Protein rotational diffusion and lipid/protein interactions in recombinants of bovine rhodopsin with saturated diacylphosphatidylcholines of different chain lengths studied by conventional and saturation-transfer electron spin resonance.

Bovine rhodopsin has been reconstituted in seven different saturated diacylphosphatidylcholine species of odd and even chain lengths from C-12 to C-18 at a lipid/protein ratio (60:1 mol/mol) comparable to that in the native rod outer segment disk membrane. All recombinants were found to be photochemically active, in that optical bleaching produced a temperature- and lipid chain-length-dependent mixture of species absorbing at 480 and 380 nm. Both the rotational diffusion of rhodopsin and lipid-protein interactions in the various recombinants were studied by saturation transfer and conventional electron spin resonance spectroscopy of spin-labeled rhodopsin and of spin-labeled phosphatidylcholine, respectively. In the fluid lipid phase, the rotational diffusion rate of rhodopsin was found to be dependent on the lipid chain length of the different recombinants in a nonmonotonic manner. The diffusion rate in dilauroylphosphatidylcholine was found to be very slow, indicating extensive protein aggregation, whereas that in dipentadecanoylphosphatidylcholine was rapid (effective correlation time ca. 7 microseconds), consistent with the presence of monomeric protein. For recombinants with longer lipid chain lengths, the rotational diffusion rate again decreased, indicating the presence of di- or oligomeric protein. The fraction of lipid motionally restricted at temperatures in the fluid phase was also dependent on the chain length of the phosphatidylcholine used in the reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotational mobility of an erythrocyte membrane integral protein band 3 in dimyristoylphosphatidylcholine reconstituted vesicles and effect of binding of cytoskeletal peripheral proteins

Band 3 protein was isolated from human erythrocyte membranes, purified, and reconstituted into a well-defined phospholipid bilayer matrix (dimyristoylphosphatidylcholine). The preparation yielded uniform single-bilayered vesicles of the diameter 40--80 nm. The rotational motion of band 3 was studied by saturation transfer electron spin resonance (ESR) spectroscopy of covalently attached maleimide spin-labels. The rotational mobility changed in response to the host lipid phase transition. The rotational correlation time was in a range from 73 (37 degrees C) to 94 microseconds (26 degrees C) in the fluid phase and from 240 (15 degrees C) to 420 microseconds (5 degrees C) in the solid phase. The motion was analyzed based on the anisotropic rotation of band 3 in the reconstituted vesicles. To obtain information on the rotational diffusion constant around the axis parallel to the membrane normal, we made an attempt to measure the angle between the spin-label magnetic axis and the membrane normal. The result gave 3.9 x 10(4) s-1 at 37 degrees C as a rough estimate for the diffusion constant. This is compatible to anisotropic rotation of a cylinder of radius 3.3 nm in a two-dimensional matrix with inner viscosity 2 P and inner thickness 4 nm. The cytoskeletal peripheral proteins caused a definite increase in the rotational correlation time (from 73 to 180 microseconds at 37 degrees C, for example). The restriction of the rotational mobility was shown to be due to the ankyrin-linked interaction between band 3 and spectrin-actin-band 4.1 proteins in the reconstituted membranes.     

Functional reconstitution of rhodopsin into tubular lipid bilayers supported by nanoporous media

We report on a novel reconstitution method for G-protein-coupled receptors (GPCRs) that yields detergent-free, single, tubular membranes in porous anodic aluminum oxide (AAO) filters at concentrations sufficient for structural studies by solid-state NMR. The tubular membranes line the inner surface of pores that traverse the filters, permitting easy removal of detergents during sample preparation as well as delivery of ligands for functional studies. Reconstitution of bovine rhodopsin into AAO filters did not interfere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored by UV-vis spectrophotometry, was indistinguishable from rhodopsin in unsupported unilamellar liposomes. The rhodopsin in AAO pores is G-protein binding competent as shown by a [35S]GTPgammaS binding assay. The lipid-rhodopsin interaction was investigated by 2H NMR on sn-1- or sn-2-chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as a matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed to spectral density of motions with correlation times in the range of nano- to microseconds, detected as a significant reduction in spin-spin relaxation times. The change in lipid chain order parameters due to interaction with rhodopsin was insignificant.     

Rotational motion of yeast cytochrome oxidase in phosphatidylcholine complexes studied by saturation-transfer electron spin resonance

Cytochrome oxidase from yeast has been covalently labeled with a nitroxide derivative of maleimide and reconstituted in lipid-substituted complexes with dimyristoyl-, dioleoyl-, or dielaidoyl-phosphatidylcholine. The rotational mobility of the enzyme in the complexes has been studied as a function of temperature and time, and of lipid/protein ratio, using saturation-transfer electron spin resonance spectroscopy. For complexes with dimyristoylphosphatidylcholine, the rotational mobility of the protein decreases abruptly below the gel-to-fluid-phase transition. This change is accompanied by a lateral segregation of the protein, as seen by freeze-fracture electron microscopy, and by an increase in the activation energy for the enzymatic activity. A time-dependent decrease in the rotational motion of the protein is observed on incubating at temperatures in the fluid phase of the lipid. This corresponds with a time-dependent loss of enzyme activity observed on incubation at temperatures in the fluid phase, but not at temperatures in the gel phase, over a period of 3 h. The rotational mobility decreases with increasing protein concentration in the complexes, both in the fluid and in the gel phases. The dependence of the protein mobility on lipid/protein ratio can be interpreted quantitatively in terms of the effect of increased random protein-protein contacts in the fluid phase. The maximum limiting rotational correlation time for the protein diffusion at high lipid/protein ratios in the fluid phase is tau R[[ approximately equal to 25 microseconds, suggesting that the protein is present as either a monomer or more probably a dimer in the reconstituted membrane.     

Distribution of charge on photoreceptor disc membranes and implications for charged lipid asymmetry.

A novel spin labeling technique is used to determine both the inner and outer surface potentials of isolated rod outer segment disc membranes and of reconstituted membranes containing rhodopsin with defined lipid compositions. It is shown that these potentials can be accounted for in a consistent manner by the accepted model of rhodopsin, the known lipid composition, and the Gouy-Chapman theory, provided the charged lipid is asymmetric in the membrane, with approximately 75% on the external surface.     

Effect of packing density on rhodopsin stability and function in polyunsaturated membranes

Rod outer segment disk membranes are densely packed with rhodopsin. The recent notion of raft or microdomain structures in disk membranes suggests that the local density of rhodopsin in disk membranes could be much higher than the average density corresponding to the lipid/protein ratio. Little is known about the effect of high packing density of rhodopsin on the structure and function of rhodopsin and lipid membranes. Here we examined the role of rhodopsin packing density on membrane dynamic properties, membrane acyl chain packing, and the structural stability and function of rhodopsin using a combination of biophysical and biochemical techniques. We reconstituted rhodopsin into large unilamellar vesicles consisting of polyunsaturated 18:0,22:6n3PC, which approximates the polyunsaturated nature of phospholipids in disk membranes, with rhodopsin/lipid ratios ranging from 1:422 to 1:40. Our results showed that increased rhodopsin packing density led to reduced membrane dynamics revealed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, increased phospholipid acyl chain packing, and reduced rhodopsin activation, yet it had minimal impact on the structural stability of rhodopsin. These observations imply that densely packed rhodopsin may impede the diffusion and conformational changes of rhodopsin, which could reduce the speed of visual transduction.     

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using saturation-transfer electron spin resonance

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.     

Aggregation of rhodopsin molecules during damaging exposure of photoreceptor membranes to light

Injuring light induced structural changes in rod outer segment (ROS) membranes are studied using "ST EST spectroscopy" for spin labelled rhodopsin, ESR of lipid spin label and SDS gel-electrophoresis. Free SH-group content of rhodopsin and lipid peroxidation level were simultaneously determined as well. A decrease of rotational mobility of rhodopsin in ROS induced by prolonged illumination is shown to result from irreversible protein aggregation caused by disulfide bond formation between "hydrophobic" SH-groups of rhodopsin. Some decrease of lipid microviscosity and degree of order are found, in contrast to considerable rise in microviscosity due to Fe2+-ascorbate induced lipid peroxidation of ROS membranes. Lipid oxidation is found to accelerate protein aggregation which in its turn influences the state of lipid bilayer.     

Rotational mobility and orientational stability of a transport protein in lipid membranes

A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide spin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin resonance. Bovine rhodopsin was also selectively spin-labeled and studied to aid the interpretation of the measurements. Observations of spin-labeled proteins in macroscopically aligned bilayers indicated that the spin label tends to orient so as to reflect the transmembrane orientation of the protein. Rotational correlation times of 1-2 micros for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers of dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the specific mixture of lipids used to reconstitute LacS (30 degrees C). The rotational correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to decrease sharply and was determined to be 9 +/- 1 micros (mean +/- SD) for these samples. This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongated configuration within the membrane. Rather than changing its oligomeric state, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid.     

The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates.

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids.     

Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin

Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.     

Rotational dynamics of protein and boundary lipid in sarcoplasmic reticulum membrane

We have used spin labels and electron paramagnetic resonance (EPR) to study the correlation between the rotational dynamics of protein and lipid in sarcoplasmic reticulum (SR) membranes. A short-chain maleimide spin label was used to monitor the submillisecond rotational mobility of the Ca-ATPase enzyme (using saturation transfer EPR); a free fatty acid spin label was used to monitor the submicrosecond rotational mobility of the bulk lipid hydrocarbon chains (using conventional EPR); and a fatty acid spin label derivative (long-chain maleimide) attached to the enzyme was used to monitor the mobility of hydrocarbon chains adjacent to the protein (i.e., boundary lipid). In the native SR membranes, the protein was highly mobile (effective correlation time 50 microseconds). The spectra of the hydrocarbon probes both contained at least two components. For the unattached probe, the major component indicated nearly as much mobility as in the absence of protein (effective rotational correlation time 3 ns), while a minor component, corresponding to 25-30% of the total signal, indicated strong immobilization (effective correlation time greater than or equal to 10 ns). For the attached hydrocarbon probe, the major component (approximately 70% of the total) was strongly immobilized, and the mobile component was less mobile than that of the unattached probe. When the lipid-to-protein ratio was reduced 55% by treatment with deoxycholate, protein mobility decreased considerably, suggesting protein aggregation. A concomitant increase was observed in the fraction of immobilized spin labels for both the free and attached hydrocarbon probes. The observed hydrocarbon immobilization probably arises in part from immobilization at the protein-lipid boundary, but protein-protein interactions that trap hydrocarbon chains may also contribute. When protein aggregation was induced by glutaraldehyde crosslinking, submillisecond protein mobility was eliminated, but there was no effect on either hydrocarbon probe. Thus protein aggregation does not necessarily cause hydrocarbon chain immobilization.     

Rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy

We have investigated the microsecond rotational motions of the Ca-ATPase in rabbit skeletal sarcoplasmic reticulum (SR), by measuring the time-resolved phosphorescence anisotropy of erythrosin 5-isothiocyanate (ERITC) covalently and specifically attached to the enzyme. Over a wide range of solvent conditions and temperatures, the phosphorescence anisotropy decay was best fit by a sum of three exponentials plus a constant term. At 4 degrees C, the rotational correlation times were phi 1 = 13 +/- 3 microseconds, phi 2 = 77 +/- 11 microseconds, and phi 3 = 314 +/- 23 microseconds. Increasing the solution viscosity with glycerol caused very little effect on the correlation times, while decreasing the lipid viscosity with diethyl ether decreased the correlation times substantially, indicating that the decay corresponds to rotation of the protein within the membrane, not to vesicle tumbling. The normalized residual anisotropy (A infinity) is insensitive to viscosity and temperature changes, supporting the model of uniaxial rotation of the protein about the membrane normal. The value of A infinity (0.20 +/- .02) indicates that each of the three decay components can be analyzed as a separate rotational species, with the preexponential factor Ai equal to 1.25X the mole fraction. An empirically accurate measurement of the membrane lipid viscosity was obtained, permitting a theoretical analysis of the correlation times in terms of the sizes of the rotating species. At 4 degrees C, the dominant correlation time (phi 3) is too large for a Ca-ATPase monomer, strongly suggesting that the enzyme is primarily aggregated (oligomeric).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotational dynamics of erythrocyte spectrin

The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere and rigid rod. The data indicate that the ribbon-like spectrin molecule possesses considerable torsional and segmental flexibility. These motions are restricted, but not abolished, when spectrin is reconstituted into cross-linked cytoskeletal protein networks, or bound to spectrin-actin depleted erythrocyte membrane vesicles.     

Microaggregation of hormone-occupied epidermal growth factor receptors on plasma membrane preparations.

The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time-resolved polarization of phosphorescence of erythrosin-labeled hormone. The measured rotational correlation times of 16-20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association.     

Effects of alpha-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

The effect of alpha-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of alpha-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. beta-, gamma-, and delta-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 +/- 0.12 microM) of the interaction of alpha-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of alpha-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 microM ascorbic acid and 10 microM Fe2+. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by alpha-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 +/- 0.16 to 18.5 +/- 0.51 microseconds after addition of 25 microM alpha-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by alpha-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl+. Based on these results, the effect of alpha-tocopherol on the lipid fluidity of the membranes is discussed.     

The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-alpha-dipalmitoylglycerophosphoethanolamine (Pam2 GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator. The temperature dependence of the anisotropy decay showed that above 25 degrees C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (eta 1). Extrapolation to eta 1 = 0 Pa.s yielded a correlation time of phi = 20 +/- 5 ns, t = 30 degrees C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr = 2.0 x 10(9) rad2.s-1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was eta 2 approximately 12 mPa.s. The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of eta 1 = 1 mPa.s yielded a value of phi r approximately 450 ns corresponding to Dr = 8.8 x 10(7) rad2.s-1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross-sectional area of the protein part embedded in the membrane was calculated to be approximately 9 nm2. This cross-sectional area is large enough to include at most 14 membrane-spanning helices. Our results also indicated that at lipid/protein molar ratios greater than or equal to 1.5 x 10(4): 1 aggregation occurred in the model membranes below 30 degrees C. However, above 30 degrees C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.     

Self-association of class I major histocompatibility complex molecules in liposome and cell surface membranes.

Fluorescent derivatives of a human MHC class I glycoprotein, HLA-A2, were reconstituted into dimyristoylphosphatidylcholine (DMPC) liposomes. Measurements of lateral diffusion of fluorescein-(Fl-) labeled HLA-A2 by fluorescence photobleaching recovery (FPR), of rotational diffusion of erythrosin-(Er-) labeled HLA-A2 by time-resolved phosphorescence anisotropy (TPA), and of molecular proximity by flow cytometric fluorescence resonance energy transfer (FCET) showed that these class I MHC molecules self-associate in liposome membranes, forming small aggregates even at low surface concentrations. The lateral diffusion coefficient (Dlat) of Fl-HLA-A2 decreases with increasing surface protein concentration over a range of lipid:protein molar ratios (L/P) between 8000:1 and 2000:1. The reduction in Dlat of HLA molecules in DMPC liposomes is found to be sensitive to time and temperature. The rotational correlation time for Er-HLA-A2 in DMPC liposomes at 30 degrees C is 87 +/- 0.8 microseconds, at least 10 times larger than that expected for an HLA monomer. There is also significant quenching of donor (Fl-HLA) fluorescence at 37 degrees C in the presence of acceptor-labeled (sulforhodamine-labeled HLA) protein indicating proximity between HLA molecules even at L/P = 4000:1. FPR and FCET measurements with another membrane glycoprotein, glycophorin, give no evidence for its self-association. HLA aggregation measured by FPR, FCET, and TPA was blocked by beta 2-microglobulin, b2m, added to the liposomes. The aggregation of HLA-A2 molecules is not an artifact of their reconstitution into liposomes. HLA aggregates, defined by FCET, were readily detected on the surface of human lymphoblastoid (JY) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Influenza Virus Fusion Peptide with Lipid Membranes: Effect of Lysolipid

The effect of lysophosphatidylcholine (LPC) on lipid vesicle fusion and leakage induced by influenza virus fusion peptides and the peptide interaction with lipid membranes were studied by using fluorescence spectroscopy and monolayer surface tension measurements. It was confirmed that the wild-type fusion peptide-induced vesicle fusion rate increased several-fold between pH 7 and 5, unlike a mutated peptide, in which valine residues were substituted for glutamic acid residues at positions 11 and 15. This mutated peptide exhibited a much greater ability to induce lipid vesicle fusion and leakage but in a less pH-dependent manner compared to the wild-type fusion peptide. The peptide-induced vesicle fusion and leakage were well correlated with the degree of interaction of these peptides with lipid membranes, as deduced from the rotational correlation time obtained for the peptide tryptophan fluorescence. Both vesicle fusion and leakage induced by the peptides were suppressed by LPC incorporated into lipid vesicle membranes in a concentration-dependent manner. The rotational correlation time associated with the peptide’s tryptophan residue, which interacts with lipid membranes containing up to 25 mole % LPC, was virtually the same compared to lipid membranes without LPC, indicating that LPC-incorporated membrane did not affect the peptide interaction with the membrane. The adsorption of peptide onto a lipid monolayer also showed that the presence of LPC did not affect peptide adsorption.     

Reconstitution of an electrically active conformational transition in rhodopsin-containing membranes

An electrically active event that has been observed in native rod outer segment disk membranes can be reconstituted into membrane vesicles containing purified rhodopsin and defined phospholipids. The magnitude of this charge-transfer event, as estimated using spin-labeled derivatives of hydrophobic ions, is a function of the phospholipid composition. In reconstituted membranes containing rhodopsin and egg phosphatidylcholine, the charge transferred during this event is approximately 10% that measured in the native system. The addition of 20 mol% egg phosphatidylethanolamine, phosphatidic acid or brain phosphatidylserine returns the magnitude of the charge transfer to within 60 to 100% of the native activity. The response seen in the reconstituted membrane system is consistent with a previously proposed interfacial charge-transfer mechanism.