Dynamical theory of fluorescence polarization in a planar array of oriented pigment molecules.

A theory is developed appropriate for the analysis of fluorescence polarization experiments with pigment molecules in a planar array (plane membrane). Especially rotatory and oscillatory dynamics of the pigment molecules are considered. Three model calculations are performed, which describe the following different situations: (a) Rotational diffusion of molecules around the normal to the plane membrane. (b) Oscillatory diffusion of molecules with respect to this normal. (c) As a two-dimensional example the independent superposition of both types of motion. Central point of these model calculations is the determination of an intensity of emission function, from which in practical application the measured fluorescence intensities may uniquely be calculated.     

Fluorescence energy transfer between Ca2+ transport ATPase molecules in artificial membranes.

The purified ATPase of sarcoplasmic reticulum was covalently labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) or with iodoacetamidofluorescein (IAF). In reconstituted vesicles containing both types of ATPase molecules fluorescence energy transfer was observed from the IAEDANS (donor) to the IAF (acceptor) fluorophore as determined by the ratio of donor and acceptor fluorescence intensities, and by nanosecond decay measurements of donor fluorescence in the presence or absence of the acceptor. The observed energy transfer may arise by random collisions between ATPase molecules due to Brownian motion or by formation of complexes containing several ATPase molecules. Experimental distinction between these two models of energy transfer is possible based on predictions derived from mathematical models. Up to tenfold dilution of the lipid phase of reconstituted vesicles with egg lecithin had no measurable effect upon the energy transfer, suggesting that random collision between ATPase molecules in the lipid phase is not the principal cause of the observed effect. Addition of unlabeled ATPase in five- to tenfold molar excess over the labeled molecules abolished energy transfer. These observations together with electron microscopic and chemical cross-linking studies support the existence of ATPase oligomers in the membrane with sufficiently long lifetimes for energy transfer to occur. A hypothetical equilibrium between monomeric and tetrameric forms of the ATPase governed by the membrane potential is proposed as the structural basis of the regulation of Ca uptake and release by sarcoplasmic reticulum membranes during muscle contraction and relaxation.     

A fluorescence decay study of parinaroyl-phosphatidylinositol incorporated into artificial and natural membranes

Phosphoinositide metabolism in the plasma membrane is linked to transmembrane signal transduction. In this study we have investigated some physical properties (e.g. molecular order and dynamics) of phosphatidylinositol (PI) in various membrane preparations by time-resolved fluorescence techniques, using a synthetic PI derivate with a cis-parinaroyl chain on the sn-2 position. Phospholipid vesicles, normal and denervated rat skeletal muscle sarcolemmal membranes, and acetylcholine receptor rich membrances from Torpedo marmorata were investigated both at 4°C and 20 °C. For comparison we have also included 2-parinaroyl-phosphatidylcholine (PC) in this study. The fluorescent lipids were incorporated into the membrane preparations by way of specific phospholipid transfer proteins, to ensure an efficient and non-perturbing insertion of the lipid-probes. In the Torpedo membranes the order parameters measured for the parinaroyl derivatives of both PC and PI were higher than in phospholipid vesicles. For the Torpedo membrane preparations the acyl chain order for the PI was lower than that for PC, whereas the opposite was true for the vesicles. This inversion strongly suggests that PI has different interactions with certain membrane components as compared to PC. This is also suggested by the significantly higher rate of restricted rotation of PI as compared to PC. In contrast to the order parameters, the correlation times were almost identical for both probes and showed little difference between vesicles and the Torpedo membranes. In contrast to Torpedo membranes, the time-dependent fluorescence anisotropy of the two lipid probes in the sarcolemmal membranes showed, after an initial fast decay, a subsequent gradual increase. This phenomenon was satisfactorily analyzed by assuming two populations of probe lipids with distinct lifetimes, rotational correlation times and molecular order. The order parameter of the population with a short lifetime compared with that of phospholipid vesicles, whereas the population with a long lifetime agreed with that of the Torpedo membranes.Abbreviations PI phosphatidylinositol - PC phosphatidylcholine - PA phosphatidic acid - PE phosphatidylethanolamine - PS phosphatidylserine, PnA, cis-parinaric acid: cis,trans,trans,cis-9,11,13,15-octadecatetraenoic acid - 2-PnA-PC 1-acyl, 2-parinaroyl-PC - 2-PnA-PI 1-acyl,2-parinaroyl-PI - DPH diphenylhexatriene - POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene - NMR nuclear magnetic resonance - ESR electron spin resonance - I parallel fluorescence intensity component - I perpendicular fluorescence intensity component - SET-buffer 0.25 M Sucrose, 1 mM EDTA, 10mM Tris-HCl, pH 7.4     

Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach.

We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.     

Incorporation of integrins into artificial planar lipid membranes: characterization by plasmon-enhanced fluorescence spectroscopy

An optimized peptide-tethered artificial lipid membrane system has been developed. Integrins (cell adhesion receptors) were functionally incorporated into this membrane model and integrin-ligand interactions were analyzed by surface plasmon-enhanced fluorescence spectroscopy (SPFS). The transmembrane receptors alpha(v)beta(3) and alpha(1)beta(1) of the integrin superfamily were incorporated into a lipid-functionalized peptide layer by vesicle spreading. Consecutive layer formations were monitored by surface plasmon spectroscopy (SPS). Orientation and accessibility of the membrane receptor alpha(v)beta(3) was reliably assessed by specific and reproducible binding of selective antibodies. Moreover, full retention of the functional properties of this receptor was verified by specific and reversible binding of natural ligands. Functional integrity of incorporated integrins was maintained over a time period of 72 h. The integrin/extracellular matrix ligand complexes, whose formations are known to depend on the presence of divalent cations, were lost upon addition of ethylenediaminetetraacetate. Therefore, regeneration of the surface for further binding experiments with minimized unspecific ligand association was possible. These results demonstrate that integrins can be functionally incorporated into peptide-tethered artificial membranes. In combination with the SPS/SPFS method, this artificial membrane system provides a reliable experimental platform for investigation of isolated membrane proteins under experimental conditions resembling those of their native environment.     

Theory of fluorescence polarization of oriented pigment molecules in spherical arrays.

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 fluroescence 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.     

Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers

Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k _d 3nm.B _max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl^– and Ca²⁺, respectively. The reconstituted Ca²⁺ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca²⁺ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca²⁺/TRIS⁺ permeability ratio of 11.0. They also conducted other divalent cations (Ba²⁺>Ca²⁺>Sr²⁺>Mg²⁺) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca²⁺ channels, sharing some of the characteristics of Ca²⁺ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca²⁺ release from internal membrane stores.     

Voltage dependence of liver gap-junction channels reconstituted into liposomes and incorporated into planar bilayers.

The voltage dependence of rat liver gap junctions was investigated using non-denaturing solubilization and reconstitution of gap-junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single-channel conductance was voltage independent, and its magnitude was 700-1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed-state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage-sensitive conformational changes.     

Fluorescence polarization study of lipids and membranes prepared from brain hemispheres of a hibernating mammal

The physical behavior of total lipids, microsomes and microsomal lipids prepared from brain hemispheres of European Hamsters ($\text{Cricetus cricetus}$) was approached by the measure of the fluorescence polarization of the probe 1,6-diphenyl 1,3,5-hexatriene. We compare in this study the results obtained for two critical periods for a hibernator: winter (torpid state) and summer (active state). An increase in fluidity was noticed in the winter lipid and membrane preparations. The difference was however of very low magnitude, suggesting that only the microenvironment of some proteins was involved, rather than the bulk membrane fluidity.     

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied ‘food grade’ sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC₅₀ value of 5.521 mg/mL is high compared with 0.2811 μg/mL (182.5 ρM or 1.825 × 10⁻¹⁰ M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E^® is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%, 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease.     

Fluorescence studies of chlorophyll a incorporated into lipid mixtures, and the interpretation of "phase" diagrams.

The fluorescence of chlorophyll a incorporated into liposomes of mixtures of phosphatidylcholines and phosphatidylethanolamines is reported. Plots of fluorescence intensities against temperature show breaks at characteristic temperatures which can be attributed to the onset and completion of solid phase lipid formation. These temperatures can be plotted to give diagrams analogous to the phase diagrams obtained for macroscopic systems. Complications due to "small-system effects" are discussed, and the experimental diagrams are compared with theoretical phase diagrams calculated for ideal mixing. Introduction of cholesterol leads to a reduction in fluorescence intensity, most readily explained by a 1:1 lipid:cholesterol interaction with exclusion of monomeric, fluorescent, chlorophyll a. Interaction of divalent ions with mixtures of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylserine leads to exclusion of chlorophyll a from the phosphatidylserine.     

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane

Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. II. Binding of cytochrome c to oxidase-containing cardiolipin/phosphatidylcholine membranes.

Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, whereas the final saturation level of bound protein is independent of salt concentration and corresponds to approximately 53% of the total cco molecules present in the membrane. This suggests a 1:1 binding stoichiometry between the two proteins. The nonspecific binding component is governed by electrostatic interactions between cyt c and the membrane lipids and results in a partially ionic strength-reversible protein-membrane association. Thus, hydrophobic interactions between cyt c and the membrane, which are the predominant mode of binding in the absence of cco, are greatly suppressed. Both the amount of nonspecifically bound protein and the binding affinity can be varied over a broad range by changing the ionic strength and the extent of CL incorporation into the membrane. Under conditions approximating the physiological state in the mitochondrion (i.e., 20 mol% CL and medium ionic strength), 1-1.5 cyt c molecules are bound to the lipid phase per molecule of cco, with a dissociation constant of 0.1 microM. The possible physiological significance of these observations is discussed.     

Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations.

Depth-dependent fluorescence quenching in membranes is playing an increasingly important role in the determination of the low resolution structure of membrane proteins. This paper presents a graphical way of visualizing membrane quenching caused by lipid-attached bromines or spin labels with the help of a depth-dependent fluorescence quenching profile. Two methods are presently available to extract information on membrane penetration from quenching: the parallax method (PM; ) and distribution analysis (DA; A. S. Biophys. J. 64:290a (Abstr.); A. S. Methods Enzymol. 278:462-473). Analysis of various experimental and simulated data by these two methods is presented. The effects of uncertainty in the local concentration of quenching lipids (due to protein shielding or nonideality in lipid mixing), the existence of multiple conformations of membrane-bound protein, incomplete binding, and uncertainty in the fluorescence in nonquenching lipid are described. Regardless of the analytical form of the quenching profile (Gaussian function for DA or truncated parabola for PM), it has three primary characteristics: position on the depth scale, area, and width. The most important result, not surprisingly, is that one needs three fitting parameters to describe the quenching. This will keep the measures of the quenching profile independent of each other resulting in the reduction of systematic errors in depth determination. This can be achieved by using either DA or a suggested modification of the PM that introduces a third parameter related to quenching efficiency. Because DA utilizes a smooth fitting function, it offers an advantage for the analysis of deeply penetrating probes, where the effects of transleaflet quenching should be considered.     

A theoretical analysis of binding to the Ca2+-specific sites on troponin incorporated into thin filaments.

Recent data on the binding of Ca2+ to the specific sites on troponin, alone, in regulated actin, and in regulated actomyosin, as well as data on the Ca2+ activation of the actomyosin ATPase (Grabarek, Z., J. Grabarek, P.C. Leavis, and J. Gergely, 1983, J. Biol. Chem., 258:14098-14102.), are analyzed on the basis of a model used previously for qualitative theoretical studies of the Ca2+ activation of muscle contraction (Shiner and Solaro, 1982). The data allow and require an extension of the model to consider the effects of tropomyosin explicitly. Three major results of the analysis are at variance with previous investigations. A repulsive interaction between tropomyosins; and an attractive interaction between actins (or myosin heads attached to actin) are found, whereas others have found or assumed an attractive tropomyosin-tropomyosin interaction and no actin-actin interaction. The parameter values found here predict hysteresis under the conditions of the ATPase experiments; no other existing model for the interactions manifest in the Ca2+ activation of contraction can predict hysteresis. The prediction is of increased interest in light of experimental reports of hysteresis in the Ca2+ activation of isometric force (Ridgeway, E. B., A. M. Gordon, and D. A. Martyn, 1983, Science (Wash. DC), 219:1075-1077; Gordon, A. M., E. B. Ridgeway, and D. A. Martyn, 1984, Plenum Publishing Corp., New York, 553-563; Brandt, P. W., B. Gluck, M. Mini, and C. Cerri, 1985, J. Mus. Res. Cell Motil. 6:197-205.).     

An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single-channel behavior and conductance properties

Summary An anion channel of sarcoplasmic reticulum vesicle has been incorporated into planar lipid bilayers by means of a fusion method and its basic properties were investigated. Analysis of fusion processes suggested that one SR vesicle contained approximately one anion channel. The conductance of this channel has several substates and shows a flickering behavior. The occupation probability of each substate was voltage dependent, which induced an inward rectification of macroscopic currents. Further, the anion channel was found to have the following properties. (1) The single-channel conductance is about 200 pS at 100mm Cl^–. (2) The channel does not select among monovalent anions but SO ₄ ^2– hardly permeates through the channel. (3) SO ₄ ^2– added to thecis side (the side to which SR vesicles were added) inhibits Cl^– current competitively in a voltage-dependent manner. (4) An analysis of this voltage dependence suggests that the binding site of SO ₄ ^2– is located at about 36% of the way across the channel from thecis entrance.     

The electrical response to light of bacteriorhodopsin in planar membranes.

We have measured the light-induced short-circuit current generated by a planar membrane containing bacteriorhodopsin incorporated by vesicle fusion. The experimental results are consistent with an equivalent electrical circuit analogue that assumes that the vesicles remain intact after fusion and that the current generator equivalent of the light-driven proton pump is linearly dependent on bias voltage. The transient response to light of the planar membrane has also been examined. Slow response times are seen to be associated with the capacitive charging and discharging of the fused vesicles. A study of the leading edge of the light response curve of the planar membrane yields information about the transient response of the light-driven proton pump. We propose that the translocation of protons across the membrane is associated with a first-order process characterized by a rate constant lambda.