Association of bacteriorhodopsin with lipid-impregnated filters. Evidence for fusion of bacteriorhodopsin-containing vesicles with the lipid phase of the filter.

Bacteriorhodopsin vesicles were associated with cellulose-nitrate filters impregnated with a solution of phospholipids in hexadecane. The generation of (photo)potentials upon illumination of the filter was studied in the absence and presence of ionophores, phospholipase A2, EDTA or polyene antibiotics. From these experiments the following conclusions are drawn. 1. Upon illumination of the filter, bacteriorhodopsin pumps protons into aqueous compartments located in the filter. 2. These aqueous compartments possibly do not originate from the compartments enclosed by the bacteriorhodopsin vesicles. Evidence is obtained that aqueous compartments are present in the surface layers of the lipid-impregnated filters. 3. The results are explained most easily by a mechanism, whereby fusion occurs between the vesicles and the lipids of the filter.     

Thermal acoustic radiation from multilamellar vesicles in lipid phase transition

A new acoustical method for the investigation of lipid phase transition is introduced based on the measurement of the thermal acoustic radiation (TAR) inherent in lipids. The TAR of multilamellar vesicles from dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) was measured in the megahertz range and the variations in the radiation intensity during the lipid phase transition were recorded. Two types of variations are possible: if the temperature of the vesicles decreases (in the process of transition from the liquid crystalline state to the gel state) then the TAR intensity increases, and if the temperature increases (in the reverse transition) then the TAR intensity decreases. These effects are connected with an increase in the ultrasonic absorption in the vesicles under lipid phase transition. Basing on the results of the TAR investigation, a new theoretical estimate has been developed of the variation in the absorption coefficient during the lipid phase transition. In this estimate, the variation is equated to the ratio of the phase transition entropy to the gas constant.     

A new method for membrane reconstitution: Fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature

A new method of membrane reconstitution was developed by fusion of channel protein containing vesicles with planar bilayer membranes. The fusion process only occurred below and near the phase transition temperature of the lipid used. We obtained the following results: 1. Our system is solvent-free and vesicles do not come into contact with the air/water interface. This obviates a possible denaturation of hydrophobic proteins. 2. Channel forming proteins and protein complexes, respectively, are active in a frozen lipid matrix. 3. We detected an unknown channel in cilia fragments. 4. Purified acetylcholine receptors form fluctuating channels in a membrane consisting of a pure synthetic lecithin (two component system).     

Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition.

The existence of distinct regions of mismatch in molecular packing at the interfaces of the fluid and ordered domains during the phase transition of dimyristoylphosphatidylcholine vesicles has been demonstrated by measuring the temperature dependence of the permeability to a spin-label cation and comparing this with a statistical mechanical calculation of the fraction of interfacial lipid. The kinetics of uptake and release of the 2,2,6,6-tetramethylpiperidinyl-1-oxycholine (Tempo-choline) spin label by single-bilayer dimyristoylphosphatidylcholine vesicles were measured using electron spin resonance spectroscopy to quantitate the amount of spin label present within the vesicles after removal of the external spin-label by ascorbate at 0 degrees C. Both the uptake and release experiments show that the Tempo-choline permeability peaks to a sharp maximum at the lipid-phase transition, the vesicles being almost impermeable to Tempo-choline below the transition and having a much reduced permeability above. The temperature profile of the permeability is in reasonable quantitative agreement with calculations of the fraction of interfacial boundary lipid from the Zimm and Bragg theory of cooperative transitions, which use independent spin-label measurements of the degree of transition to determine the cooperativity parameter. The relatively high intrinsic permeability of the interfacial regions (P approximately 0.2-1.0 X 10(-8) cm/s) is attributed to the mismatch in molecular packing of the lipid molecules at the ordered-fluid boundaries, which could have important implications not only for permeability in natural membranes (e.g., in transmitter release), but also for the function of membrane-bound enzymes and transport proteins.     

Negative hydrophobic ions as transport-mediators for positive ions. Evidence for a carrier mechanism

The permeability of hydrophobic cations, such as tetraphenylarsonium across biological membranes and artificial lipid membranes is strongly increased in the presence of trace amounts of hydrophobic anions like tetraphenylborate (Liberman, Y.A. and Topaly, V.P. (1969) Biofizika 14, 452–461). Voltage-jump relaxation experiments performed on thin lipid membranes support the idea that the anions, A^?, act as carriers for the cations, B⁺, by the formation of neutral ion pairs, A^?B⁺. Their permeability is not affected by the electric dipole potential, which hinders the movement of free cations, B⁺.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

Effects of electric field on the photocycle of bacteriorhodopsin

The photoconversion of bacteriorhodopsin and the effects of an applied electric field (5 · 10⁷ V · m^?1) were studied in dry films of purple membranes from Halobacterium halobium. The electric field was found to cause at least two different effects: (1) it blocks in part the formation of the batho-bacteriorhodopsin (K), most probably due to electrically-induced dark transition of some bacteriorhodopsin molecules into the photochemically inactive form; (2) it decreases the rate of the intermediate M decay, the rise time of the M formation being unaffected by electric field. The observed phenomena may suggest a feedback control mechanism for the regulation of the bacteriorhodopsin photocycle in purple membranes.     

An EPR study of the interfacial properties of phosphatidylcholine vesicles with different lipid chain lengths

The hydrophobic spin probe 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl octadecanoate (TEMPO-stearate) is used to study the interfacial properties of a variety of phosphatidylcholine vesicles. Since the spin probe exhibits a fast motional electron paramagnetic resonance (EPR) spectrum above the phase transition, the EPR spectrum of the spin probe is analyzed by nonlinear least-squares spectral fitting. EPR spectral line fitting provides high precision spectral parameters, which can be used to construct a detailed picture of the dynamics of the probe and its environment. The hyperfine coupling spacing is used to estimate the effective water concentration in the polar shell of vesicles, while the rotational correlation times give the information on the motion of the spin probe. The effective water concentration of the polar shell of dimyristoyl-phosphatidylglycerol (DMPG) vesicles is greater on average by about 4.0M than the effective water concentration of the polar shell of dimyristoyl-phosphatidylcholine (DMPC) vesicles. The effective water concentration decreases by about 0.5M for an increase of two carbons in the chain, and increases noticeably with hydrocarbon chain unsaturation, which is in good agreement with literature values. The nitroxide moiety rotates preferentially along the N-O bond, that is, parallel to its hydrocarbon chain.     

Simulations of frequency-dependent photoacoustic magnitude signals and their implications for bacteriorhodopsin photocycle energetics

The modulation frequency dependence of photoacoustic signals obtained from photoactive samples can provide information on the time-dependent enthalpy changes occurring during the light-induced process. The experimental requirements for this type of calorimetry, and the interpretation ot the results, are critically examined with reference to the light-driven proton pump bacteriorhodopsin. For a three-step unbranched model of the bacteriorhodopsin photocycle we derive an expression for the photoacoustic magnitude signal as a function of frequency. Simulations are performed for various values of the rate constants and energetic changes. It is shown that the net heat uptake during a low, final step postulated by some workers should be reflected in the photoacoustic magnitude frequency spectrum, giving rise to a characteristic maximum. However, this effect, which has been observed experimentally, may also be produced by a fast, strongly endothermic step occurring earlier. The precise chronology of an endothermic transition cannot be resolved unambiguously by magnitude measurements alone, although they are free from assumptions regarding difficult-to-measure phase relationships. Hence, the published photoacoustic observations showing the effect are consistent with a cyclic sequence of events in which the bacteriorhodopsin system first undergoes an increase of entropy, followed by a decrease on returning to the initial state, as well as the reverse. It is argued that the molecular disorder-order sequence is more probable.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Interactions of La3+ with phosphatidylserine vesicles binding,phase transition,leakage and fusion

The interaction of La³⁺ with phosphatidylserine vesicles is elucidated by binding studies, differential scanning calorimetry, X-ray diffraction, freeze fracture electron microscopy, and release of vesicle contents. La³⁺ effectively competes with Ca²⁺ for phosphatidylserine binding sites. The saturation level is close to a La/lipid ratio of 1:3. A concentration of 0.1 mM of La³⁺ is sufficient to induce fusion between sonicated vesicles.     

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (T_c) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [¹⁴C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T_c of the lipid and increases with temperature above the T_c. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.     

Studies on the mechanism of capacitation II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro

1. 1. Evidence has been provided for the transfer of phosphatidyl[¹⁴C]choline and [³H]cholesterol between bovine serum albumin and cauda epididymal rat spermatozoa in Krebs-Ringer bicarbonate medium, which can promote sperm capacitation.

2. 2. An analysis of the lipid composition in both albumin and spermatozoa revealed that phospholipid levels decreased in the protein and increased by roughly comparable amounts in sperm cells during incubation in vitro.

3. 3. Cholesterol (free + ester) increased in albumin and decreased in spermatozoa. Changes in the amount of esterified cholesterol were solely responsible for the increase associated with albumin, whereas whole sperm cell extracts showed a significant decline in free cholesterol.

4. 4. The composition of albumin-bound fatty acids did not alter appreciably as a result of incubation with spermatozoa.

5. 5. Rates of [¹⁴C]palmitic acid utilization by spermatozoa suggest that lipid synthesis accounted for less than 5% of the changes observed under the conditions of this study.

6. 6. These results are interpreted as broadly supporting our previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro. Specifically, the results are compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.

In vitro interaction of zajdela ascites hepatoma cells with lipid vesicles

We studied the in vitro interaction between Zajdela ascites hepatoma cells and small unilamellar vesicles, consisting of ¹⁴C-labeled phosphatidylcholine, cholesterol, and phosphatidylserine (molar ratio 5: 4: 1), containing high intravesicular concentrations of carboxyfluorescein or fluorescein isothiocyanate tagged dextran.The entrapped markers were found to be associated with the cells to a lesser degree than the vesicle membrane marker. This discrepancy, which is slightly less pronounced for fluorescein isothiocyanate tagged dextran than for carboxyfluorescein, increases with incubation time and decreases with increasing vesicle lipid concentration in the incubation mixture. Vesicle-plasma membrane exchange of the vesicle lipid marker could not entirely explain the observed discrepancy. It is tentatively concluded that the gap mainly arises from a selective loss of entrapped dyes from vesicles actually interacting with the cell surface. Both spectrofluorimetric and fluorescence microscopic observations, as well as the relative insensitivity of vesicle uptake towards the presence of metabolic inhibitors, exclude a major contribution of endocytosis as a vesicle uptake route. We therefore conclude that vesicles are primarily internalized by a vesicle-cell fusion-like process. The observed discrepancy in uptake between entrapped materials and vesicle lipid is discussed in terms of a two-site vesicle cell surface interaction model.     

Interactions of La with phosphatidylserine vesicles binding, phase transition, leakage and fusion

The interaction of La³⁺ with phosphatidylserine vesicles is elucidated by binding studies, differential scanning calorimetry, X-ray diffraction, freeze fracture electron microscopy, and release of vesicle contents. La³⁺ effectively competes with Ca²⁺ for phosphatidylserine binding sites. The saturation level is close to a La/lipid ratio of 1:3. A concentration of 0.1 mM of La³⁺ is sufficient to induce fusion between sonicated vesicles.     

Fusion of bacteriorhodopsin with submitochondrial particles yields a new system with retention of energy coupling and acquisition of photophosphorylation activity

Submitochondrial particles were fused with purple membranes of Halobacterium halobium cells by means of a freeze-thawe sonication procedure. It is reported that fusion of inner mitochondrial membranes with a bacterial membrane yields a new particle which shows not only retention of redox- and photon-linked energy-coupling activities, but also creation of an additional energy-coupling process, light-driven ATP synthesis.     

Interaction of influenza virus proteins with planar bilayer lipid membranes II. Effects of rimantadine and amantadine

The dependence of the surface potential difference (ΔU), transversal elasticity module (E₁) and membrane conductivity (G₀) on the concentrations of the antiviral drugs, rimantadine and amantadine was studied in the planar bilayer lipid membrane system. The method used was based on independent measurements of the second and third harmonics of the membrane capacitance current. The binding constants of bilayer lipid membranes obtained from the drug adsorption isotherms were 2.1 · 10⁵ M^?1 and 1.3 · 10⁴ M^?1 for rimantadine and amantadine, respectively. The changes in G₀ took place only after drug adsorption saturation had been achieved. The influence of rimantadine and amantadine on the interaction of bilayer lipid membranes with matrix protein from influenza virus was also investigated. The presence of 70 μg/ml rimantadine in the bathing solution resulted in an increase in the concentration of M-protein at which the adsorption and conductance changes were observed. The effects of amantadine were similar to those of rimantadine but required a higher critical concentration of amantadine. The results obtained suggest that the antiviral properties of rimantadine and amantadine may be related to the interaction of these drugs with the cell membrane, which can affect virus penetration into the cell as well as maturation of the viral particle at the cell membrane.     

My remembrances of H.G. Khorana: exploring the mechanism of bacteriorhodopsin with site-directed mutagenesis and FTIR difference spectroscopy

H.G. Khorana’s seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced vibrational spectroscopic techniques such as FTIR difference spectroscopy to explore the mechanism of bacteriorhodopsin and other integral membrane proteins. In this review, I provide a personal perspective of my collaborative research and interactions with Gobind, from 1982 to 1995 when our groups published over 25 papers together which resulted in an early picture of key features of the bacteriorhodopsin proton pump mechanism. Much of this early work served as a blueprint for subsequent advances based on combining protein bioengineering and vibrational spectroscopic techniques to study integral membrane proteins.     

Destabilization of a lipid non-bilayer phase by high pressure

Pressure is found to destabilize the non-bilayer phase with respect to the bilayer in a model lipid system. The lamellar to inverted hexagonal (H₁₁) phase transition of aqueous egg phosphatidylethanolamine is shifted to higher temperatures by hydrostatic pressure. The slope of the increase in transition temperature is constant to beyond 300 bar, and is greater than that seen for other lipid phase transitions. This behavior is consistent with the hypothesis that increasing chain disorder drives the conversion from the bilayer into the hexagonal phase. If this non-bilayer lipid phase is an intermediate in membrane fusion, then pressure should inhibit the process. This may explain the inhibition of chemical transmission at neural synapses by pressure.     

Phase transition and lateral phase separation in planar lipid membranes as sensed by the lipophilic ion dipicrylamine

The permeation of the lipophilic ion dipicrylamine through planar lipid membranes formed from dipalmitoylphosphatidylcholine in n-decane shows an anomaly near the main phase transition of this system. Both the rate constant, k_i, of ion translocation across the membrane interior and the interfacial concentration, N, of this ion have a maximum at about 36°C. Analogous experiments were performed with tetraphenylborate. A considerably lesser effect of the phase transition was found. The addition of cholesterol leads to a broadening of the maxima for k_i and N. The time course of the current following a voltage jump shows a characteristic change below a temperature of about 45°C, if the molar ratio cholesterol/ phosphatidylcholine in the membrane forming solution exceeds 1. While the current transient decays exponentially above 45°C, a sum of two exponential terms yields an adequate fit below that temperature. This is regarded as evidence for a lateral phase separation below 45°C into structurally different domains, which provide two different pathways for dipicrylamine.