Phospholipids chiral at phosphorus. Dramatic effects of phosphorus chirality on the deuterium NMR properties of the choline head group of phospholipids in the liquid crystalline phase

To probe the motional and conformational properties of the choline head group of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC), the Rp, Sp, and Rp + Sp isomers of [alpha-D2]DPPsC, [beta-D2]DPPsC, and [delta-D9]DPPsC in the subgel, gel, and liquid crystalline phases were investigated with deuterium NMR, and the results were compared with those of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) labeled at the same positions. In the subgel phase (5 degrees C) all isomers of [alpha-D2]DPPsC and [beta-D2]DPPsC displayed amorphous line shapes characteristic of a restricted and disordered motional environment, whereas [delta-D9]DPPsC showed narrower and symmetric line shapes indicating substantial motions. For all three labeled positions the apparent line width of the Rp isomer is larger than those of Sp and Rp + Sp isomers, and the amorphous line shape of the Rp isomer also persists at 25 and 35 degrees C, which confirm the previous observation that the Rp isomer is unusually stable in the subgel phase and suggest that the Rp isomer is more rigid than the other isomers in the choline head group. In the gel phase (25 and 35 degrees C) narrower and symmetric line shapes were observed for Sp and Rp + Sp isomers, and the apparent line widths were comparable to those of DPPC. In the liquid crystalline phase there are dramatic differences between the spectra of DPPC and different isomers of DPPsC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipids chiral at phosphorus. Characterization of the sub-gel phase of thiophosphatidylcholines by use of X-ray diffraction, phosphorus-31 nuclear magnetic resonance, and Fourier transform infrared spectroscopy

A recent study using differential scanning calorimetry (DSC) showed that the thermotropic phase behavior of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) is sensitive to the configuration at phosphorus and that the Rp isomer displayed only a broad transition at 45.6 degrees C [Wisner, D. A., Rosario-Jansen, T., & Tsai, M.-D. (1986) J. Am. Chem. Soc. 108, 8064-8068]. We have employed X-ray diffraction, 31P NMR, and Fourier transform infrared (FT-IR) spectroscopy to characterize various phases of the isomers of DPPsC, to compare the structural differences between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and isomers of DPPsC, and to identify structural factors responsible for the unique behavior of the RP isomer. The results from all three techniques support the previous proposal based on DSC studies that (SP)- and (RP + SP)-DPPsC undergo a subtransition, a pretransition, and a main transition analogous to those of DPPC, while (RP)-DPPsC is quite stable at the subgel phase and undergoes a direct subgel----liquid-crystalline transition at 46 degrees C. Quantitative differences between DPPC and DPPsC (i.e., the effect of sulfur substitution rather than the configurational effect) in the subgel phase have also been observed in the chain spacing, the motional averaging, and the factor group splitting (revealed by X-ray diffraction, 31P NMR, and FT-IR, respectively). In particular, DPPsC isomers are motionally rigid and show enhanced factor group splitting in the subgel phase. These results suggest that DPPsC is packed in different subcells relative to DPPC in the subgel phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipids chiral at phosphorus. Stereochemical mechanism of reactions catalyzed by phosphatidylinositide-specific phospholipase C from Bacillus cereus and guinea pig uterus

(Rp)- and (Sp)-1,2-dipalmitoyl-sn-glycero-3-thiophosphoinositol (DPPsI) were synthesized as a mixture and their configurations assigned on the basis of the stereospecific hydrolysis catalyzed by phospholipase A2 (PLA2) from bee venom. PLA2 is known to be stereospecific to the Rp isomer of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) and 1,2-dipalmitoyl-sn-glycero-3-thiophosphoethanolamine (DPPsE). Since the configurations of (Rp)- and (Sp)-DPPsI correspond to those of (Sp)- and (Rp)-DPPsC, respectively, due to a change in priority, the isomer specifically hydrolyzed by PLA2 was assigned to (Sp)-DPPsI. The DPPsI analogues were then used to probe the mechanism and to elucidate the steric course of the reaction catalyzed by phosphatidylinositide-specific phospholipase C (PI-PLC) from Bacillus cereus and for both isozyme I and isozyme II of PI-PLC from guinea pig uterus. It was found that the Rp isomer of DPPsI is the preferred substrate for all three PI-PLCs. Thus PI-PLC shows the same stereospecificity as phosphatidylcholine-specific PLC (PC-PLC), which prefers the Sp isomer of DPPsC. The ratio of the two products inositol 1,2-cyclic phosphorothioate (cIPs) and inositol phosphorothioate (IPs) was not significantly perturbed by the use of phosphorothioate analogue for all three PI-PLCs, which implies that IPs is not produced by enzyme-mediated ring opening of cIPs and supports a parallel pathway for the formation of both products. In order to elucidate the steric course of the cyclization reaction, exo and endo isomers of cIPs were synthesized and their absolute configurations at phosphorus were determined by nuclear magnetic resonance and other techniques. It was found that exo-cIPs is the product produced by all three PI-PLCs. Thus the steric course of the conversion DPPsI to cIPs catalyzed by all three PI-PLCs was inversion of configuration at phosphorus. These results taken together suggest that the reaction catalyzed by PI-PLC most likely proceeds via direct attack by the 2-OH group to generate the cyclic product, and parallelly by water to generate the noncyclic inositol phosphates, without involving a covalent enzyme-phosphoinositol intermediate.     

Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

Separate diastereomers of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine ( DPPsE ) were prepared in 97% diastereomeric purity and characterized by 31P, 13C, and 1H nuclear magnetic resonance (NMR). The isomers hydrolyzed by phospholipases A2 and C specifically were designated as isomer B (31P NMR delta 59.13 in CDCl3 + Et3N ) and isomer A (59.29 ppm), respectively, analogous to the isomers B and A of 1,2-dipalmitoyl-sn-glycero-3- thiophosphocholine ( DPPsC ) [ Bruzik , K., Jiang , R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. Phospholipase D from cabbage was shown to be specific to isomer A of DPPsC in transphosphatidylation . The product DPPsE was shown to be isomer A. The absolute configuration of chiral DPPsE at phosphorus was elucidated by bromine-mediated desulfurization in H2 18O to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphoethanolamine ( [18O]DPPE) followed by 31 P NMR analysis [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The absolute configuration of chiral DPPsC was elucidated by desulfurization in H2 18O mediated by bromine or cyanogen bromide to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphocholine ( [18O]DPPC), which was then converted to [18O]DPPE by phospholipase D with retention of configuration [ Bruzik , K., & Tsai, M.-D. (1984) Biochemistry (preceding paper in this issue)]. The results indicate that isomer A of both DPPsE and DPPsC is SP whereas isomer B is RP.     

Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Phospholipids chiral at phosphorus. Use of chiral thiophosphatidylcholine to study the metal-binding properties of bee venom phospholipase A2

It has been shown recently by 31P nuclear magnetic resonance (NMR) that phospholipase A2 (PL A2) from bee venom shows a high degree of stereoselectivity toward the "isomer B" of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) [Bruzik, K., Jiang, R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. We now report a quantitative kinetic study of PL A2 using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and (RP)-, (SP)-, and (RP + SP)-DPPsC by a spectrophotometric assay. The substrates were mixed with Triton X-100 to form mixed micelles, and steady-state kinetic theories were applied. The enzyme was activated by Ca2+, which induced a conformational change of the enzyme, as shown by UV difference spectra. The apparent dissociation constant of Ca2+/PL A2 is 2.5 mM. In the presence of Ca2+, large substrate specificity and stereospecificity in Vmax (in mumol min-1 mg-1) were observed: DPPC, 1850; (RP)-DPPsC, 7.6; (RP + SP)-DPPsC, 64; (SP)-DPPsC, 0.044. On the other hand, relatively small variation in Km was observed, which suggests that the interfacial interaction is relatively nonspecific among the substrates studied. (SP)-DPPsC and Cd2+ were shown as competitive inhibitors for the hydrolysis of DPPC by Ca2+/PL A2. Binding of Cd2+ with apo-PL A2 was also demonstrated by UV difference spectra, with a dissociation constant of 0.59 mM. Activation of apo-PL A2 by Cd2+ was unequivocally demonstrated for (SP)-DPPsC by use of 31P NMR. The Vmax values of Cd2+/PL A2 were DPPC/(RP)-DPPsC/(SP)-DPPsC = 17.6/0.069/0.0044 mumol min-1 mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

Abstract

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (F_s), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when F_s is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.     

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.     

Phospholipids chiral at phosphorus. Stereochemical mechanism for the formation of inositol 1-phosphate catalyzed by phosphatidylinositol-specific phospholipase C.

The phosphatidylinositol-specific phospholipase C (PI-PLC) from mammalian sources catalyzes the simultaneous formation of both inositol 1,2-cyclic phosphate (IcP) and inositol 1-phosphate (IP). It has not been established whether the two products are formed in sequential or parallel reactions, even though the latter has been favored in previous reports. This problem was investigated by using a stereochemical approach. Diastereomers of 1,2-dipalmitoyl-sn-glycero-3-(1D- [16O,17O]phosphoinositol) ([16O,17O]DPPI) and 1,2-dipalmitoyl-sn-glycero-3-(1D-thiophosphoinositol) (DPPsI) were synthesized, the latter with known configuration. Desulfurization of the DPPsI isomers of known configurations in H2(18)O gave [16O,18O]DPPI with known configurations, which allowed assignment of the configurations of [16O,17O]DPPI on the basis of 31P NMR analyses of silylated [16O,18O]DPPI and [16O,17O]DPPI (the inositol moiety was fully protected in this operation). (Rp)- and (Sp)-[16O,17O]DPPI were then converted into trans- and cis-[16O,17O]IcP, respectively, by PI-PLC from Bacillus cereus, which had been shown to proceed with inversion of configuration at phosphorus [Lin, G., Bennett, F. C., & Tsai, M.-D. (1990) Biochemistry 29, 2747-2757]. 31P NMR analysis was again used to differentiate the silylated products of the two isomers of IcP, which then permitted assignments of IcP with unknown configuration derived from transesterification of (Rp)- and (Sp)-[16O,17O]DPPI by bovine brain PI-PLC-beta 1. The results indicated inversion of configuration, in agreement with the steric course of the same reaction catalyzed by PI-PLCs from B. cereus and guinea pig uterus reported previously. For the steric course of the formation of inositol 1-phosphate catalyzed by PI-PLC, (Rp)- and (Sp)-[16O,17O]DPPI were hydrolyzed in H2(18)O to afford 1-[16O,17O,18O]IP, which was then converted to IcP chemically and analyzed by 31P NMR. The results indicated that both B. cereus PI-PLC and the PI-PLC-beta 1 from bovine brain catalyze conversion of DPPI to IP with overall retention of configuration at phosphorus. These results suggest that both bacterial and mammalian PI-PLCs catalyze the formation of IcP and IP by a sequential mechanism. However, the conversion of IcP to IP was detectable by 31P NMR only for the bacterial enzyme. Thus an alternative mechanism in which IcP and IP are formed by totally independent pathways, with formation of IP involving a covalent enzyme-phosphoinositol intermediate, cannot be ruled out for the mammalian enzyme. It was also found that both PI-PLCs displayed lack of stereo-specifically toward the 1,2-diacylglycerol moiety, which suggests that the hydrophobic part of phosphatidylinositol is not recognized by PI-PLC.     

Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (RP)- and (SP)-[18O]DPPE, respectively, as indicated by 31P nuclear magnetic resonance (NMR) analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configuration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[ 16O , 17O, 18O]phosphate ( [ 16O , 17O, 18O] DPPA ) with unknown configuration. The latter compound was then converted to 1-[ 16O , 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The configuration of the phosphopropane -1,2-diol was determined as RP by 31P NMR analysis following ring closure and methylation [ Buchwald , S. L., & Knowles, J. R. (1980) J. Am. Chem. Soc. 102, 6601-6603]. The results indicated that hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. Our results therefore support a two-step mechanism involving a phosphatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.     

Phase characteristics of positional isomers of 1,2-di(heptacosadiynoyl)-sn-glycero-3-phosphocholine; tubule-forming phosphatidylcholines

We have examined the phase behavior of positional isomers of a polymerizable diacetylenic phospholipid, 1,2-di(heptacosadiynoyl)-sn-glycero-3-phosphocholine which has the diacetylene in varying position along the acyl chains. Upon cooling multilamellar vesicles (MLVs) through the liquid-crystalline to gel phase transition, all isomers examined spontaneously formed hollow, cylindrical microstructures (or tubules). Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) have been used to characterize positional isomers of this lipid in an effort to understand the effect of diacetylenic position on the molecular characteristics of tubule formation. Calorimetric results indicate that moving the position of the diacetylene along the acyl chain results in the alternation of the exotherm observed for the hydrated transition temperature associated with tubule formation, with higher transition temperatures (Tm) observed from isomers with an even number of methylenes between the diacetylene groups and the glycerol backbone. As the diacetylene is moved toward either end of the acyl chain, even with the observed alternation, the Tm was observed to increase. Calorimetric results of dry members of this series reveal an exotherm during cooling, the same temperature at which fully hydrated samples form tubules. This suggests that there is little difference in the phase behavior observed upon cooling the hydrated tubules and the dry diacetylenic material. FTIR results support the high degree of conformational order observed in tubules of this isomer series as a very strong CH2 wagging progression is observed between 1375 and 1200 cm-1. In addition, the C-H stretch region (3000 cm-1 to 2800 cm-1) indicates tight acyl chain packing with many all-trans segments. These results provide further evidence that tubules are uniquely crystalline microstructures and that this inherent crystallinity, and the formation of tubules is not affected by diacetylenic position.     

Effect of hydrophobic structure on the catalysis of nitric oxide release from zwitterionic diazeniumdiolates in surfactant and liposome media.

The effect of phospholipid liposomes and surfactant micelles on the rate of nitric oxide release from zwitterionic diazeniumdiolates, R1R2N[N(O)NO]-, with significant hydrophobic structure, has been explored. The acid-catalyzed dissociation of NO has been examined in phosphate-buffered solutions of sodium dodecylsulfate (SDS) micelles and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-(1-glycerol)] sodium salt (DPPG) phospholipid liposomes. The reaction behavior of dibenzylamine-, monobenzylamine-, and dibutylamine-derived substrates [1]: R1 = C6H5CH2, R2 = C6H5CH2 NH2+(CH2)2, 2: R1 = C6H5CH2, R2 = NH3+(CH2)2, and 3: R1 = n-butyl, R2 = n-butyl-NH2+(CH2)6] has been compared with that of SPER/NO, 4: R1 = H2N(CH2)3, R2 = H2N(CH2) 3NH2+(CH2)4]. Catalysis of NO release is observed in both micellar and liposome media. Hydrophobic interactions contribute to micellar binding for 1-3 and appear to be the main factor facilitating catalysis by charge neutral DPPC liposomes. Binding constants for the association of 1 and 3 with SDS micelles were 3-fold larger than those previously obtained with comparable zwitterionic substrates lacking their hydrophobic structure. Anionic DPPG liposomes were much more effective in catalyzing NO release than either DPPC liposomes or SDS micelles. DPPG liposomes (at 10 mM total lipid) induced a 30-fold increase in the NO dissociation rate of SPER/NO compared to 12- and 14-fold increases in that of 1 and 3.     

Cholesterol-lipid interactions: An infrared and raman spectroscopic study of the carbonyl stretching mode region of 1,2-dipalmitoyl phosphatidylcholine bilayers

Infrared and Raman spectra were obtained for the 1690–1770 cm^?1 carbonyl stretching mode region for 1,2-dipalmitoyl phosphatidylcholine (DPPC) bilayers in the anhydrous, partially hydrated and completely hydrated states. Spectral features at approx. 1740 and 1721 cm^?1 are assigned to CO stretching modes associated with the 1- and 2-chain carbonyl groups, respectively. Splittings of the primary transitions at 1743, 1738, ～1731 and ～1721 cm^?1 are attributed to rotational isomers involving the entire chain. Hydrogen bond formation between the fatty acid carbonyl and 3βOH cholesterol groups was investigated for anhydrous DPPC bilayers. Examination of frequencies, intensities and half-widths of the carbonyl bands indicates that no hydrogen bonding occurs at either of the two carbonyl sites. However, the addition of cholesterol to completely hydrated DPPC dispersions reduces the conformational inequivalence between the two fatty acid carbonyl groups by specifically perturbing the 2-chain. For cholesterol containing systems the carbonyl stretching mode transitions were also used to monitor lattice effects within the interface region as water binds to the bilayer head groups. Specifically, the addition of approx. 2 molecules of water per lipid molecule orders the lipid lattice and increases the bilayer packing density, while the subsequent addition of 4 molecules of water per lipid molecule releases the packing constraints within the interface region and thereby decreases the packing density.     

The phase transition behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membrane influenced by 2,4-dichlorophenol--an FT-Raman spectroscopy study

The effect of 2,4-dichlorophenol (DCP) on the structures and phase transitions of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes was studied using FT-Raman spectroscopy. Whereas the Raman frequency shifts of the most frequently investigated bands of C-C and C-H stretching regions only indicate the main phase transition (P(beta')-L(alpha)) of the pure DPPC/water system, the Raman shift of C-H scissoring vibration at 1440 cm(-1) was found to be able to reveal the pretransition (L(beta')-P(beta')) as well. Analyzing the spectral parameters of the trans band at 1128 cm(-1), which does not overlap with DCP vibrational modes, a continuous decrease of trans conformations was found with increasing DCP concentration at 26 degrees C accompanying the phase transitions L(beta')-P(beta') and P(beta')-L(alpha). The intensity ratio of the symmetrical and asymmetrical methylene stretching bands (at 2850 cm(-1) and 2880 cm(-1)), defined as the disorder parameter by Levin [Levin, I.W., 1985. Two types of hydrocarbon chain interdigitation in sphingomielin bilayers. Biochemistry 24, 6282-6286], indicated that in the interdigitated phase (L(I)) the order is markedly high and comparable with that of L(beta). Both the phase transition P(beta')-L(alpha) in the DCP/DPPC molar ratio range of 10/100-50/100 and the phase transition L(I)-L(alpha) led to a significant increase of disordered chains and the presence of DCP molecules induced a more disordered chain region than that observed in the L(alpha) phase of DPPC. Nevertheless, it was found that the L(alpha) phase with DCP contains approximately the same amount of trans conformers than that without DCP.     

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electric field increases the phase transition temperature in the bilayer membrane of phosphatidic acid

The effect of the electric field on the phase transition temperature (Tc) of acidic 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl-sn-glycero-3-thionphosphate (thion-DPPA) and zwitterion, i.e. 1,2-dipalmitoyl-rac-3-phosphocholine and 1,2-distearoyl-rac-glycero-3-phosphocholine (DPPC and DSPC), lipids has been investigated. The phase transition was detected using the jump-like increase effect in the conductance of the planar bilayer membrane. A voltage increase to 150 mV has been shown to increase the phase transition temperature in a bilayer lipid membrane (BLM) of phosphatidic acids (DPPA and thion-DPPA) by 8-12 degrees C while the transition temperature in the bilayer of zwitterion lipids (DPPC and DSPC) increases insignificantly. The increasing of Tt in BLM of acidic lipids is attributed to the voltage-induced changes in the molecule packing density.     

Raman spectroscopic study of polycrystalline mono- and polyunsaturated 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines: bilayer lipid clustering and acyl chain order and disorder characteristics

Polycrystalline lipid samples of a series of mono- and polyunsaturated, double bond positional isomers of 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines [C(20-d(39)):C(20:1 Delta(j))PC, with j = 5, 8, 11, or 13; C(20-d(39)):C(20:2 Delta(11,14))PC; and C(20-d(39)):C(20:3 Delta(11, 14,17))PC] were investigated using vibrational Raman spectroscopy to assess the acyl chain packing order-disorder characteristics and putative bilayer cluster formation of the isotopically differentiated acyl chains. Perdeuteration of specifically the saturated sn-1 acyl chains for these bilayer systems enables each chain's intra- and intermolecular conformational and organizational properties to be evaluated separately. Various saturated chain methylene CD(2) and carbon-carbon (C&bond;C) stretching mode peak height intensity ratios and line width parameters for the polycrystalline samples demonstrate a high degree of sn-1 chain order that is unaffected by either the double bond placement or number of unsaturated bonds within the sn-2 chain. In contrast, the unsaturated sn-2 chain spectral signatures reflect increasing acyl chain conformational disorder as either the cis double bond is generally repositioned toward the chain terminus or the number of double bonds increases from one to three. The lipid bilayer chain packing differences observed between the sn-1 and sn-2 chains of this series of monounsaturated and polyunsaturated 20 carbon chain lipids suggest the existence of laterally distributed microdomains predicated on the formation of highly ordered, saturated sn-1 chain clusters.     

Magnetic alignment and orientational order of dipalmitoylphosphatidylcholine bilayers containing palmitoyllysophosphatidylcholine

Mixed bilayers of 1-palmitoyl-sn-glycero-3-phosphocholine (palmitoyllysophosphatidylcholine; PaLPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoyl phosphatidylcholine; DPPC) have been investigated by 2H-NMR and 31P-NMR spectroscopy. Binary phospholipid mixtures were studied in which the acyl chains of one or the other component were perdeuterated. At temperatures below the main order-disorder phase transition, the mixed PaLPC/DPPC bilayers appear to coexist with PaLPC micelles. The micelles disappear at temperatures above the phase transition, where mixed bilayers in the liquid-crystalline state are formed. The orientational order of the alkyl chains of the PaLPC component is essentially identical to that of the DPPC component in the mixed bilayers, both in the low temperature and liquid-crystalline phases. However, the presence of PaLPC perturbs the segmental ordering of DPPC as compared to the pure system. The order is increased in the low-temperature phase, where effective diffusion of the chains about their long axes occurs, but is decreased in the liquid-crystalline phase compared to pure DPPC bilayers. The mixed liquid-crystalline bilayers orient preferentially with their director axes perpendicular to the magnetic field. This alignment is easily observed in 31P- and 2H-NMR spectra, where the intensity of the perpendicular edges of the lineshapes is pronounced. One possible explanation of the magnetic alignment involves alteration of the curvature free energy of the DPPC bilayer due to incorporation of PaLPC in the mixed membranes.