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Types of Lipid Clustering in Phospholipid Membranes as Classified by Nearest-Neighbor Recognition Analysis Takehisa Dewa,† Yasuhito Miyake,† Ferenc J. Ke´zdy,‡ and Steven L. Regen*,† Department of Chemistry and Zettlemoyer Center for Surface Studies, Lehigh University, Bethlehem, Pennsylvania 18015, and The Laboratory of Biochemistry, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49001 Received October 21, 1999 The nearest-neighbor recognition method is applied to the problem of phospholipid clustering in bilayers composed of di[1,2-dimyristoyl-sn-glycero-3-phosphoethanol(3′-thio)propionamide] (I), di[1,2-distearoylsn-glycero-3-phosphoethanol(3′-thio)propionamide] (II), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanol(3′-thio)propionamide-1,2-distearoyl-sn-glycero-3-phosphoethanol(3′-thio)propionamide (III). The value and the composition dependency of the apparent equilibrium constant, K, defined by the equilibrium concentrations of homodimers (I and II) and the corresponding heterodimer (III), allow one to distinguish among three fundamentally different classes of clustering: (i) random clustering, (ii) cooperative clustering, and (iii) nonrandom-noncooperative clustering. Experimental results indicate that random clustering of these phospholipids is pervasive in fluid bilayers, whereas cooperative clustering exists in the gel-fluid coexistence region. In the physiologically relevant fluid phase, these same lipids give rise to nonrandomnoncooperative clustering when cholesterol has been included in the bilayer.
Introduction The notion that phospholipids can form microdomains in fluid bilayers has attracted considerable attention in recent years.1-5 While it is generally believed that microdomains are present in functioning biological membranes, obtaining evidence for their existence has proven to be difficult. Even for the simplest of model membranes (e.g., fluid bilayers containing two different phospholipids), establishing whether microdomains are present has become a formidable challenge. Part of the difficulty has been the absence of experimental techniques that can quantify lipid clustering at the molecular level. Another part of the problem has been the lack of a clear and generally accepted definition of a “lipid microdomain”. For example, some researchers have proposed that a lipid microdomain be defined by minimal cluster sizessuch as an aggregate of at least 20 identical phospholipidssbut this definition is somewhat arbitrary and is not readily translatable into experimental criteria.2 It also does not address the cause or the mechanism of clustering, especially the selective interaction between like lipids. We have introduced a chemical method for studying phospholipid mixing and have used it to explore the relationships that exist between molecular structure and mixing behavior of phospholipids in model membranes.6-14 † ‡
Lehigh University. Pharmacia & Upjohn, Inc.
(1) Tocanne, J. F.; Cezanne, L.; Lopez, A.; Piknova, B.; Schram, V.; Tournier, J. F.; Welby, M. Chem. Phys. Lipids 1994, 73, 139. (2) Welti, R.; Glaser, M. Chem. Phys. Lipids 1994, 73, 121. (3) (a) Thompson, T. E.; Sankaram, M. B.; Biltonen, R. L. Comments Mol. Cell. Biophys. 1992, 8, 1. (b) Vaz, W. L. C. Comments Mol. Cell. Biophys. 1992, 8, 17. (c) Glaser, M. Comments Mol. Cell. Biophys. 1992, 8, 37. (d) Tocanne, J. F. Comments Mol. Cell. Biophys. 1992, 8, 53. (e) Edidin, M. Comments Mol. Cell. Biophys. 1992, 8, 73. (f) Wolf, D. E. Comments Mol. Cell. Biophys. 1992, 8, 83. (g) Jesaitis, A. J. Comments Mol. Cell. Biophys. 1992, 8, 97. (4) For a recent series of papers describing domain formation in biological membranes, see: Mol. Membr. Biol. 1995, 12, 1-162. (5) Gennis, R. B. Biomembranes: Molecular Structure and Function; Springer-Verlag: New York, 1989; p 165. (6) Davidson, S. K. M.; Regen, S. L. Chem. Rev. 1997, 97, 1269. (7) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1992, 114, 9828.
Our method consists of measuring the equilibrium composition of bilayers that are formed from either an equimolar mixture of exchangeable homodimers (AA and BB) or the corresponding heterodimer (AB). Selective nearest-neighbor recognition, reflecting structure-specific molecular interactions, is then detectable by a higher than statistical equilibrium concentration of homodimers (Scheme 1). In contrast to other lipid dimer-based approaches that have been used to probe lipid mixing, the nearest-neighbor recognition (NNR) method reflects, directly, the force that drives two identical phospholipids to become nearest neighbors.1,2 It provides, therefore, a direct thermodynamic measure of nearest-neighbor interactions. In previous studies, the NNR technique revealed that liposomal membranes made from a 1/1 mixture of di[1,2-dimyristoylsn-glycero-3-phosphoethanol(3′-thio)propionamide] (I) and di[1,2-distearoyl-sn-glycero-3-phosphoethanol(3′-thio)propionamide] (II) or pure 1,2-dimyristoyl-sn-glycero-3phosphoethanol(3′-thio)propionamide-1,2-distearoyl-snglycero-3-phosphoethanol(3′-thio)propionamide (III) produce a purely statistical equilibrium mixture of dimers at 60 °C, where the bilayer is in the fluid state.7 In contrast, analogous membranes containing 29 mol % cholesterol yielded an equilibrium mixture of dimers that favored the homodimer.8 The fact that inclusion of a fluid-phase phospholipid, having alkyl chains of intermediate length (e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphocholine), effectively eliminated such recognition has provided strong evidence that intramolecular and intermolecular forces (8) Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 7838. (9) Dewa, T.; Vigmond, S. J.; Regen, S. L. J. Am. Chem. Soc. 1996, 118, 3435. (10) Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1996, 118, 7069. (11) Uragami, M.; Dewa, T.; Inagaki, M.; Hendel, R. A.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 3797. (12) Inagaki, M.; Shibakami, M.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 7161. (13) Shibakami, M.; Inagaki, M.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 12354. (14) Shibakami, M.; Inagaki, M.; Regen, S. L. J. Am. Chem. Soc. 1998, 120, 3758.
10.1021/la991386y CCC: $19.00 © 2000 American Chemical Society Published on Web 03/07/2000
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are similar in magnitude and that lipid mixing is nonideal in the absence of the diluent. Of particular relevance to the present work, however, is the question of whether the associative interactions between “like” phospholipids are cooperative. Measuring only the equilibrium composition of an equimolar mixture does not provide the answer to this question. However, as we propose to show in this paper, by extending the analysis of the equilibrium compositions to a series of different molar ratios, the NNR method can also provide information about the extent of lipid clustering. Specifically, the method allows one to differentiate between three fundamentally different types of clustering, namely, (i) random clustering, (ii) cooperative clustering, and (iii) nonrandom-noncooperative clustering. We also propose that the hallmark of a lipid microdomain is cooperative clustering, which is now an experimentally accessible property of the bilayer in the fluid phase. Materials and Methods Exchangeable Phospholipid Dimers. The chemical syntheses of di[1,2-dimyristoyl-sn-glycero-3-phosphoethanol(3′-thio)propionamide] (I), di[1,2-distearoyl-sn-glycero-3-phosphoethanol(3′-thio)propionamide] (II), and 1,2-dimyristoyl-sn-glycero-3phosphoethanol(3′-thio)propionamide-1,2-distearoyl-sn-glycero3-phosphoethanol(3′-thio)propionamide (III) have previously been reported.7 Preparation of Liposomes and Initiation of ThiolateDisulfide Interchange. In a typical preparation, dichloromethane solutions of I and II (0.45 µmol of each dimer) were transferred to a test tube. The dichloromethane was then evaporated under a stream of argon over the solution, thereby leaving a thin film of the lipid mixture. The resulting thin film was then dissolved with 150 µL of chloroform and then diluted by 400 µL of diisopropyl ether. Subsequent addition of 50 µL of
3.3 mM borate buffer (47 mM NaCl and 0.7 mM NaN3, pH 7.4) results in an emulsion. After the emulsion was sonicated for 3 min by use of a mild (bath-type) sonicator, the organic phase was removed by gentle evaporation at 60 °C to afford a white gel in the bottom of the test tube. The gel was then collapsed by vigorous vortex mixing for 5 min, and 3.0 mL of additional buffer (10 mM borate, 140 mM NaCl, and 2.0 mM NaN3, pH 7.4) was added dropwise with vortex mixing. The vesicle dispersion was then degassed with an aspirator for 20 min and the residual trace of organic solvent was removed by an 18 h dialysis (Spectra/Por Membrane, MWCO 6000-8000) under an argon atmosphere against two 200 mL portions of degassed 10 mM borate buffer, pH 7.4. After the sample had thermally equilibrated at the desired temperature, the thiolate-disulfide interchange reaction was initiated by increasing to pH 8.5 (addition of 20 µL of 0.15 M NaOH) followed by injection of 166 µL of an aqueous solution of 8.7 mM dithiothreitol (1.6 equiv relative to moles of lipid) and brief vortex mixing. All dispersions were maintained under an argon atmosphere throughout the course of the interchange reaction. Aliquots of 0.45 mL were removed at desired time intervals and quenched with 120 µL of 0.01 M HCl to give a final pH of 5.0. After removal of water under reduced pressure, the resulting white salt was triturated with 2 mL of chloroform and centrifuged. The chloroform was then removed under reduced pressure to yield a clear film. Samples were dissolved in 5 µL of chloroform plus 95 µL of the mobile phase (HPLC) prior to injection. Analysis of Dimer Distributions by High-Performance Liquid Chromatography. Mixtures of lipid dimers were analyzed by HPLC using a Beckman Ultrasphere C18 reverse phase column (4.6 × 250 mm, 5 µm particle size). In general, the premixed mobile phase contained 80% 10 mM tetrabutylammonium acetate (TBA) in denatured ethanol, 12% water, and 8% hexane (v/v/v). The flow rate was 0.9 mL/min, and the column was maintained at 31.2 °C. Peaks were monitored at 205 nm using a Waters 996 photodiode array detector. Data were collected and processed using a Millennium workstation (Waters Corp.). In all cases, nearest-neighbor recognition values were determined from the integrated areas for the C18 homodimer relative to the corresponding C14/18 heterodimer.
Results and Discussion To test for the presence of cooperative forces in bilayers composed of I-III and cholesterol, we have varied the initial mole fraction of II (and the corresponding thiol monomer) and have analyzed the resulting equilibrium product mixtures. Specific protocols that were used for the synthesis of the phospholipids, the formation of vesicles, and the analysis of the dimers were similar to those previously described.7-9 A summary of our results is shown in Table 1. Do these results reveal anything about microdomain formation? To address this question, we first need to define a microdomain in experimentally accessible terms. A microdomain consists of a cluster of identical molecules held together by noncovalent intermolecular forces where at least some of the component molecules interact with more than one neighbor. For those molecules, the interac-
Scheme 1
Types of Lipid Clustering in Phospholipid Membranes
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Table 1. Equilibrium Composition as a Function of the Initial Mole Fraction of IIa initial mole fraction II
equilibrium mole fraction (B) I
equilibrium mole fraction (A) II
equilibrium mole fraction (M) III
K
0.192 0.287 0.387 0.499 0.564 0.669 0.775
0.658 ( 0.003 0.522 ( 0.008 0.390 ( 0.005 0.278 ( 0.004 0.218 ( 0.010 0.129 ( 0.005 0.071 ( 0.007
0.042 ( 0.002 0.095 ( 0.005 0.164 ( 0.006 0.275 ( 0.008 0.346 ( 0.005 0.466 ( 0.007 0.620 ( 0.007
0.300 ( 0.002 0.383 ( 0.012 0.446 ( 0.003 0.447 ( 0.011 0.436 ( 0.006 0.405 ( 0.009 0.309 ( 0.009
3.25 ( 0.21 2.96 ( 0.38 3.11 ( 0.20 2.61 ( 0.24 2.52 ( 0.22 2.73 ( 0.27 2.17 ( 0.36
a Equilibrium mole fractions were determined by HPLC, using procedures similar to those previously described. Constant ratios of dimers were generally reached in ca. 3 h.
Table 2. Enhancement of the Same Nearest Neighbor as a Function of the Initial Mole Fractions initial mole fraction II
equilibrium mole fraction II (A)
enhancement of the same nearest neighbor Ea
initial mole fraction I
equilibrium mole fraction I (B)
enhancement of the same nearest neighbor Eb
0.192 0.287 0.387 0.499 0.564 0.669 0.775
0.042 0.095 0.164 0.275 0.346 0.466 0.620
1.138 1.153 1.093 1.104 1.088 1.040 1.032
0.808 0.713 0.613 0.501 0.436 0.331 0.225
0.658 0.522 0.390 0.278 0.218 0.129 0.071
1.008 1.028 1.037 1.108 1.147 1.183 1.392
tion is cooperative; i.e., the interaction with one neighbor conditions the interaction with the others. Hence, our proposed definition of a microdomain is as follows: a noncovalent aggregate of more than two molecules held together by cooperative forces. Theoretical Considerations: Statistical Mixing and Enhancement of Nearest-Neighbor Interactions. In a homogeneous mixture of compounds A and B, the mole fractions A0 and B0 also represent the probability of choosing, randomly, one molecule of A or B, respectively. The probability of choosing, randomly, compound A twice in a row is, therefore, A0A0. Similarly, the probability of choosing, randomly, compound B twice in a row is, therefore, B0B0. Finally, the probability of choosing A followed by B is A0B0, and the probability of choosing B first, followed by A, is B0A0. If one thinks of two molecules of A in a row as dimer X, two molecules of B in a row as dimer Y, and molecules of A and B together as dimer Z, then an ideal, purely random mixture of A and B can be treated as an ideal, randomly mixed system of X, Y, and Z, having mole fractions equaling Xm,ideal ) A0A0, Ym,ideal ) B0B0, and Zm,ideal ) A0B0 + B0A0 ) 2A0B0, respectively. If the equilibrium mole fractions of the homodimers, X and Y, are higher than those of an ideal mixture, then Xm ) A0pa-a, where pa-a is the probability of finding an identical molecule as a nearest neighbor, with pa-a > A0. Similarly, Ym ) B0pb-b, with pb-b > B0. The enhancements of identical nearest neighbors with respect to that of the ideal mixture are then given by Ea ) pa-a/A0 ) Xm/A02 ) Xm/Xm,ideal and Eb ) pb-b/B0 ) Ym/B02 ) Ym/Ym,ideal. These enhancements, given in Table 2, show that there is, indeed, a significant preference for identical nearest-neighbors at all initial compositions. However, the enhancement for both components is inversely related to their initial molar fraction, whereas if the nearest neighbor recognition were cooperative and extending beyond the next molecule, then the enhancement, for at least for one of the components, would increase with increasing mole fraction. Interactions Limited to One Nearest Neighbor: Formation of Noncovalent Dimeric Complexes. If nearest-neighbor interactions are not cooperative, then an informative way of treating these interactions observed by the NNR method is to consider them as formal bonds between pairs of adjacent lipids, vide ante. Thus, a membrane that is composed of A and B molecules may
then be described as an equilibrium mixture of homodimers and heterodimers as indicated by eqs 1 and 2. K
AA + BB {\} 2AB
(1)
K ) M2/AB
(2)
Here A, B, and M represent equilibrium concentrations of the AA homodimer, BB homodimer, and AB heterodimer, respectively, and K is the equilibrium constant. In an ideal mixture, where no structure-specific interactions occur, K is equal to 4.0 because one molecule of either homodimer leads to two molecules of heterodimer, but one molecule of heterodimer yields only half of a homodimer molecule. Thus, for a membrane that contains an equimolar amount of AA and BB, the molar ratio of AA/AB/BB would be 1/2/1. When forces exist that lead to the recognition of identical nearest neighbors, then K will be less than 4.0 because homodimers are favored. Nevertheless, as long as the association is limited to dimer formation, K should remain constant and independent of the initial ratio of AA/BB. In contrast, a system displaying cooperative clustering should not conform to eqs 1 and 2, and changes in the initial ratio of AA/BB should produce a variation in the apparent equilibrium constant. The magnitude of this variation would depend on the magnitude of the cooperativity that is present. In principle, therefore, a determination of K as a function of the initial mole ratio of AA/BB should allow one to distinguish between ideal mixtures, nonideal mixtures, and microdomains. Experimentally, K may be directly calculated from the observed values of A, B, and M (Table 1). It appears that within our experimental error K is constant over at least the interval of the initial mole fraction of II from 0.287 to 0.669. Thus, for at least that interval, these results are most consistent with the experimental system being governed by forces limited to interaction between nearest neighbors. They are clearly inconsistent with the system being an ideal mixture, or containing microdomains. The average value of the equilibrium constant for the constant interval is 2.79, reflecting a difference in free energy of 238 cal/mol from that of the ideal mixture. This rather modest free energy change represents the energy necessary to break four methylene-methylene interactions when going from AA + BB to 2AB.
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Alternatively, a best-fit value of K may be computed by curve fitting, using equations in which A, B, and M are expressed in terms of K and the initial concentration of A: The stoichiometric relationships are expressed in eqs 3-5, where A0 and B0 are the initial mole fractions of AA and BB, respectively.
A0 ) A + M/2
M ) 2(A0 - A)
and
(3)
1 ) A 0 + B0 ) A + B + M
(4)
B ) 1 + A - 2A0
(5)
Substitution into eq 2 then yields
K)4
(A0 - A)2
(6)
A(1 + A - 2A0)
Rearrangement of eq 6 yields a second-order equation for A as a function of A0, which has the following solution:
A)
x[
4 -1 1 + 2A0 K
(
4 -1 1 + 2A0 K 4 2 -1 K
)
( )
(
)]
2
16A02 4 -1 K K
[
]
(7) Similarly, K may be defined according to eq 8, which then
M2
K)
(
A0 -
M M 1 - A0 2 2
)(
)
(8)
allows one to define M as a function of A0 (Eq 9). Finally,
M)
1 2
x14 - 4A (1 - A )(41 - K1 ) 0
1 1 4 K
(
2
0
)
we needed to find a positive example of such a system. For this purpose, we chose a system of lipid bilayers made from I-III in the gel-fluid coexistence region, at 33 °C.7 We used two different initial mole fractions of II and measured the resulting equilibrium mole fractions. With the initial mole fraction of II being 0.5, K was found to be equal to 0.58. In sharp contrast, when the initial mole fraction of II was lowered to 0.22, K increased to 2.7. Also, for the long-chain component, the enhancement of recognition of the same nearest neighbor Ea-a decreased from 1.45 to 1.25. Thus, both the very large change in K and the directsinstead of an inversesdependence of Ea-a on the initial mole fraction of II are consistent with the presence of microdomains while inconsistent with either an ideal mixture or recognition limited to one nearest neighbor.
(9) Conclusions
by use of eq 9, the value of B as a function of A0 is given in eq 10.
B ) 1 - A0 - M/2
Figure 1. Plot of equilibrium fraction (Xeq) of I (4), II (O), and III (b) as a function of the initial mole fraction of II (XII); the experimental error is indicated by the size of each symbol. The solid lines that are drawn represent a best fit for a nonlinear least-squares fit of the data according to eqs 7, 9, and 10, where K ) 2.73 ( 0.12. The dashed lines represent fully random distributions.
(10)
A nonlinear least-squares fit of the data in Table 1, using eq 7, yields a best-fit value of K ) 2.73 ( 0.12 (Figure 1), the same as the average of the individual equilibrium constants calculated for the range of initial mole fractions of II from 0.287 to 0.669. The reason for the nonconstancy of K for the initial mole fractions of II equaling 0.192 and 0.775 is not presently clear. It is most likely the consequence of secondary effects that result from changes in the thickness in the bilayer as one proceeds from a majority of shortchain molecules to a majority of long-chain ones. Indeed, increased bilayer thickness results in more extended fatty acyl chains and, hence, more contact between neighboring molecules. In a sense, one could claim that this effect, however diffuse, is also a cooperative effect involving the whole membrane. Even so, this cooperativity is not at the molecular level, and it certainly does not reflect the formation of microdomains. To be able to claim that the NNR method is able to identify systems where cooperative clustering does exist,
The results reported herein indicate that, in a bilayer composed of two phospholipids of different chain lengths, at least three different types of molecular clusters can be experimentally detected by use of the nearest-neighbor recognition method: (a) Random clusteringsclustering that is the result of statistical fluctuations in nearestneighbor distributions that involve no special recognition between lipids having the same fatty acyl chain length. This clustering is characterized by an equilibrium constant K ) 4 which is independent of composition. (b) Microdomainssclustering that involves the selective association of more than two identical lipids, where cooperative forces are significant. This cooperative clustering is characterized by a K that is lower than 4, by the dependence of the value of K on the composition of the bilayer, and by an Ei-i value that increases as the mole fraction of the ith component increases. (c) Nonrandomnoncooperative clusteringsclustering that results from noncooperative forces, which are limited to nearest neighbors. This type of clustering is characterized by a K that is lower than 4 and by the independence of K from the composition of the bilayer. Whereas previous NNR experiments could not distinguish between cooperative clustering and nonrandom-noncooperative clustering (both were referred to simply as “nonrandom distributions” or “lateral heterogeneity”), the extended NNR method that
Types of Lipid Clustering in Phospholipid Membranes
is described in this paper is capable of making a clear distinction between them. Nearest-neighbor recognition experiments, of the type reported herein, provide an opportunity for probing phospholipid clustering in ways that have not previously been possible. As such, they represent a simple yet powerful approach that should help to define the relationships that exist between lipid
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structure and membrane organization within the physiologically relevant fluid phase. Acknowledgment. This research was supported by a grant from the National Institutes of Health (PHS Grant GM56149). LA991386Y
