Ching-hsien Huang

Calorimetric and molecular mechanics studies of the thermotropic phase behavior of membrane phospholipids.

In this review, we summarize the results of recent studies on the main phase transition behavior of phospholipid bilayers using the combined approaches of molecular mechanics simulations and high-resolution differential scanning calorimetry. Following a brief overview of the phase transition phenomenon exhibited by the lipid bilayer, we begin with the review by showing how several structural parameters underlying various phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol are defined and determined. Specifically, these structural parameters are obtained with saturated lipids packed in the gel-state bilayer using computer-based molecular mechanics calculations. Then we proceed to present the calorimetric data obtained with the lipid bilayer composed of saturated phospholipids as it undergoes the gel-to-liquid-crystalline phase transition in excess water. The general equations that can correlate the gel-to-liquid-crystalline phase transition temperature (T(m)) of the lipid bilayer with the structural parameters of the lipid molecule constituting the lipid bilayer are subsequently presented. From these equations, two tables of predicated T(m) values for well over 400 molecular species of saturated phosphatidylcholine and saturated phosphatidylethanolamine are generated. We further review the structure and chain-melting behavior of a large number of sn-1 saturated/sn-2 unsaturated phospholipids. Two T(m)-diagrams are shown, from which the effects of the number and the position of one to five cis carbon-carbon double bonds on T(m) can be viewed simultaneously. Finally, in the last part of this review, simple molecular models that have been invoked to interpret the characteristic T(m) trends exhibited by lipid bilayers composed of unsaturated lipids with different numbers and positions of cis carbon-carbon double bonds as seen in the T(m)-diagram are presented.

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.

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.

EFFECTS OF VARIOUS NUMBERS AND POSITIONS OF CIS DOUBLE BONDS IN THE SN-2 ACYL CHAIN OF PHOSPHATIDYLETHANOLAMINE ON THE CHAIN-MELTING TEMPERATURE

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novosynthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into thesn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature,T m , of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position ofcis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel T m diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds onT m can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristicT m profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.

Thermotropic phase behavior of mixed-chain phosphatidylglycerols: implications for acyl chain packing in fully hydrated bilayers.

In this communication we report the first systematic investigation of the thermodynamic properties of fully hydrated mixed-chain phosphatidylglycerols (PG) using high-resolution differential scanning calorimetry (DSC). The crystal structure of dimyristoylphosphatidylglycerol shows an acyl chain conformation that is nearly opposite to that of phosphatidylcholine (PC). In PC, the sn-1 chain is straight while the sn-2 chain contains a bend; for PG, the sn-1 contains a bend while the sn-2 chain is in the all-trans conformation (R.H. Pearson, I. Pascher, The molecular structure of lecithin dihydrate, Nature, 281 (1978) 499-501; I. Pascher, S. Sundell, K. Harlos, H. Eibl, Conformational and packing properties of membrane lipids: the crystal structure of sodium dimyristoylphosphatidylglycerol, Biochim. Biophys. Acta, 896 (1987) 77-88). If the structure of PG found in the single crystal can be extrapolated to that in the fully hydrated gel-state bilayer, the observed difference in acyl chain conformations implies that modulation of the acyl chain asymmetry will have an opposite effect on the thermotropic phase behavior of PG and PC. For example, it is expected, based on the crystal structures, that C(15):C(13)PG should have a higher main phase transition temperature (Tm) than C(14):C(14)PG, and C(13):C(15)PG should have a lower Tm than C(14):C(14)PG. However, our DSC studies show clearly that the expectation is not borne out by experimental data. Rather, the Tm values of C(15):C(13)PG, C(14):C(14)PG, and C(13):C(15)PG are 18.2 degrees C, 23.1 degrees C, and 24.4 degrees C, respectively. Several other PGs, each with a unique acyl chain composition, have also been studied in this laboratory using high-resolution DSC. It is shown that the acyl chain conformation of fully hydrated PG in general is nearly opposite to that seen in the PG crystal structure.

Bilayer packing characteristics of mixed chain phospholipid derivatives: Raman spectroscopic and differential scanning calorimetric studies of 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine (C(18) : C(10)PC) and 1-stearoyl-2-capryl-sn-glycero-3-phospho-N-trimethylpropanolamine (C(18) : C(10)TMPC)

Raman spectroscopy and high-sensitivity differential scanning calorimetry (DSC) were used to compare the effects of headgroup conformation on the acyl chain packing arrangements in two highly asymmetric phosphatidylcholine (PC) analogues, 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine (C(18):C(10)PC) and a polar headgroup derivative of C(18):C(10)PC, 1-stearoyl-2-capryl-sn-glycero-3-phospho-N-trimethylpropanolami ne (C(18):C(10)TMPC), which contains an additional methylene group within the choline moiety; namely, -P-O-(CH2)3-N(CH3)3. The C(18):C(10)TMPC headgroup exhibits an extended trans conformation which is independent of bilayer phase. A comparison of gel phase spectral order parameters of the two lipid species indicates a mixed interdigitated state characteristic of three chains per headgroup for C(18): C(10)TMPC. A more intermolecularly ordered liquid crystalline phase is observed, however, for the C(18):C(10)TMPC bilayers. The phase transition cooperative unit size estimated for the C(18):C(10)PC bilayers (approximately 140 molecules per unit) is about 7-fold greater than that for the C(18):C(10)TMPC dispersions (approximately 20 molecules per unit). We suggest that the extended headgroup for C(18):C(10)TMPC induces a slight tilt in the gel phase packing arrangements for the acyl chains, which may persist in the partially interdigitated liquid crystalline phase bilayer. Macroscopically, tighter packed multilamellar dispersions of C(18):C(10)TMPC occur for systems prepared first in the presence of a higher ionic strength medium. The stacked bilayers may then be transferred to a lower ionic strength environment without loss of their more closely packed adjacent lamellae.

Interdigitated bilayer packing motifs: Raman spectroscopic studies of the eutectic phase behavior of the 1-stearoyl-2-caprylphosphatidylcholine/dimyristoylphosphatidylcholine binary mixture.

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of binary mixtures of 1-stearoyl-2-caprylphosphatidylcholine (C(18):C(10)PC), an asymmetric chain species, and dimyristoylphosphatidylcholine (C(14):C(14)PC), a symmetric chain lipid, were monitored by vibrational Raman spectroscopy. In order to examine each component of the binary mixture separately, the acyl chains of the symmetric chain species were perdeuterated. As shown by differential scanning calorimetry, the mismatch in the gel phase bilayer thickness between the two lipid components generates a lateral phase separation resulting in two distinct gel phases, G(I) and G(II), which coexist over much of the composition range. The Raman data demonstrate that the mixed interdigitated phase (three chains per headgroup), analogous to single component phase behavior, is retained when the C(18):C(10)PC component act as a host for the G(I) gel phase. In contrast, the C(18):C(10)PC molecules exhibit partial interdigitation (two chains per headgroup) when they are included as guests within the C(14):C(14)PC host matrix to form the G(II) gel phase. Compared to pure C(14):C(14)PC bilayers at equivalent reduced temperatures, the host G(II) gel phase C(14):C(14)PC molecules exhibit an increased acyl chain order, while for the host G(I) gel phase the C(14):C(14)PC lipid species show increased intrachain disorder.

CALORIMETRIC STUDIES OF PHOSPHATIDYLETHANOLAMINES WITH SATURATED SN-1 AND DIENOIC SN-2 ACYL CHAINS

We have semi-synthesized 18 species of mixed chain phosphatidylethanolamines (PE) in which the sn-1 acyl chain is derived from stearic, arachidic, and behenic acids, and thesn-2 acyl chain is originated fromcis,cis-octadecadienoic andcis,cis-eicosadienoic acids with the two methylene-interrupted double bonds located at various positions. These PEs constituting the bilayers in the aqueous dispersion were subjected to differential scanning calorimetric experiments. TheT m values associated with the gel-to-liquid crystalline phase transitions for these PEs are found to be significantly smaller than those of the saturated counterparts. Moreover, the magnitude of the T m -lowering effect of acyl chain diunsaturation depends critically on the positions of the two methylene-interrupted cis double bonds in thesn-2 acyl chain. Specifically, if the sn-2 acyl chain is derived from cis,cis-octadecadienoic acid, the T m -lowering effect has the following decreasing order: Δ9,12 > Δ6,9 > Δ12,15. For cis,cis-eicosadienoyl acyl chain, the T m -lowering effect is stronger in the order of Δ10,13 > Δ11,14 > Δ8,11 > Δ5,8 > Δ14,17. Finally, a refined molecular model is presented that can adequately explain the T m -lowering effect of sn-2 acyl chain diunsaturation. Moreover, this same refined molecular model can also be invoked to better interpret theT m -lowering effect observed for sn-1 saturated/sn-2 monoenoic PE.