Glycophorin-induced cholesterol-phospholipid domains in dimyristoylphosphatidylcholine bilayer vesicles.

Glycophorin has been incorporated into unilamellar cholesterol-containing dimyristoylphosphatidylcholine vesicles that were reconstituted by the freeze and thaw technique. Evidence was obtained for a protein-induced structural reorganization of these mixed membranes. By differential scanning calorimetry, we were able to construct a phase diagram for the phospholipid/cholesterol mixture consisting of a liquid-ordered, a solid-ordered, and a liquid-disordered phase. Glycophorin at low molar fractions (XG less than 3 X 10(-3)) increases the relative amount of lipid in the liquid-ordered phase, which is interpreted as an enrichment of cholesterol in the vicinity of the protein. The formation of such steroid-enriched domains could be demonstrated directly by electron paramagnetic resonance using a spin-labeled cholesterol analogue. A drastic increase of the spin-spin interaction of the labeled steroid was observed in the presence of glycophorin.     

Lecithin:cholesterol acyltransferase regulation. Effect of fluidity of dimyristoylphosphatidylcholine vesicles

The regulation of lecithin:cholesterol acyltransferase by changes in phospholipid bilayer fluidity was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity of dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles was decreased by the addition of up to 20% (mol/mol) cholesterol and increased by the addition of up to 10% (mol/mol) lysoDMPC. When both cholesterol and lysoDMPC are present in the bilayer, their individual effects on fluidity are altered. These changes can be explained by complex formation between cholesterol and phospholipid as in the model of Presti et al. (Presti, F.C., Pace, R.J. and Chan, S.I. (1982) Biochemistry 21, 3831-3335). Lecithin:cholesterol acyltransferase activity with these vesicles as substrates was measured to determine whether activity can be modulated by the fluidity changes of the bilayer on which the enzyme acts. When 10% lysoDMPC, a known lecithin:cholesterol acyltransferase inhibitor, is added to the vesicles, inhibition of activity is observed. When 7.5% lysoDMPC is added to vesicles which contain either 5 or 10% cholesterol, lecithin:cholesterol acyltransferase activity increases. This increase in lecithin:cholesterol acyltransferase activity due to vesicle-fluidity increase is sufficient to overcome the decrease in activity due to lecithin:cholesterol acyltransferase inhibition. This is the first report of the ability of lysoDMPC to increase lecithin:cholesterol acyltransferase activity.     

Local anesthetic-phospholipid interactions. The pH dependence of the binding of dibucaine to dimyristoylphosphatidylcholine vesicles

The interaction of the local anesthetic dibucaine with unilamellar vesicles of dimyristoylphosphatidylcholine was studied by equilibrium dialysis. Saturating binding profiles (as a function of dibucaine) were found, with apparent association constant ranging from 1.26 X 10(3)M-1 to 2.57 X 10(3)M-1 as pH is increased from 5.0 to 7.5. The number of phospholipid molecules comprising a binding site was found to be about 5 at each pH. Analysis of the data was also achieved using the Stern model, which takes into account the electrostatic effect on binding of the cationic drug due to the build up of a surface potential.     

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.     

Effect of ethanol on dimyristoylphosphatidylcholine large unilamellar vesicles investigated by quasi-elastic light scattering and vibrational spectroscopy

The gel-like liquid phase transition of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles prepared by reverse phase evaporation has been investigated in buffers containing ethanol by quasi-elastic light scattering (QLS) and vibrational (infrared and Raman) spectroscopy. With the QLS technique, the relative change in the vesicles area (which is related to the molecular cross-sectional area of lipid molecules) was followed versus both temperature and ethanol concentration. When the latter was low, the depression of the transition point was a linear function of the alcohol concentration, c, but the vesicles area was practically unmodified. At alcohol concentration 10% v/v, an abrupt change of the vesicles area was observed and for c greater than 10% the depression of the transition point was a non-linear function of c. The infrared and Raman spectra showed a perturbation of the hydrophobic regions, including the terminal methyl groups of the acyl tails.     

The interaction of potential-sensitive molecular probes with dimyristoylphosphatidylcholine vesicles investigated by 31P-NMR and electron microscopy

The effect of a number of commonly employed potential-sensitive molecular probes on the 31P-NMR properties of dimyristoylphosphatidylcholine vesicles at two field strengths has been investigated in order to obtain information on the location and effect of these probes on the membrane bilayer. In comparison to the control dye-free vesicle spectrum, the probes diS-C3-(5) and diS-C4-(5), when added to a vesicle suspension, cause a substantial broadening of the 31P resonance with no detectable chemical shift within an uncertainty of +/- 0.05 ppm at 24 MHz. The spin-lattice and spin-spin relaxation times are also reduced when the cyanines are present by well over 20% relative to those of the control vesicle preparation. The addition of anionic probes, including several oxonol derivatives and merocyanine 540, causes no chemical shift, line broadening, or changes in the relaxation times. Possible explanations for the failure of the anionic probes to alter the vesicle 31P-NMR properties include charge repulsion between these dyes and the phosphate group that prevents the probes from penetrating the bilayer to a depth sufficient to alter the local motion of the phosphate moiety. The 31P resonance broadening and reduction in the relaxation times caused by the two cyanines is at least in part due to an increase in vesicle size as judged by electron microscopy measurements, although an inhibition of the local phosphate motion as well cannot be completely eliminated. The cyanine-mediated increase in vesicle size appears to be due to an irreversible vesicle-fusion process possibly initiated by the screening of surface charge by these probes. The implications of these observations in relation to functional energy-transducing preparations is discussed.     

The effect of loperamide on the thermal behavior of dimyristoylphosphatidylcholine large unilamellar vesicles

The effect of loperamide, a drug belonging to the opiate family, on dimyristoyl phosphatidylcholine large unilamellar vesicles (DMPC LUV) was investigated by quasielastic light scattering (QLS) and Fourier transform infrared spectroscopy (FT-IR). Both techniques show that, in the presence of loperamide, DMPC LUV undergoes a two step transition in cooling: one step around the transition point of pure lipid vesicles, the other at a lower temperature. The temperature of the latter step transition is different for the head and tail regions of the drug-containing vesicles: FT-IR spectra demonstrate that the hydrophobic acyl chains transition starts at a temperature well above that of the interfacial region whereas the transition of the entire vesicle, explored by QLS, is broad and covers both temperature ranges. These transitions are thermally reversible in the FT-IR which measures local order but aggregation effects prevent the thermal reversibility of the QLS results. The nature of the drug-lipid interaction is also discussed.     

Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Transbilayer and interbilayer phospholipid exchange in dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine large unilamellar vesicles

The rates of spontaneous interbilayer and transbilayer exchange of [3H]dimyristoylphosphatidylcholine ([3H]DMPC) were examined in DMPC and DMPC/dimyristoylphosphatidylethanolamine (DMPE) large unilamellar vesicles in the liquid-crystalline-, gel-, and mixed-phase states. DMPC desorption rates from either gel or liquid-crystalline phases containing DMPE are very similar to the corresponding rates from pure DMPC gel or liquid-crystalline phases. This is not the case for DMPC desorption from distearoylphosphatidylcholine (DSPC)-containing gel phases, where the desorption rates are significantly faster than from a pure DMPC gel phase [Wimley, W. C., & Thompson, T. E. (1990) Biochemistry 29, 1296-1303]. We proposed that the DMPC/DSPC behavior results from packing defects in gel phases composed of both DMPC and DSPC molecules because of the four-carbon difference in the acyl chain lengths of the two species. The present results strongly support this hypothesis because no such anomalous behavior is observed in DMPC/DMPE, which is similar to DMPC/DSPC in phase behavior but does not have the chain length difference. The inclusion of 10-30 mol % DMPE in DMPC bilayers was also found to have a significant effect on the rate of transbilayer movement (flip-flop) of [3H]DMPC in the liquid-crystalline phase. Between 10 and 30 mol % DMPE, flip-flop of DMPC is slowed by at least 10-fold relative to flip-flop in DMPC bilayers, and the entropy and enthalpy of flip-flop activation are both substantially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of an interfacial methylene in dimyristoylphosphatidylcholine vesicles using carbon-13 spin relaxation

The dynamic and conformational properties of the 2-methylene on the sn-2 chain of dimyristoylphosphatidylcholine have been investigated in small unilamellar vesicles. An analysis of the spin relaxation of a proton-coupled 13C nucleus has been used to provide the additional information necessary to propose a specific geometry for motion. The results suggest a model with three motions in addition to vesicle tumbling: (1) a slow axial rotation of the entire molecule about the bilayer normal (tau congruent to 2 X 10(-8) s); (2) torsional oscillations about C-C bonds on a very fast time scale; and (3) rapid jumps (tau = 6 X 10(-10) s) between two conformers having approximate gauche+ and gauche- conformations about the C2-C3 bond of the sn-2 chain. The proposed conformations are compared to those previously predicted on the basis of crystal structures, spectroscopic data, and energy-minimization calculations.     

Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.     

Topography of the C terminus of cytochrome b5 tightly bound to dimyristoylphosphatidylcholine vesicles

Cytochrome b5 holoenzyme was bound asymmetrically in the tightly bound form to small unilamellar dimyristoylphosphatidylcholine vesicles. [3H]Taurine, a membrane-impermeant nucleophile, was added to the external medium and was then cross-linked to cytochrome carboxyl residues by the addition of a water-soluble carbodiimide. Nonpolar peptide was isolated after trypsin digestion of taurine-labeled apocytochrome b5 and contained 1.7-1.9 residues of taurine. The C-terminal tetrapeptide containing residues Thr130-Asn133 was generated by chymotryptic hydrolysis of radiolabeled nonpolar peptide and was purified by gel filtration and ion exchange chromatography. Amino acid analysis of the C-terminal tetrapeptide showed that about 1.6 mol of taurine was cross-linked per mol of peptide. When the experiment was performed with taurine trapped inside the vesicles, no cross-linking was observed. The results suggest that when cytochrome b5 holoenzyme is bound to vesicles in the tight binding form, the C terminus is located on the external surface of the vesicles.     

Unfolding of apomyoglobin examined by synchrotron footprinting

Hepatitis E virus is a human RNA virus containing three open reading frames. Of these, ORF2 encodes the major capsid protein (pORF2) and may possess regulatory functions, in addition to a structural one. In this study, we have shown using the yeast two-hybrid system and in vitro immobilization experiments that full-length pORF2 is capable of self-association, thus forming a homodimer. Using mutational analysis we have studied dimerization of various truncated versions of the ORF2 capsid protein using the yeast two-hybrid system and supported our findings with in vitro immobilization experiments. Deletions of pORF2 reveal a loss of the dimerization potential for all deletions except an N-terminal 127-amino-acid deletion. Our studies suggest that the dimerization property of pORF2 may not be amino-acid sequence dependent but instead a complex formation of a specific tertiary structure that imparts pORF2 its property to self-associate.     

Characterization of "native" apomyoglobin by molecular dynamics simulation.

We have used molecular dynamics simulation methods to study the structure and fluctuations of "native" apomyoglobin in aqueous solution for a period of greater than 0.5 nanosecond. This work was motivated by the recent attempts of Hughson et al. to characterize the structure and motion of both this molecule and the less compact, acid stabilized I stage, using methods of pulsed H/2H exchange. The study of these systems provides new insights into protein folding intermediates and our simulation has yielded a detailed model for structure and fluctuations in apomyoglobin which complements the experimental studies. We find that local (short-time) fluctuations agree well with fluctuations observed for the holoprotein in aqueous solution, as well as results from the crystallographic B-factors. In addition, the structural features we observe for native apomyoglobin are very similar to the holoprotein, in basic agreement with the findings of Hughson et al. By examining larger-scale motions, developing only over timescales in excess of a 100 picoseconds, we are able to identify conformationally "labile" and "non-labile" regions within native apomyoglobin. These regions correspond extremely well to those identified in the nuclear magnetic resonance experiments as unstable and stable "folding subdomains" in the I state of apomyoglobin. Overall we find that helices A, B, E, G and H show the least amount of motion and helices C, D and F move substantially over the timescales examined. The major motions, and the primary difference between the holo and apo structures as we have observed them, are due to the shifting motion of helices C, D and F into the vacant heme cavity. We also find that motions at the interface of helical segments can be large, with one important exception being the chain segment connecting helices G and H. This segment of chain interacts with the conformationally "non-labile" helix A to form a relatively rigid subdomain composed of helices A, G and H. We believe that these findings provide direct support for the suggestion of Hughson et al. that helices A, G and H constitute a compact subdomain that remains in a native-like conformation as the protein begins to unfold in environments of decreasing pH.     

Calorimetric determination of the partition coefficient for chlorpromazine hydrochloride in aqueous suspensions of dimyristoylphosphatidylcholine vesicles

Lipid suspensions containing from 0.1 to 0.2% by weight dimyristoylphosphatidylcholine were mixed in a flow calorimeter with equal volumes of chlorpromazine hydrochloride at concentrations ranging from 6×10^?5 to 1.2×10^?4 M. The vesicle bilayer volume fraction of the suspension was determined by density measurements. Linear relationships were obtained between heat production per ml suspension and chlorpromazine concentration at each level of lipid volume. Using phase partitioning as a model, the values of the partition coefficient and the enthalpy change were found to be K_c′=1300 and ΔH=?30 kJ·mol^?1 at 25°C.Heat outputs at slightly higher concentrations of chlorpromazine increased less than linearly because of repulsive forces between neighboring chlorpromazine cations absorbed in the bilayer phase. At still higher concentrations the slope increased again but partition coefficients became variable, which indicated a change in the nature of the interactions.In batch calorimeter titrations at higher concentrations a sharp increase in heat output was observed at the critical micelle concentrations of chloropromazine (4 mM) and a final levelling off at 6 mM. Enthalpies of dilution of chlorpromazine obtained in separate experiments were large and endothermic, but no break in the curve could be detected at the critical micelle concentrations.