 0.05 and 0.05, respectively), in cardiac mitochondria from rats which were made to swim bearing a weight representing 3% of body weight for 3 or more hours in water at 35 degrees C. The ratio of cholesterol to phospholipid did not change. Membrane viscosity tended to decrease very slightly. Steady-state anisotropy at infinite time, fluorescence life time and wobbling angle of phospholipids showed no significant change. Electron-microscopy showed no clear morphological damage, swelling or hypertrophy of cardiac mitochondria after long lasting exercise. The number of mitochondria was found to be increased by 19% in the long lasting exercise group compared with the control group. It was noteworthy that the dynamic microstructure and electron-microscopic structure of the cardiac mitochondria remained unaltered despite the remarkable changes in the phospholipid constituents.     

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. I. Theory.

It is shown that fluorescence anisotropy from lipidlike probes in the hexagonal HII phase gives information of (a) orientational order parameters, (b) the wobbling diffusion constant, and (c) the hopping diffusion constant of the probe, DH, equals DL/R2, the lateral diffusion constant over the square of the radius of the hexagonal tubes. Here we consider only lipidlike probes having the absorption transition movement and/or the emission transition moment along the long axis of the molecule. Three models are introduced for analysis of time-resolved data: the "WOBHOP," the "reduced WOBHOP," and the "P2P4HOP" model. The fluorescence anisotropy in response to a very short excitation pulse in each of the three models is a constant plus a number of exponentials. The WOBHOP and reduced WOBHOP models have 3 and 2 exponentials, respectively, and both contain four fitting parameters: r0 (the fundamental anisotropy), (P2) (the second rank orientational order parameter), DW (the wobbling diffusion constant), and DH (the hopping diffusion constant). The P2P4HOP model has eight exponentials and five fitting parameters: the four parameters listed above and (P4) (the fourth rank orientational order parameter). Analysis of fluorescence anisotropy data in the hexagonal HII phase using one of these models allows for obtaining the hopping diffusion constant, and, if the lateral diffusion constant is known, the radius of the hexagonal tubes. Substitution of DH = 0 in each of the three models yields an expression for the fluorescence anisotropy that is used in the literature for lamellar (L alpha or L beta) phases. The fluorescence anisotropy in coexisting L alpha/HII phases is discussed.     

Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes.

The theory of fluorescent emission anisotropy [r(t)] of a cylindrical probe in a membrane suspension is developed. It is shown, independent of any model, that the limiting anisotropy [r(infinity)] is proportional to the square to the order parameter of the probe. The order parameter determines the first nontrivial term in the expansion of the equilibrium orientational distribution function of the probe in a series of Legendre polynomials. Following Kinosita, Kawato, and Ikegami, the motion of the probe is described as diffusion ("wobbling") within a cone of semiangle theta 0. Within the framework of this model, an accurate single-exponential approximation for r(t) is considered. An analytic expression relating the effective relaxation time, which appears in the above approximation, to theta 0 and the diffusion coefficient for wobbling is derived. The model is generalized to the situation where the probe is attached to a macromolecule whose motion cannot be neglected on the time scale of the fluorescence experiment. Finally, by exploiting the formal similarity between the theory of fluorescence depolarization and 13C-NMR dipolar relaxation, expressions for T1, T2, and the nuclear Overhauser enhancement are derived for a protonated carbon which is nonrigidly attached to a macromolecule and undergoes librational motion described as diffusion on a spherical "cap" of semiangle theta 0.     

Selective labeling and rotational diffusion of the ADP/ATP translocator in the inner mitochondrial membrane

Submitochondrial particles were labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide. On sodium dodecyl sulfate-polyacrylamide gels, eosin fluorescence occurred in a single band of Mr approximately 30,000. The labeled band was identified as the ADP/ATP translocator, since EMA binding was completely inhibited by carboxyatractylate. Furthermore, the EMA-labeled polypeptide had the same molecular weight as the purified carboxyatractylate-bound translocator and the purified EMA-labeled translocator. Rotational diffusion of the translocator around the membrane normal in submitochondrial particles was measured by observing flash-induced absorption anisotropy of EMA. The translocator rotates with a time constant which varied from approximately 240 microseconds at 5 degrees C to approximately 100 microseconds at 37 degrees C. However, it is likely that only a fraction of the translocator rotates, the remainder being immobile over the measurement time of 500 microseconds. The mobile fraction of the translocator decreased with decrease in temperature. The observed fluorescence anisotropy of 0.24 indicates that EMA undergoes subnanosecond rapid wobbling in the binding site of the ADP/ATP translocator.     

Independent flexible motion of submolecular domains of the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum measured by time-resolved fluorescence depolarization of site-specifically attached probes

The Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum was site-specifically labeled with either N-(1-anilinonaphth-4-yl)maleimide (ANM) or 5-[[(iodoacetamido)-ethyl]amino]naphthalene-1-sulfonate (IAEDANS), and the segmental motion of submolecular domains of the ATPase molecule was examined by means of time-resolved and steady-state fluorescence anisotropy measurements. The ANM-binding domain showed wobbling with a rotational relaxation time phi = 69 ns in the absence of free Ca2+ without any independent wobbling of the ANM moiety. The IAEDANS-binding domain showed a significantly slower wobbling with phi = 190 ns in the absence of Ca2+. The present results demonstrated for the first time that the ATPase molecule is composed of distinct domains whose mobilities are considerably different from each other. The binding of Ca2+ to the transport site increased the segmental motion of ANM-labeled domain, leading to a phi value of 65 ns. Solubilization of the ANM-labeled SR membranes by deoxycholate led to a further increase in the segmental flexibility (phi = 48 ns in the absence of free Ca2+), indicating that the mobility of the ANM-binding domain was considerably restricted through interaction with the membrane. The mobility of the ANM-binding domain of solubilized ATPase was also increased to some extent upon binding of Ca2+.     

Analysis for the molecular motion of phospholipid bilayer with picosecond fluorometry

The viscosity and the molecular motion of phospholipid molecule in biological and artificial phospholipid bilayers were studied using picosecond fluorescence depolarization method with rod-like fluorophore, DPH. From the relationship between the viscosity in the lipid bilayer and the free space of phospholipid acyl-chain, it is concluded that the viscosity is determined mainly by the range of wobbling motion of the acyl-chain. Motion of polar head group was also measured by the same method with a newly synthesized fluorescent phospholipid, dipalmitoyl-phosphatidyl-umbelliferone. The rate and the range in the motion of head group were faster and larger than those of acyl-chain and gave the viscosity of head group layer to be 0.03 poise, which was about one tenth of that of acyl-chain layer in the liquid crystalline phase. This fact indicates that the head group layer would not resist the lateral diffusion of molecules in membrane and that the lateral diffusion rate of molecules could be estimated from the viscosity in the acyl-chain layer.     

大鼠心肌线粒体内、外膜磷脂动态结构的研究

我们以DPH为荧光探针.用毫微秒荧光分光光度计测定了大鼠心肌线粒体及线粒体内、外膜的动态微细结构;用HPLC分析了磷脂组成.实验结果提示.完整线粒体膜流动性主要反映了线粒体外膜的运动状态.线粒体内膜微粘度及磷脂分子摇动角大于外膜,扩散速率小于外膜.除去了蛋白质的线粒体内、外膜磷脂脂质体膜流动性无明显差异.提示线粒体内膜的高微粘度与膜中所含有的多量蛋白有关.     

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.     

Gangliosides asymmetrically alter the membrane order in cultured PC-12 cells

Exogenous gangliosides readily associate with the cell membranes and produce marked effects on cell growth and differentiation. We have studied the effect of bovine brain gangliosides (BBG) on the membrane dynamics of intact cells. The structural and dynamic changes in the cell membrane were monitored by the fluorescence probes DPH, TMA-DPH and laurdan. Incorporation of BBG into the cell membrane decreased the fluorescence intensity, lifetime and the steady state anisotropy of TMA-DPH. Analysis of the time resolved anisotropy decay by wobbling in the cone model revealed that BBG decreased the order parameter, and increased the cone angle without altering the rotational relaxation rate. The fluorescence intensity and lifetime of DPH were unaffected by BBG incorporation, however, a modest increase was observed in the steady state anisotropy. BBG incorporation reduced the total fluorescence intensity of laurdan with pronounced quenching of the 440-nm band. The wavelength sensitivity of generalized polarization of laurdan manifested an ordered liquid crystalline environment of the probe in the cell membrane. BBG incorporation reduced the GP values and augmented the liquid crystalline behavior of the cell membrane. BBG incorporation also influenced the permeability of cell membranes to cations. An influx of Na+ and Ca2+ and an efflux of K+ was observed. The data demonstrate that incorporation of gangliosides into the cell membrane substantially enhances the disorder and hydration of the lipid bilayer region near the exoplasmic surface. The inner core region near the center of the bilayer becomes slightly more ordered and remains highly hydrophobic. Such changes in the structure and dynamics of the membrane could play an important role in modulation of transmembrane signaling events by the gangliosides.     

Analysis of internal motion of single tryptophan in Streptomyces subtilisin inhibitor from its picosecond time-resolved fluorescence.

A mode of internal motion of single tryptophan, Trp 86, of Streptomyces subtilisin inhibitor, was analyzed from its time-resolved fluorescence. The intensity and anisotropy decays of Trp 86 were measured in the picosecond range. These decays were analyzed with theoretical expressions derived assuming that the indole ring of tryptophan as an asymmetric rotor rotates around covalent bonds connecting indole with the peptide chain and an effective quencher of fluorescence of Trp 86 is the nearby SS bond of Cys 35-Cys 50. First, the intensity decays at 6 degrees, 20 degrees, and 40 degrees C were analyzed, and then the both decays of the intensity and anisotropy at 20 degrees C were simultaneously simulated with common parameters. Constants concerning geometrical structures of the protein used for the analysis were obtained from x-ray crystallographic data. Best fit between the observed and calculated decay curves was obtained by a nonlinear least squares method by adjusting a quenching constant averaged over the rotational angles, koq height of the potential energy, p, and three of six diffusion coefficients, Dxx, Dyy, Dzz, Dxy, Dyz, and Dzx, as variable parameters. The obtained results revealed that the internal motion of the indole ring became faster, the quenching rate of the fluorescence of Trp 86 was enhanced and the height of potential energy became lower at higher temperatures, and suggested that Trp 86 was wobbling around the long axis of the indole ring in the protein.     

The fluorescence studies of the sol-gel transition by styrylpyridine derivative

The gelation process of tetraethylorthosilanes in acid environment was monitored with the trans-4-(p-N,N-dimethylaminostyryl)-N-vinylbenzylopyridinium chloride (vbDMASP) fluorescent probe. The fluorescence steady-state and anisotropy measurements of material during sol-gel transition are reported. The results are compared with fluorescence studies of the probe in a modeled viscous system of water-glycerol mixtures. A strong increase of anisotropy, from 0.1 to 0.9, with gelation time as well with wavelength, was observed. Although the increase of anisotropy with wavelength is due to specificity of the compounds exhibiting charge transfer properties, the increase of the anisotropy with gelation time is due to an increase of microviscosity. On this basis, suitability of the applied fluorophore in recording of viscosity changes during sol-gel transition is discussed. The molecular structure of vbDMASP in the excited states in dependence on environmental polarity was optimized using the HyperChem and Amsol program. The dynamics of torsional angle C35-C34-N31-C28 of the multichromophore dye in correlation with micropolarity and microviscosity of the network formation during the sol-gel transition is discussed.     

Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30 degrees for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.     

On the wobbling-in-cone analysis of fluorescence anisotropy decay.

Interpretation of fluorescence anisotropy decay for the case of restricted rotational diffusion often requires a model. To investigate the extent of model dependence, two models are compared: a strict cone model, in which a fluorescent probe wobbles uniformly within a cone, and a Gaussian model, where the stationary distribution of the probe orientation is of a Gaussian type. For the same experimental anisotropy decay, analysis by the Gaussian model predicts a smaller value for the rate of wobbling motion than the strict cone analysis, but the difference is 35% at most; the cone angle obtained by the strict cone analysis agrees closely with the effective width of the Gaussian distribution. The results suggest that, when only two parameters (the rate and the angular range) are extracted from an experiment, the choice of a model is not crucial as long as the model contains the essential feature, e.g., the more-or-less conical restriction, of the motion under study. Model-independent analyses are also discussed.     

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from noninterdigitated gel phases of symmetric diacylphosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Dynamics of membrane structure of frog erythrocyte ghosts measured with a nanosecond fluorometer

The dynamics of membrane microstructure was studied as molecular motions of phospholipids for bullfrog erythrocyte ghosts by the DPH fluorescence depolarization technique with a nanosecond fluorometer. The bullfrog erythrocyte ghosts were obtained by hypotonic lysis and collagenase treatment. The constituents of membrane proteins were confirmed by the disk gel electrophoresis. The viscosity of erythrocyte membrane ghosts was estimated to be 3.3 +/- 1.0 at 10 degrees C, and 2.1 +/- 0.1 at 20 degrees C and 1.3 +/- 0.2 at 30 degrees C in the unit of poise and the wobbling angle of lipid molecule was 35 +/- 1, 41 +/- 1 and 43 +/- 1 degree at the respective temperatures on an average and +/- S.D. The viscosity is lower than that of human erythrocytes. The relatively low viscous phospholipid bilayer may be one of the factors for the deformability of bullfrog erythrocytes.     

Cofactor-induced orientation of the DNA bases in single-stranded DNA complexed with RecA protein. A fluorescence anisotropy and time-decay study

The structure of the RecA-single-stranded DNA complex was investigated by studying the fluorescence emission of poly(deoxy-1,N6-ethenoadenylic acid (poly(d epsilon A)), a fluorescent derivative of poly(dA), under various viscosity conditions. The fluorescence intensity and average lifetime of poly(d epsilon A) are much smaller than those of nonpolymerized monoethenonucleotides (1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenine deoxyribose 5'-monophosphate) at low viscosity and reflect intramolecular base-base collisions in the polymer. They considerably increased upon RecA binding, both in the presence and absence of cofactor ATP or adenosine 5'-O-(3-thiotriphosphate). This increase, as well as the increase in fluorescence anisotropy upon RecA binding, was very similar to that which resulted from sucrose addition to free poly(d epsilon A). These observations point to a decrease in the mobility of DNA bases upon RecA binding. In the presence of cofactor, the fluorescence features became independent of viscosity. This strongly suggests the absence of base motion of significant amplitude on the time scale of the fluorescence lifetime (about 10 ns). In the absence of cofactor, however, these features remained sensitive to viscosity, implying residual local motions of the bases. Such cofactor-dependent rigid attachment of DNA bases to stiff phosphate backbone could facilitate the search for homology between two DNA molecules during recombination.