Effects of polypeptide-phospholipid interactions on bilayer reorganizations Raman spectroscopic study of the binding of polymyxin B to dimyristoylphosphatidic acid and dimyristoylphosphatidylcholine dispersions

The interactions of the antibiotic polymixin B, a polycationic cyclic polypeptide containing a branched acyl side chain, with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidic acid (DMPA) bilayers were investigated by Raman spectroscopy for a wide range of lipid/polypeptide mole fractions. Temperature profiles, constructed from peak height intensity ratios derived from the lipid methylene C-H stretching and acyl chain C-C stretching mode regions, reflected changes originating from lateral chain packing effects and intrachain trans / gauche rotamer formation, respectively. For DMPC/polymyxin B bilayers the temperature dependent curves indicate a broadening of the gel-liquid crystalline phase transition accompanied by an approx. 3 C deg. increase in the phase transition temperature from 22.8°C for the pure bilayer to 26°C for the polypeptide complex. For a 10:1 lipid/polypeptide mole ratio the temperature profile derived from the C-C mode spectral parameters displays a second order/disorder transition, at approx. 35.5°C, associated with the melting behavior of approximately three bilayer lipids immobilized by the antibiotic's charged cyclic headgroup and hydrophobic side chain. For the 10:1 mole ratio DMPA/polypeptide liposomes, the temperature profiles indicate three order/disorder transitions at 46, 36 and 24°C. Pure DMPA bilayers display a sharp lamellar-micellar phase transition at 51°C.     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of proteins in protection against radiation-induced damage in membranes

The effect of gamma irradiation on liposomes in the presence of a large number of commercially available proteins has been studied. Experiments were designed to demonstrate that the configuration of both acyl chain and cis C = C bonds created by lipid-protein associations are crucial in autocatalyzed radiation-induced lipid peroxidation. Raman spectroscopy was used to characterize these states. Raman spectra in the C-C stretching region show three prominent bands at 1064, 1090, and 1125 cm-1, assigned to trans, gauche, and trans C-C bonds, respectively. A single symmetrical C = C stretching band assigned to the cis isomer occurs at 1660 cm-1. The intensity ratios (I1064/I1090) and (I1660/I1440) are used as Raman probes to define the conformational states of acyl chains and C = C bonds, respectively. Our data show that the ratio (I1064/I1090) decreases in the presence of proteins, indicating that these proteins induce more gauche structures. Upon irradiation, the ratio (I1064/I1090) increases by about 30% in the absence of proteins and by about 15% in the presence of proteins. This shows that proteins retain the gauche structures in irradiated samples. The ratio (I1660/I1440) decreases in liposomes containing proteins, showing that proteins modify the configuration of cis C = C bonds. Upon irradiation, this ratio decreases by about 45-50% in samples without proteins and by about 10% in samples with proteins. These data show that proteins inhibit the radiation-induced configurational changes in the cis C = C bonds. The determination of radiation-induced peroxides (as malondialdehyde equivalents) in liposomes reveals that proteins inhibit the formation of peroxide products at low molar ratio and that the preventive capacity of different proteins is different. We conclude that proteins alter the conformation of both acyl chains and cis C = C bonds in liposomes and that these altered states are less sensitive to radiation-induced peroxidation.     

Dynamics of lipid-protein interactions. Interaction of apolipoprotein A-II from human plasma high density lipoproteins with dimyristoylphosphatidylcholine

ApoA-II and dimyristoylphosphatidylcholine (DMPC) spontaneously associate to give three different complexes whose structures are determined by the initial reactant concentration and by the reaction temperature with respect to Tc (23.9 degrees C), the gel to liquid crystalline transition temperature of DMPC. At an initial lipid to protein ratio of 45/1, a single complex (2.29 x 10(5) daltons) is quantitatively formed at all temperatures between Tc - 4 degrees C and Tc + 6 degrees C. When the 45/1 complex is mixed with DMPC liposomes there is lipid exchange but no net transfer of lipid, so that the structure of the complex remains unaltered. At an initial molar ratio of 100 to 300:1, the reaction scheme is more complex. At 24 degrees C a 240/1 complex (1.5 x 10(6) daltons) is formed from a precursor 75/1 complex (3.43 x 10(5) daltons) if excess (approximately 300 mol/mol) lipid is present. The 75/1 complex exhibits lipid exchange in the presence of added DMPC liposomes at 24 degrees C, and both the 75/1 and the 240/1 complex can be converted to smaller protein-rich complexes in the presence of added apoA-II. These results suggest that the initial lipid/protein ratio and the physical state of a lipid or lipid . protein complex determines the composition and structure of the resulting complex and support the view that lipid-protein interactions are stronger than protein-protein or lipid-lipid interactions.     

Interaction of ferricytochrome c with zwitterionic phospholipid bilayers: a Raman spectroscopic study

The vibrational Raman spectra of both pure L-alpha-dipalmitoylphosphatidylcholine (DPPC) liposomes and DPPC multilayers reconstituted with ferricytochrome c under varying conditions of pH and ionic strength are reported as a function of temperature. Total integrated band intensities and relative peak height intensity ratios, two spectral scattering parameters used to determine bilayer disorder, are invariant to changes in pH and ionic strength but exhibit a sensitivity to the bilayer concentration of the ferricytochrome c. Protein concentrations were estimated by comparing the 1636 cm-1 resonance Raman line of known ferricytochrome c solutions to intensity values for the reconstituted multilayer samples. Temperature-dependent profiles of the 3100-2800 cm-1 C-H stretching, 1150-1000 cm-1 C-C stretching, 1440 cm-1 CH2 deformation, and 1295 cm-1 CH2 twisting mode regions characteristic of acyl chain vibrations reflect bilayer perturbations due to the weak interactions of ferricytochrome c. The DPPC multilamellar gel to liquid-crystalline phase transition temperature, TM, defined by either the C-H stretching mode I2935/I2880 or the C-C stretching mode I1061/I1090 peak height intensity ratios, is decreased by approximately 4 degrees C for the approximately 10(-4) M ferricytochrome c reconstituted DPPC liposomes. Other spectral features, such as the increase in the 2935 cm-1 C-H stretching mode region and the enhancement of higher frequency CH2 twisting modes, which arise in bilayers containing approximately 10(-4) M protein, are interpreted in terms of protein penetration into the hydrophobic region of the bilayer.     

Thermodynamics of lipid protein associations. Thermodynamics of helix formation in the association of high density apolipoprotein A-I (apoA-I) to dimyristoyl phosphatidylcholine.

The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.     

Low levels of irradiation modify lipid domains in model membranes: a laser Raman study

We have used Raman spectroscopy to study the effects of ionizing radiation on thermal transitions of dipalmitoyl lecithin + polyunsaturated fatty acid liposomes. Raman spectra in the CH (2800-3000 cm-1), C = C (1600-1680 cm-1), and C-C (1000-1150 cm-1) stretching regions are sensitive to ionizing radiation. The CH stretching of acyl chains yields three strong bands around 2850, 2880, and 2930 cm-1. The ratios of the relative intensities of 2880 and 2850 cm-1 bands, i.e., I2880/2850, when plotted against temperature show multiple infection points which correspond to multiple spectroscopic transitions. These are ascribed to a separate phase with distinctive proportions of lecithin and polyunsaturated fatty acids. We find these transitions sensitive to low levels of ionizing radiation. Doses as low as 5-15 rad after 48 h of 60Co gamma irradiation and 60 kVp X irradiation drastically broaden and shift the polyunsaturated rich phase which occurs at lower temperatures (-7 to +5 degrees C) than that of pure dipalmitoyl lecithin (39 degrees C). In addition a new transition around 46 degrees C also emerges upon irradiation (48 h postirradiation). These irradiation effects can be accelerated by the presence of catalytic amounts of Fe2+/EDTA +H2O2. The membrane transition modification is more sensitive to 60 kVp X rays in comparison to 60Co gamma rays owing to the high LET component of the former. The intensity of 1660 cm-1 band, assigned to C = C stretching in the cis-configuration, loses intensity upon irradiation. Concomitantly, a new band around 1675 cm-1, assigned to trans-configuration, emerges. Similarly the increase in the "order parameter" as calculated from the relative intensities of C--C stretching bands indicates rigidification of membrane. Various factors such as reduction in unsaturation, increase in trans-configuration, and the formation of multiple peroxidation products are invoked as lipid phase modifiers.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Raman spectroscopy of selectively deuterated dimyristoylphosphatidylcholine: studies on dimyristoylphosphatidylcholine-cholesterol bilayers

Dimyristoylphosphatidylcholine (DMPC), selectively deuterated in the sn-2 chain at the 3, 6, and 10 positions is used to probe DMPC-cholesterol interactions in multilamellar dispersions. Using the Raman spectral linewidths of the 2100 cm-1 C2H2 stretching modes as an index of membrane disorder, we demonstrate that cholesterol tends to order, or increase the number of trans carbon-carbon bonds within the DMPC acyl chain near the headgroup region at all temperatures. At low temperatures, cholesterol disorders the acyl chains near the methyl termini by inducing gauche conformers; cholesterol orders the entire chain at higher temperatures. These determinations are qualitatively consistent with conclusions drawn from deuterium nuclear magnetic resonance studies, but specifically reflect acyl chain trans/gauche isomerization on the 10(-12)-10(-13) s vibrational time scale.     

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).     

Lipid-binding studies of human apolipoprotein A-I and its terminally truncated mutants

Apolipoprotein A-I (apoA-I, 243 amino acids) is the major protein of high-density lipoproteins (HDL) that plays an important structural and functional role in lipid transport and metabolism. The central region of apoA-I (residues 60-183) is predicted to contain exclusively amphipathic alpha-helices formed from tandem 22-mer sequence repeats. To analyze the lipid-binding properties of this core domain, four terminally truncated mutants of apoA-I, Delta(1-41), Delta(1-59), Delta(1-41,185-243), and Delta(1-59,185-243), were expressed in baculovirus infected Sf-9 cells. The effects of mutations on the ability of apoA-I to form bilayer disk complexes with dimyristoyl phosphatidylcholine (DMPC) that resemble nascent HDL were analyzed by density gradient ultracentrifugation and electron microscopy (EM). The N-terminal deletion mutants, Delta(1-41) and Delta(1-59), showed altered lipid-binding ability as compared to plasma and wild-type apoA-I, and in the double deletion mutants, Delta(1-41, 185-243) and Delta(1-59, 185-243), the lipid binding was abolished. Thermal unfolding of variant apoA-I/DMPC complexes monitored by circular dichroism (CD) showed hysteresis and a shift in the melting curves by about -12 degrees C upon reduction in the heating rate from 1.0 to 0.067 K/min. This indicates an irreversible kinetically controlled transition with a high activation energy E(a) = 60 +/- 5 kcal/mol. CD and EM studies of the apoA-I/DMPC complexes at different pH demonstrated that changes in the net charge or in the charge distribution on the apoA-I molecule have critical effects on the conformation and lipid-binding ability of the protein.     

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.     

Methyl group substitution at C(1), C(2) or C(3) of the glycerol backbone of a diether phosphocholine: a comparative study of bilayer chain disorder in the gel and liquid-crystalline phases

Alterations in the inter- and intramolecular packing characteristics of aqueous dispersions of methyl derivatives of di-O-hexadecylglycerophosphocholine (DHPC), an ether lipid in which the methyl group is substituted at the 1, 2 or 3 position of the glycerol backbone, were monitored by changes in the vibrational frequencies and intensities of selected spectral features by Raman spectroscopy. Temperature profiles constructed from spectra reflecting intermolecular order/disorder rearrangements (C-H stretching mode region) and intramolecular order/disorder processes (C-C stretching mode region) provide insight into several important structural properties of diether lipid bilayers. The introduction of a methyl group into any position of the glycerol backbone alters both the characteristics of the DHPC pretransition and the temperature of the gel to liquid-crystalline phase transition. The main gel to liquid-crystalline phase transitions are 42.8 degrees C in the pure diether lipid, 41.6 degrees C for 3-Me-DHPC, 40.5 degrees C for 2-Me-DHPC and 38.1 degrees C for 1-Me-DHPC. Temperature profiles indicate that the degree of disordering for both the gel and liquid-crystalline states follows the sequence 2-Me-DHPC less than 3-Me-DHPC less than DHPC less than 1-Me-DHPC. Phase transition widths, delta T, determined from the spectroscopic temperature profiles, are discussed in terms of van't Hoff enthalpy functions involving both interchain and trans/gauche effects.     

Evidence for acyl chain trans/gauche isomerization during the thermal pretransition of dipalmitoyl phosphatidylcholine bilayer dispersions

In order to clarify, in dipalmitoyl phosphatidylcholine multilayers, the effect of the 34 degrees C thermal pretransition on the acyl chain intramolecular disordering process, Raman spectra of dipalmitoyl phosphatidylcholine gels at 20 and 34 degrees C were compared in the 1000--1200 cm-1 skeletal C-C stretching region. In addition to an overall intensity decrease associated with a change in chain packing characteristics, the growth of intensity in the 1080--1090 and 1122 cm-1 regions in the 34--20 degrees C) difference spectrum clearly indicates that the thermal pretransition is accompanied by an increase in the population of hydrocarbon chain gauche rotamers toward the center of the bilayer.     

Calcium-induced lateral phase separations in phosphatidylcholine-phosphatidic acid mixtures. A Raman spectroscopic study

The effects of calcium ions on mixed membranes of dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidylcholine (DMPC) with either the PA or the PC component deuterated have been studied by Raman spectroscopy. The spectra of the pure components show that the acyl chains of hydrated DMPA bilayers are less tightly packed and have more trans bonds than those of DMPC. This behavior appears to be due to the particular arrangement of the polar head groups of DMPA for which the glycerol chain is oriented parallel to the bilayer surface. In agreement with the calorimetrically determined phase diagram [Graham, I., Gagné, J., & Silvius, J. R. (1985) Biochemistry (preceding paper in this issue)], the Raman results show that, in the absence of calcium, DMPA and DMPC are completely miscible at an equimolar ratio but undergo extensive phase separation in the presence of excess calcium. DMPC in phase-separated DMPC-DMPA (Ca2+) mixtures has a conformation that is very similar to that of pure DMPC bilayers, but it is packed more tightly since, depending on the temperature, it is at least partly incorporated into either a solid solution in DMPA or a DMPA-Ca2+-rich "cochleate" phase. This latter shows the same characteristics as the cochleate phase of pure DMPA-Ca2+ which is highly ordered and does not give rise to a thermotropic transition between 5 and 100 degrees C. However, the cochleate phase in DMPA (Ca2+)-DMPC mixtures contains some 20 mol % of DMPC trapped in small domains. These clusters do not melt cooperatively but become as fluid as pure DMPC at 50 degrees C.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Lateral distribution of phospholipid and cholesterol in apolipoprotein A-I recombinants

The influence of cholesterol on the assembly and structure of model high-density lipoproteins (HDL) has been investigated. Model HDL composed of apolipoprotein A-I (apoA-I) and 1,2-dimyristoylphosphatidylcholine (DMPC) formed spontaneously at the transition temperature (Tc) of the lipid. Those composed of apoA-I and 1-palmitoyl-2-oleoylphosphatidylcholine were formed by a cholate dialysis method. At low cholesterol/phospholipid ratios both lipids and assembly methods yielded a model HDL whose composition was identical with that of the initial mixture; as the cholesterol/phospholipid ratio of the initial mixture was increased, the fraction of cholesterol appearing in the model HDL decreased, and a negative correlation between the cholesterol and protein contents of the model HDL was observed. At high cholesterol/phospholipid ratios the association of apoA-I and phospholipids appeared to be thermodynamically unfavorable. The effects of cholesterol content on the thermal properties of a model HDL composed of DMPC and apoA-I were further investigated by differential scanning calorimetry, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, fluorescence energy transfer, and excimer fluorescence of pyrenyl derivatives of phosphatidylcholine (PC) and cholesterol. The addition of cholesterol decreased the transition enthalpy of DMPC, raised the midpoint of the transition, and modulated motional freedom in the phospholipid matrix. The amount of cholesterol required to produce these effects was lower in the model HDL than in multilamellar liposomes. In a model HDL composed of DMPC and apoA-I, the lateral diffusion of a pyrene-labeled cholesterol was dramatically changed at the Tc whereas little change was observed in that of a pyrene-labeled PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

An infrared spectroscopic study of specifically deuterated fatty-acyl methyl groups in phosphatidylcholine liposomes.

The region of the infrared spectrum corresponding to C-2H stretching vibrations (2050-2250 cm-1) has been examined for liposomes composed of dimyristoylphosphatidylcholine deuterated specifically at the methyl ends of either one (sn-2) or both the fatty acyl chains. This label is intended to provide information on lipid dynamics in the contact region between monolayers. The two most prominent bands observed correspond, respectively, to antisymmetric (2212 cm-1) and symmetric (2075 cm-1) C-2H stretching vibration. The antisymmetric band consists of two overlapping peaks, whose positions vary with the gel or liquid-crystalline state of the lipid. The separation between the peaks making up the antisymmetric band increases with temperature, and is maximum above the Tc transition temperature; this rules out the previously proposed assignment of these two peaks to different rotational modes of the methyl group relative to the adjacent methylene. The position and width of the symmetric band at 2075 cm-1 are also sensitive to the physical state of the lipid. The presence of cholesterol at an equimolar ratio with the phospholipid abolishes all the phase-dependent changes observed. The intrinsic polypeptide gramicidin A, at a 5:1 lipid/peptide mol ratio, is seen to enlarge the lipid thermotropic transition, with small effects above Tc. Cytochrome c, an extrinsic protein, at a 10:1 mole ratio, does not modify the phase-dependent behaviour of the terminal methyl groups, but consistently shifts all the observed bands to lower-frequency positions, which suggests a long-range effect of the protein along the phospholipid fatty acyl chains.     

Incorporation of cholesterol into apolipoprotein A-I-dimyristoylphosphatidylcholine recombinants

Apolipoprotein A-I (apoA-I) spontaneously associates with dimyristoylphosphatidylcholine (DMPC) liposomes to form discoidal high-density lipoprotein (HDL) recombinants. The uptake of cholesterol by this model HDL was studied by incubation with Celite-dispersed cholesterol. Separation of the resulting complexes by gradient centrifugation and gel filtration showed a heterogeneous distribution of particle size and composition as a consequence of the disruption and rearrangement of the recombinants. Quantitation of the amount of cholesterol taken up gave values between about 28 and 40 mol% cholesterol for the fractions within the protein peaks; the fractions with the lowest DMPC/apoA-I ratios had the lowest cholesterol contents. In another set of experiments, the association of apoA-I with DMPC-cholesterol liposomes was shown to result in complexes with characteristics similar to those obtained by the cholesterol-uptake experiments. Low concentrations of cholesterol in the liposomes enhanced the rate of lipid-protein association, but larger amounts decreased the yield of complexes by making the process thermodynamically and kinetically unfavorable. The enthalpy of recombinant formation increased with decreasing lipid/protein ratio and increasing cholesterol content, and became endothermic at about 23 mol% cholesterol. The effect of cholesterol on the thermal properties of HDL recombinants suggests that cholesterol is partially excluded from the boundary region adjacent to apoA-I. It is concluded that discoidal HDL recombinants, as a model for 'nascent' HDL, can acquire substantial amounts of cholesterol, which may be of great physiological importance for the reverse cholesterol transport and prevention of atherosclerosis.     

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy.

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.