Hai-nan Lin

Topology of gel-phase domains and lipid mixing properties in phase-separated two-component phosphatidylcholine bilayers

The influence of the lipid mixing properties on the lateral organization in a two-component, two-phase phosphatidylcholine bilayer was investigated using both an experimental (fluorescence recovery after photobleaching (FRAP)) and a simulated (Monte Carlo) approach. With the FRAP technique, we have examined binary mixtures of 1-stearoyl-2-capryl-phosphatidylcholine/1,2-distearoyl-phosphat idylcholine (C18C10PC/DSPC), and 1-stearoyl-2-capryl-phosphatidylcholine/1,2-dipalmitoyl-phospha tid ylcholine (C18C10PC/DPPC). Comparison with the 1,2-dimyristoyl-phosphatidylcholine/1,2-distearoyl-phosphatidylcholine (DMPC/DSPC) previously investigated by FRAP by Vaz and co-workers (Biophys. J., 1989, 56:869-876) shows that the gel phase domains become more effective in restricting the diffusion coefficient when the ideality of the mixture increases (i.e., in the order C18C10PC/DSPC-->C18C10PC/DPPC-->DMPC/DSPC). However, an increased lipid miscibility is accompanied by an increasing compositional dependence: the higher the proportion of the high-temperature melting component, the less efficient the gel phase is in compartmentalizing the diffusion plane, a trend that is best accounted for by a variation of the gel phase domain shape rather than size. Computer-simulated fluorescence recoveries obtained in a matrix obstructed with obstacle aggregates of various fractal dimension demonstrate that: 1) for a given obstacle size and area fraction, the relative diffusion coefficient increases linearly with the obstacle fractal dimension and 2) aggregates with a lower fractal dimension are more efficient in compartmentalizing the diffusion plane. Comparison of the simulated with the experimental mobile fractions strongly suggests that the fractal dimension of the gel phase domains increases with the proportion of high-temperature melting component in DMPC/DSPC and (slightly) in C18C10PC/DPPC.

Interconversion of bilayer phase transition temperatures between phosphatidylethanolamines and phosphatidylcholines

High-resolution differential scanning calorimetric studies were performed to investigate the thermotropic phase behavior of 31 molecular species of phosphatidylethanolamines in excess water. Upon reheating, the aqueous dispersions of these lipids undergo the gel to liquid-crystalline phase transitions at well defined temperatures (Tm). These Tm values were shown to relate to the structural parameters of the underlying lipid molecules in a characteristic manner. Based on these observations, an interconversion of the Tm values between saturated phosphatidylethanolamines and phosphatidylcholines is established quantitatively for the first time.

The effect of ethanol on the phase transition temperature and the phase structure of monounsaturated phosphatidylcholines.

Previous studies from our laboratories have delineated the relationship between the acyl chain asymmetry of mixed-chain phosphatidylcholines, C(X):C(Y)PC, and the effect of ethanol concentration, [EtOH], on the main phase transition temperature, T(m), and the phase structure of the lipid bilayer composed of C(X):C(Y)PC using differential scanning calorimetry and X-ray diffraction techniques [Huang and McIntosh, Biophys. J. 72 (1997) 2702--2709]. In the present work, we have extended these studies to characterize the effect of [EtOH] on the T(m) and the phase structure of the lipid bilayer composed of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines with various positions of the cis double bond. Specifically, five positional isomers of 1-eicosanoyl-2-eicosenoyl-sn-glycero-3-phosphocholines, C(20):C(20:1 Delta(n))PC with n=5, 8, 11, 13 and 17, were synthesized and studied. For C(20):C(20:1 Delta(n))PC with n=5 and 8, results from the calorimetric experiments showed that in response to various concentrations of ethanol, the change in T(m) of the lipid bilayer composed of monounsaturated lipids was characterized by a sigmoidal or biphasic profile in the plot of T(m) versus [EtOH]. In contrast, a continuous depression of the T(m) by ethanol was observed calorimetrically for C(20):C(20:1 Delta(n))PC with n> or =11. The X-ray diffraction experiments further demonstrated that C(20):C(20:1 Delta(5))PC and C(20):C(20:1 Delta(8))PC can undergo the ethanol-induced gel-to-fully interdigitated phase transition at T<T(m). Such a transition, however, was not observed for C(20):C(20:1 Delta(13))PC even at a very high ethanol concentration of 100 mg/ml. These distinct different effects of [EtOH] on the phase transition temperature and the phase structure can be attributed to various positions of the cis double bond in these monounsaturated phosphatidylcholines. And the different effects of ethanol can, in fact, be explained based on the molecular structures of these monounsaturated lipids packed in the gel-state bilayer as generated by molecular mechanics simulations. To the best of our knowledge, this is the first time that the ethanol-induced fully interdigitated bilayers are observed at T<T(m) for unsaturated phospholipids with well defined double bond positions in their sn-2 acyl chains.

Identification and characterization of kink motifs in 1-palmitoyl-2-oleoyl- phosphatidylcholines: a molecular mechanics study

As a cis carbon-carbon double bond (delta) is introduced into the middle of an isolated all-trans hydrocarbon chain, it can be shown by molecular graphics that this delta-bond makes a bend of 130 degrees in the chain axis, thus producing a boomerang-like conformation. Such a bent structure, indeed, has been detected experimentally for oleic acid by x-ray crystallography (Abrahamson and Ryderstedt-Nahringbaur, 1962). Membrane diacyl phospholipids are largely mixed-chain lipids containing a saturated sn-1 acyl chain and an unsaturated sn-2 acyl chain. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the most abundant phospholipid in animal cell membranes, is a typical example in which the sn-2 acyl chain is the acyl chain of an oleic acid. However, this sn-2 acyl chain of POPC is unlikely to adopt a boomerang-like configuration in the gel-state lipid bilayer due to the steric hindrance imposed by neighboring chains. Instead, it has been suggested that the oleate chain in POPC is kinked in the shape of a crankshaft in the gel-state bilayer (Huang, 1977; Lagaly et al., 1977), because POPC with such a kinked sn-2 acyl chain, which is denoted here as the secondary structural element or motif, can pack efficiently against other neighboring phospholipids. In this communication, 16 different types of secondary structural elements or motifs are derived for POPC at T < Tm based on a single protocol guided by two-dimensional steric contour maps and computer-based molecular graphics. After subjecting these derived molecular species to energy minimization using the molecular mechanics method, the number of the secondary structural motifs is reduced to 13 as a result of conformational degeneracy. The structure and steric energy of each of the energy-minimized lipid rotomers are presented in this communication. Furthermore, these rotomers packed in small clusters are also simulated to mimic the lipid bilayer structure of 1-palmitoyl-2-oleoyl-phosphatidylcholines at T < Tm.

The influence of acyl chain-length asymmetry on the phase transition parameters of phosphatidylcholine dispersions

The influence of acyl chain-length asymmetry on the thermodynamic parameters (Tm, delta H, and delta S) associated with the reversible main phase transition of aqueous dispersions prepared from saturated diacyl phosphatidylcholines was studied by high-resolution differential scanning calorimetry. Two series of saturated diacyl phosphatidylcholines, grouped according to their molecular weights of 678 and 706, with a total number of 25 molecular species were examined. The normalized acyl chain-length difference between the sn-1 and sn-2 acyl chains for a given phospholipid molecule in the gel-state bilayer is expressed quantitatively by the structural parameter delta C/CL, and the values of delta C/CL for the two series of lipids under study vary considerably from 0.04 to 0.67. When the value of delta C/CL is within the range of 0.09-0.40, it was shown that the thermodynamic parameters are, to a first approximation, a linear function of delta C/CL with a negative slope. In addition, the experimental Tm values and the predicted Tm values put forward by Huang (Biochemistry (1991) 30, 26-30) are in very good agreement. Beyond the point of delta C/CL = 0.41, the influence of acyl chain-length asymmetry on the thermodynamic parameters deviates significantly from a linear function. In fact, within the range of delta C/CL values of 0.42-0.67, the thermodynamic parameters in the Tm (or delta H) vs. delta C/CL plot were shown to be bell-shaped with the maximal Tm (or delta H) at delta C/CL = 0.57. These results are discussed in terms of changes in the acyl chain packing modes of various phosphatidylcholine molecules within the gel-state bilayer in excess water.

Eutectic phase behavior of 1-stearoyl-2-caprylphosphatidylcholine and dimyristoylphosphatidylcholine mixtures

The thermotropic behavior of aqueous dispersions of C(18):C(10)PC/diC(14)PC mixtures with different molar ratios has been investigated by high-resolution differential scanning calorimetry. C(18):C(10)PC is a highly asymmetric lipid molecule, whereas diC(14)PC is a symmetric species with the same molecular weight. Their packing properties in the bilayer are known to be similar at T greater than Tm, but very dissimilar at T less than Tm. Calorimetric results indicate that C(18):C(10)PC and diC(14)PC are completely miscible in the liquid-crystalline state. In the gel state, however, C(18):C(10)PC and diC(14)PC are only partially miscible. The temperature-composition phase diagram for C(18):C(10)PC/diC(14)PC mixtures has the shape characteristic of a typical eutectic system.

INFLUENCE OF THE POSITIONS OF CIS DOUBLE BONDS IN THE SN-2-ACYL CHAIN OF PHOSPHATIDYLETHANOLAMINE ON THE BILAYER'S MELTING BEHAVIOR

In an attempt to examine the effects of different numbers and positions of cis double bonds in thesn-2-acyl chain of phosphatidylethanolamine (PE) on the bilayer’s melting behavior, 21 molecular species of PE were first semisynthesized, and their T m and ΔHvalues were subsequently determined by high resolution differential scanning calorimetry. In the plot of T m versus the number of the cis double bond, some characteristic profiles were observed for the various series of PEs. For instance, if the cis double bond was first introduced into the sn-2-acyl chain of C(20):C(20)PE at the Δ5-position, the T m was observed to reduce drastically. Subsequent stepwise additions of up to fivecis double bonds at the methylene-interrupted positions toward the methyl end resulted in a progressive yet smaller decrease inT m . If, on the other hand, the cisdouble bonds were introduced sequentially at the Δ11-, Δ11,14-, and Δ11,14,17-positions along thesn-2-acyl chain of C(20):C(20)PE, theT m profile in the T m versus the number of the cis double bond showed a down-and-up trend. Most interestingly, for positional isomers of C(20):C(20:3Δ5,8,11)PE, C(20):C(20:3Δ8,11,14)PE, and C(20):C(20:3Δ11,14,17)PE, an inverted bell-shapedT m profile was detected in the plot ofT m against the position of the ω-carbon for these isomers. Similar T m profiles were also observed for C(18):C(20)PE, C(20):C(18)PE, and their unsaturated derivatives. This work thus demonstrated that both the positions and the numbers ofcis double bonds in the sn-2 acyl chain could exert noticeable influence on the gel-to-liquid crystalline phase transition behavior of the lipid bilayer. Finally, a molecular model was presented, with which the behavior of the gel-to-liquid crystalline phase transition observed for lipid bilayers composed of varioussn-1-saturated/sn-2-unsaturated lipids can be rationalized.

Phase metastability and supercooled metastable state of diundecanoylphosphatidylethanolamine bilayers

Aqueous dispersons of L-alpha-phosphatidylethanolamine (PE) with identical saturated acyl chains are known to exhibit gel-state metastability. It is also known that the metastability in PE becomes more pronounced with decreasing acyl chain-length. In an attempt to study the metastable phase behavior of PE, we have synthesized diundecanoylphosphatidylethanolamine (diC11PE) and examined its polymorphic phase behavior. A single endothermic transition at 38 degrees C is detected between 10 and 55 degrees C by DSC for the nonheated sample of diC11PE in excess water. An immediate second heating scan done after cooling slowly of the same sample from the liquid-crystalline state shows a smaller endothermic transition at a lower temperature, 18 degrees C. However, the high-temperature transition at 38 degrees C can be detected, if the sample which has been heated above 38 degrees C is quench cooled from the liquid-crystalline to a temperature between 18 and 38 degrees C. Furthermore, two endothermic transitions at 18 and 38 degrees C and an exothermic transition at 19 degrees C are recorded for diC11PE after quench supercooling of the sample from the liquid-crystalline state to an appropriate temperature below 10 degrees C. The gel-state metastability of diC11PE can be most appropriately explained in terms of changes in interbilayer headgroup-headgroup interactions. It is suggested that the kinetically trapped supercooled metastable state may be a multilamellar structure with melted acyl chains but with strong interbilayer headgroup-headgroup interactions.

Dependence of the bilayer phase transition temperatures on the structural parameters of phosphatidylcholines

Most saturated diacyl phosphatidylcholines C(X):C(Y)PC (saturated 1,2-diacyl-sn-glycero-3-phosphocholine with X carbons in thesn-1 acyl chain and Y carbons in thesn-2 acyl chain), in excess water, can self-assemble into lamellae which, upon heating, may undergo multiple thermotropic phase transitions at well-defined, discrete temperatures. The transition temperature corresponding to the main or the gel to liquid-crystalline phase transition (Tm) is known for many bilayers of fully hydrated phosphatidylcholines. In this study, we have analyzed the Tm values of 44 molecular species of phosphatidylcholines in terms of their structural and packing characteristics in the gel-state bilayer. Two general equations are thus derived: Tm=162.26−3651.71 (1/N)−88.42 (ΔC/N) for C(X):C(Y)PC with X≥Y, and Tm=157.68−3525.44 (1/N)−93.28 (ΔC/N) for C(X):C(Y)PC with X<Y. Here, N is the minimal hydrophobic thickness of the dimeric C(X):C(Y)PC in the gel-state bilayer and ΔC is the effective chain length difference between thesn-1 andsn-2 acyl chains for the monomeric C(X):C(Y)PC in the gel-state bilayer. The advantage of these two equations in predicting the Tm values for phosphatidylcholines with ΔC/CL values in the range of 0.07 to 0.40 is their simplicity. A figure containing a total of 173 calculated Tm values is also presented.

Effects of alcohols on the phase transition temperatures of mixed-chain phosphatidylcholines

The biphasic effect of ethanol on the main phase transition temperature (Tm) of identical-chain phosphatidyl-cholines (PCs) in excess H2O is now well known. This biphasic effect can be attributed to the transformation of the lipid bilayer, induced by high concentrations of ethanol, from the partially interdigitated L beta, phase to the fully interdigitated L beta I phase at T < Tm. The basic packing unit of the L beta I phase has been identified recently as a binary mixture of PC/ethanol at the molar ratio of 1:2. The ethanol effect on mixed-chain PCs, however, is not known. We have thus in this study investigated the alcohol effects on the Tm of mixed-chain PCs with different delta C values, where delta C is the effective acyl chain length difference between the sn-1 and sn-2 acyl chains. Initially, molecular mechanics (MM) simulations are employed to calculate the steric energies associated with a homologous series of mixed-chain PCs packed in the partially and the fully interdigitated L beta I motifs. Based on the energetics, the preference of each mixed-chain PC for packing between these two different motifs can be estimated. Guided by MM results, high-resolution differential scanning calorimetry is subsequently employed to determine the Tm values for aqueous lipid dispersions prepared individually from a series of mixed-chain PCs (delta C = 0.5-6.5 C-C bond lengths) in the presence of various concentrations of ethanol. Results indicate that aqueous dispersions prepared from mixed-chain PCs with a delta C value of less than 4 exhibit a biphasic profile in the plot of Tm versus ethanol concentration. In contrast, highly asymmetric PCs (delta C > 4) do not exhibit such biphasic behavior. In the presence of a longer chain n-alcohol, however, aqueous dispersions of highly asymmetric C(12):C(20)PC (delta C = 6.5) do show such biphasic behavior against ethanol. Our results suggest that the delta C region in a highly asymmetric PC packed in the L beta I phase is most likely the binding site for n-alcohol.