Thermodynamic and Kinetic Stability of Discoidal High-Density

Jun 2, 2010 - from Phosphatidylcholine/Apolipoprotein A-I Mixture ... A-I (apoA-I) with transmembrane ATP-binding cassette transporter A1 (ABCA1)...
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Thermodynamic and Kinetic Stability of Discoidal High-Density Lipoprotein Formation from Phosphatidylcholine/Apolipoprotein A-I Mixture Masakazu Fukuda, Minoru Nakano,* Masakazu Miyazaki, and Tetsurou Handa Graduate School of Pharmaceutical Sciences, Kyoto UniVersity, Sakyo-ku, Kyoto 606-8501 ReceiVed: February 4, 2010; ReVised Manuscript ReceiVed: May 8, 2010

Nascent high-density lipoproteins (HDLs), which are also known as discoidal HDLs, are formed by the interaction of apolipoprotein A-I (apoA-I) with transmembrane ATP-binding cassette transporter A1 (ABCA1). However, the molecular mechanism governing disc formation is not fully understood. Here, we evaluated the thermodynamic and kinetic stability of disc formation from mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine and apoA-I by quantifying the discs and vesicles produced. Sodium cholate dialysis experiments revealed that the discs are thermodynamically more stable than the vesicle/apoA-I mixture (∆*G ) -52 kJ/disc mol at 37.0 °C) because the decrease in enthalpy (∆*H ) -620 kJ/disc mol) exceeds the decrease in entropy (T∆*S ) -570 kJ/disc mol). Circular dichroism spectral measurements ascribed 68% of the decrease in enthalpy during disc formation to the formation of helices in apoA-I. Fluorescence measurements suggested that phospholipids enclosed in the discs are more closely packed than those in the vesicles so that they are entropically destabilized. To determine if the disc could be spontaneously produced from vesicles, we measured the decrease in the turbidity of vesicles in response to the addition of apoA-I. However, the rate of disc formation was very slow, suggesting that the large kinetic barrier against disc formation makes the vesicle/ apoA-I mixtures metastable. These results raise the possibility that ABCA1 may act to lower the activation energy, thereby facilitating disc formation. Introduction High-density lipoproteins (HDLs) transport excess cholesterol from the peripheral tissues to the liver, where it is metabolically converted to bile acids and removed from the body.1 This pathway, which is known as the reverse cholesterol transport pathway, has been focused on in basic research pertaining to the development of novel drugs for arteriosclerosis owing to the inverse correlation between the plasma levels of HDLs and the probability of developing atherosclerosis.2 Nascent HDLs, which are referred to as discoidal HDLs, are formed by the interaction of apolipoprotein A-I (apoA-I) with transmembrane ATP-binding cassette transporter A1 (ABCA1).3,4 In this reaction, discoidal HDLs accept free (unesterified) cholesterol during and after their formation. In the process of cholesterol esterification by lecithin/cholesterol acyltransferase, the particles acquire an apolar core and undergo transformation to form mature spherical HDLs.5 Discoidal HDLs are lipid-apoA-I complexes in which two or more apoA-I molecules surround the hydrophobic edge of the lipid bilayer in a beltlike manner.6,7 The reported size of these discs ranges from 7 to 15 nm, depending on the lipid/protein composition.8-11 ApoA-I contains a series of highly homologous 11- and 22-residue amphipathic R-helices, which are responsible for its lipid-associating properties.12,13 In recent years, discoidal complexes have also attracted researchers’ attention as new pharmaceutical applications14-16 or as an attractive alternative protein solubilization system over micelles and vesicles.17-19 The importance of ABCA1 in HDL biogenesis is demonstrated by the fact that mutations in the ABCA1 gene lead to Tangier disease, which is characterized by low plasma levels of HDLs.20 It is well established that ABCA1 transfers phos* To whom correspondence should be addressed. Phone: +81-75-7534565. Fax: +81-75-753-4601. E-mail: [email protected].

pholipids and free cholesterol to lipid-free apoA-I, thereby triggering disc formation; however, the molecular mechanism involved in this process is not fully understood. Several models of ABCA1-mediated lipid efflux to apoA-I have been proposed, including the molecular efflux model, in which ABCA1 directly transports individual lipids to apoA-I, and the membrane solubilization model, in which membranes disrupted by the lipid translocase activity of ABCA1 are spontaneously solubilized as discrete units by apoA-I.21,22 From a thermodynamic viewpoint, it cannot be assumed that discoidal complexes are formed via the mechanism in which ABCA1 transports hundreds of lipids to apoA-I one after the other; this is because the energy required to transport individual phospholipids from the membrane to the aqueous phase (∼60 kJ/mol) is higher than that required to carry out ATP hydrolysis (∼30 kJ/ mol). The reported ATPase activity of ABCA1 is 400-900 nmol/ min/mg of protein,23 which corresponds to ∼4 ATPs/s per protein. A high-energy transition state in which membrane-bound apoA-I molecules fold a small number of lipids could not be maintained during such a slow process (a time scale of minute for the disc construction by ABCA1). Therefore, we postulate that the membranes disrupted by ABCA1 allow spontaneous lipid extraction and disc formation by apoA-I. In this study, to evaluate the possibility that spontaneous processes are involved in disc formation, we demonstrate the relatively greater thermodynamic stability of discs consisting of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and apoA-I in comparison with a mixture of POPC vesicles and apoA-I molecules. We also discuss the implications of our findings with respect to the energy-dependent processes involved in disc formation. Experimental Methods Materials. Guanidine hydrochloride and sodium deoxycholate (cholate) were purchased from Wako Pure Chemicals (Osaka,

10.1021/jp101071t  2010 American Chemical Society Published on Web 06/02/2010

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Japan). POPC and dioleoyl-sn-glycero-3-phosphoethanolamineN-(lissamine rhodamine B sulfonyl) (Rho-DOPE) were obtained from Avanti Polar Lipids (Alabaster, AL). A series of n-(9anthroyloxy)stearic acids (n-AS, n ) 2, 6, or 12) were purchased from Molecular Probes (Eugene, OR, for 2-AS and 12-AS) and Wako (Osaka, Japan, for 6-AS). ApoA-I was isolated from pig plasma using procedures that have been described previously,24 and its purity was determined to be >95% by SDS-PAGE. Pig apoA-I consists of 241 amino acids, whereas human apoA-I consists of 243 amino acids.25 Additionally, the pig apoA-I protein sequence contains a well-conserved secondary structural motif of the amphipathic helices and is very homologous (79%) to human apoA-I.25 ApoA-I was denatured in a 6 M guanidine hydrochloride solution and dialyzed against Tris-buffered saline (TBS, 10 mM Tris, 150 mM NaCl, 1 mM EDTA, and 0.01 g/mL NaN3, pH 7.4). Sample Preparation. POPC discs containing 0.1 mol % RhoDOPE were prepared by the following cholate dialysis method.26 Briefly, POPC, apoA-I, and cholate were mixed to final concentrations of 1 mM, 12.8 µM, and 5 mM, respectively. The POPC/apoA-I molar ratio (78:1) in preparations corresponds to that of POPC discs reported previously.27 The mixtures were then incubated overnight and subsequently dialyzed three times against a 1000-fold excess of TBS for over 4 h to remove the cholate. The incubation and dialysis were performed at six different temperatures (34.0, 36.5, 37.5, 38.5, 40.0, and 41.5 °C). The POPC and apoA-I concentrations were determined using an enzymatic assay kit for choline (Wako, Osaka, Japan) and a BCA protein assay kit (Pierce, Rockford, IL), respectively. Large unilamellar vesicles (LUVs) of POPC were prepared by the extrusion method using a polycarbonate membrane with a pore size of 100 nm.28 Gel Filtration Chromatography. Samples produced by the cholate dialysis methods, which contained discs, as well as the remaining portions of the samples (POPC vesicles and apoA-I molecules), were loaded onto a Superdex 200 prep grade column (GE healthcare, Uppsala, Sweden) equipped with a JASCO FP6200 spectrofluorimeter (Tokyo, Japan). The elution profiles thus obtained were monitored at room temperature on the basis of the fluorescence of Rho-DOPE at excitation/emission wavelengths of 550/590 nm. The running buffer used was TBS, and the flow rate was 0.4 mL/min. The ratio of the area of the disc fraction to the total area (r) was then determined from the fluorescence intensity profile. The Stokes diameter of the disc was determined by comparing the Kav value given by the following equation with that of standard proteins with known diameters27

Kav ) (Ve - Vo)/(Vt - Vo)

(1)

where Vo, Vt, and Ve are the void volume, column volume, and elution volume, respectively. Thermodynamics of Disc Formation. Under the experimental conditions employed in this study, the POPC/apoA-I molar ratio of the prepared discs corresponds to that of the total samples. Therefore, the residual lipid/protein mixture should also have a similar ratio. It has been reported that discs with a POPC/apoA-I ratio of 78:1 contain two apoA-I molecules.29 Therefore, the equilibrium between the discs and the vesicle/ apoA-I mixtures is given by

µD/ + RT ln XD ) 2(µ/a + RT ln Xa) +

156 / (µ + RT ln Xv) n v (2)

where µ*, X, n, R, and T are the standard chemical potential, mole fraction, number of POPC molecules comprising the vesicle, gas law constant (8.31 J K-1 mol-1), and absolute temperature, respectively. Subscripts D, a, and v refer to the disc, apoA-I, and POPC vesicle, respectively. X is calculated by dividing the mole concentration by 55.5, which is the molar concentration of water. The change in the Gibbs standard free energy (∆*G) during the formation of a disc from 2 apoA-I molecules and 156/n vesicles is expressed as

(

∆/G ) µD/ - 2µ/a +

XD 156 / µv ) -RT ln 2 156/n n Xa Xv

)

(3)

The value of Xv156/n can be approximated to 1 because n is usually a very large number. XD and Xa are expressed as Xtr/2 and Xt(1 - r), respectively, using the total mole fraction of apoA-I (Xt ) 2.30 × 10-7) and the ratio of the disc area to the total area (r) in the gel filtration profile. Therefore, ∆*G can be expressed as

∆/G = -RT ln

Xtr/2 r ) -RT ln ) (Xt(1 - r))2 2Xt(1 - r)2 - RT ln K (4)

where K ) r/(2Xt(1 - r)2) is the equilibrium constant of disc formation. The standard enthalpy change (∆*H) and standard entropy change (∆*S) during disc formation were calculated as

∂ ln K [ ∂(1/T) ]

∆/H ) -R

∆/S )

P

∆/H - ∆/G T

(5)

(6)

Circular Dichroism. The circular dichroism (CD) spectra of apoA-I incorporated into discs, which were isolated by gel filtration chromatography, and of apoA-I in the presence and absence of POPC LUVs were recorded using a Jasco J-720 spectropolarimeter (Tokyo, Japan) at 37.0 °C. Samples were condensed or diluted with TBS to a final concentration of 2 µM apoA-I. The POPC/apoA-I molar ratio was maintained at 78:1 for the disc and vesicle/apoA-I mixture. The CD spectra of apoA-I in the presence of vesicles were monitored after preincubation at 37.0 °C for 1 h. The R-helix contents were estimated using the mean residue molar ellipticity at 222 nm ([θ]222, deg cm2 dmol-1) as follows: percent R-helix ) [(-[θ]222 + 3000)/(36000 + 3000)] × 100. Fluorescence Measurements. For the fluorescent experiments, a small amount of a methanol solution of n-AS was added to the discs or LUVs (POPC/n-AS ) 200:1 (mol/mol)). The fluorophores of n-AS incorporated into the bilayer would be located in the hydrocarbon region of the bilayers, depending on the position number, n.30,31 Thus, the larger the n value, the deeper the fluorophores are located in the hydrophobic core of the bilayer. All experiments were performed using samples of 300 µM POPC at 37.0 °C. The steady-state fluorescence anisotropy was measured on a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan). The mean fluorescence lifetime was measured on a Horiba NAES-550 ns fluorometer

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Figure 2. van’t Hoff plot of POPC/apoA-I disc formation from the mixture of vesicles and apoA-I molecules. Each point represents the means ( SD of three experiments.

TABLE 1: Thermodynamic Parameters of Disc Formation at 37.0 °C Figure 1. Gel filtration profiles of POPC/apoA-I discs containing 0.1 mol % Rho-DOPE prepared by the cholate dialysis methods at different temperatures. The profiles were monitored on the basis of the fluorescence of Rho-DOPE at room temperature. The elution volume was calibrated using a previously described method.

(Kyoto, Japan). The experimental procedures employed in this study have been described in detail elsewhere.28,32 Microsolubilization of LUVs by apoA-I. The spontaneous microsolubilization of LUVs by the addition of apoA-I was measured as a time-dependent decrease in right-angle light scattering at 37.0 °C. The reduction in light scattering comes from the transformation of LUVs (d ∼ 120 nm) to small discs (d ∼ 10 nm). LUVs and apoA-I were mixed to final concentrations of 1 mM and 12.8 µM, respectively, and the change in the right-angle light scattering intensity was monitored on an F-4500 spectrophotometer using excitation and emission wavelengths of 650 nm each. Results Temperature Dependence of Disc Formation. Discs prepared by the cholate dialysis method were separated from the remaining POPC vesicles by gel filtration chromatography. The discs were believed to be in equilibrium with the remaining POPC vesicles and apoA-I molecules because cholate was gradually removed by dialysis. When the elution profile was monitored on the basis of tryptophan fluorescence (apoA-I), the peak of the disc fraction overlapped with that of lipid-free apoAI, which was eluted at Kav ) 0.53 (data not shown). Therefore, the profile was monitored on the basis of the fluorescence of Rho-DOPE (Figure 1). The size and POPC/apoA-I molar ratio of the discs obtained were ∼9.3 nm and 78:1, respectively, and these ratios were obtained regardless of the preparation temperatures, an observation that is consistent with the findings of a previous study.27 The POPC:apoA-I molar ratio of the disc fraction agreed with that of the total sample, indicating that the remaining POPC/apoA-I mixture also has a similar ratio. The peak corresponding to the remaining POPC vesicles was observed at the void volume, and its intensity increased with the preparation temperature, suggesting that disc formation was suppressed at higher temperatures. The fluorescence efficiency

disc formationb helix formationc factors other than helix formation

∆*G (kJ/mola)

∆*H (kJ/mola)

T∆*S (kJ/mola)

-52 ( 0.2 -68 16

-620 ( 8 -420 -200

-570 ( 8 -350 -220

a Per mol of disc. b Given by eqs 4-6 and represented as the means ( SD from three experiments. c ∆*Hhelix,residue ) -3.0 (kJ/ mol) per residue and ∆*Shelix,residue ) -8.1 (J/mol K) per residue.35

of Rho-DOPE incorporated in the discs was nearly equal to that of the vesicles (data not shown); therefore, the ratio of the areas of the disc and vesicle fractions can be assumed to coincide with the ratio of the POPC concentrations involved in these fractions. The equilibrium constant (K) of disc formation was calculated from the value of r, the ratio of the disc area to the total area. The van’t Hoff plot showed a linear relationship between 1/T and ln K over the temperature range examined (Figure 2). The calculated values of ∆*G, ∆*H, and T∆*S during disc formation at 37.0 °C are listed in Table 1. The value of ∆*G is significantly negative, indicating that the disc formation process is thermodynamically favorable. Both ∆*H and T∆*S are negative, indicating that disc formation is an enthalpy-driven process. The reproducibility of the gel filtration profiles suggests that equilibrium between discoidal complexes and vesicle/apoA-I mixtures was achieved by the cholate dialysis method. Conformational Change of ApoA-I. To evaluate the contribution of the conformational change of apoA-I to the thermodynamic stability of the discs, we used CD spectroscopy to analyze the secondary structures of apoA-I in discs in the presence of vesicles and in the lipid-free form (Figure 3). The CD spectra of the secondary structures of apoA-I represented the typical pattern of an R-helix. The R-helical contents of apoA-I in discs and in the lipid-free form were c ∼84 and 53%, respectively, which were slightly higher than the reported values of 78% for discs and 46% for apoA-I in the lipid-free form. However, the difference in the helicity (31%) was consistent with the reported value of 32%,33 presumably because of small differences in the experimental conditions. The R-helical content of apoA-I in the presence of vesicles (55%) was slightly larger than that in the lipid-free form. This result suggests that apoA-I

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Figure 3. CD spectra of POPC/apoA-I disc, apoA-I in the presence of POPC LUVs, and lipid-free apoA-I at 37.0 °C at an apoA-I concentration of 2 µM. The POPC/apoA-I molar ratio was 78:1 for the disc and vesicle/apoA-I mixture.

is not strongly bound to LUVs and is in agreement with the results of a previous report.34 The increase in helicity during disc formation from vesicle/apoA-I mixtures was found to be ∼29%. It is generally accepted that the formation of helices is driven by a decrease in enthalpy (∆*Hhelix,residue ) -3.0 kJ/mol per residue at 30 °C) but opposed by a decrease in entropy (∆*Shelix,residue) -8.1 J/(mol K) per residue at 30 °C).35 The thermodynamic parameters for the formation of helices in apoA-I at 37 °C were calculated from the increase in the helicity (29%), assuming that ∆*Hhelix,residue and ∆*Shelix,residue are independent of temperature, and are listed in Table 1. The large decrease in enthalpy that occurred during disc formation was found to be primarily attributed (68%) to the formation of helices in apoA-I. This result indicates that the thermodynamic stability of the disc relies on the formation of helices in apoA-I. Contributions from processes other than the formation of helices in apoA-I represent the positive value of ∆*G with the decrease in entropy that exceeds the decrease in enthalpy, raising the possibility that lipids may possess a lower entropy in discs than in vesicles. Fluorescence Anisotropy and Fluorescence Lifetime of n-AS. To compare the properties of POPC in discs to those in LUVs, the fluorescence anisotropy and fluorescence lifetime were measured. The steady-state fluorescence anisotropy of n-AS (2-, 6-, and 12-AS) for discs and LUVs was measured to evaluate the fluidity of the membranes (Figure 4A). In both particles, the fluorescence anisotropy decreased with an increase in the n number (indicating a deeper location of the fluorophore), which suggests increased fluidity at the bilayer center when compared with the membrane interface. Each of the fluorescent probes clearly showed a much higher anisotropy in discs than in LUVs. These findings reflected the higher acyl chain order in discs and could probably be attributed to the closer lipid packing or the ordering effect of the apoA-I on the “boundary lipids” that are in direct contact with apoA-I and are essentially immobilized. The difference in the anisotropy values between discs and LUVs was larger for probes with a smaller n, indicating a significantly closer packing at shallower regions of the disc membranes. Figure 4B shows the mean fluorescence lifetime of n-AS for discs and LUVs. The fluorescence lifetime of the fluorophores is sensitive to their surrounding polarity and can be a measure of the penetration of water.32,36,37 The lifetime of n-AS for discs and LUVs increased with n because the fluorophores were located deep in the hydrophobic core. Each

Figure 4. Steady-state anisotropy (A) and mean fluorescence lifetime (B) of n-AS (n ) 2, 6, and 12) in POPC LUVs and POPC/apoA-I discs at 37.0 °C. Each point represents the means ( SD of three experiments.

of the fluorescent probes showed a longer lifetime in discs than in LUVs, indicating that there was reduced access of water to the acyl chain regions of the discs due to the closer packing. The deviation of the lifetime for discs from that for LUVs was significant in the case of probes located at shallower regions, which supports the concept of closer packing in the shallower regions of the membranes. Spontaneous Disc Formation from LUVs by apoA-I. Because a negative ∆*G during disc formation indicates that the disc is thermodynamically more stable than the mixture of POPC vesicles and apoA-I molecules, we conducted additional experiments to determine if the disc could be spontaneously produced from LUV. Figure 5 shows the time-dependent decrease in the turbidity of LUVs in response to the addition of apoA-I, representing the spontaneous formation of discs. However, the rate of disc formation was very slow (only a 15% decrease was observed over 4 days). This result suggests that the activation barrier of disc formation is markedly high and that the mixtures of POPC vesicles and apoA-I molecules are metastable. Discussion Thermodynamics of Disc Formation. In this study, we demonstrated that the POPC/apoA-I disc is thermodynamically more stable than the mixture of POPC vesicles and apoA-I molecules due to the large decrease in enthalpy that exceeds the decrease in entropy during disc formation. The entropically unfavorable property of discs suggests that they are destabilized as the temperature is increased, which is in agreement with the results of a previous study in which the disc structure was found to be disrupted at temperatures above ∼80 °C.38 The large decrease in enthalpy that occurs during disc formation is

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Figure 5. Reduction in light-scattering intensity of POPC LUVs by apoA-I at 37.0 °C. The POPC and apoA-I concentrations were 1 mM and 12.8 µM, respectively (POPC/apoA-I ) 78/1 (mol/mol)).

attributed primarily to the formation of helices in apoA-I, which presumably occurs in the C-terminal domain. Lipid-free apoA-I consists of two amphipathic helical domains: (1) a four-helix antiparallel bundle formed by the N-terminal and 75% of apoA-I and (2) a two-helix bundle adopted by the C-terminal quarter of the molecule,39 which exists as a discrete, less organized structure.34 Many studies have reported that deletions in the C-terminal domain lead to a marked reduction in the binding affinity of apoA-I to model membranes40-43 and in the lipid efflux from cells by apoA-I via ABCA1,44-48 indicating that the formation of helices in the C-terminal domain is the driving force behind disc formation. Conversely, factors other than the formation of helices in apoA-I that contribute to disc formation lead to the positive value of ∆*G that occurs with the unfavorable entropy and favorable enthalpy changes, and these factors most probably originate from the closer packing of lipids enclosed in discs and the hydrophobic hydration at the bilayer edge.27,49 We previously reported that the phospholipid headgroups at the lipid-protein boundary of the bilayer edge are more exposed to water, leading to a decrease in the fluorescence lifetime of a fluorophore located at the phospholipid headgroups.27 Fluorescence measurements suggested that lipids in discs are entropically less favorable due to the closer packing (but enthalpically more favorable due to the enhanced van der Waals interactions). We previously reported that the molecular area of dimyristoylphosphatidylcholine (DMPC) in discs (50 Å2) is much smaller than that in vesicles (65.7 Å2) and that it is equal to the molecular area of DMPC dihydrate, corresponding to the smallest possible area.49 These entropic constraints have been shown to reduce the energy barrier of the desorption of DMPC from membranes, leading to faster (more than 20-fold) interparticle transfer of DMPC in discs than in vesicles.49 Furthermore, Shaw et al. reported that the phase transition temperature of DMPC and dipalmitoylphosphatidylcholine (DPPC) was shifted 3-4 °C higher when DMPC and DPPC were incorporated into discs, representing the higher rigidity of lipids in discs.38 These results suggest that lipids in discs are more closely packed by apoA-I molecules surrounding the bilayer and are thus entropically destabilized; this consideration correlates well with the results of the present study. The closer packing of lipids in discs may be necessary to repress the packing defects and stabilize the bilayers. In the case of vesicles, lipids diffuse cooperatively so that the packing defects are not produced. In

Fukuda et al. the case of discs, however, the “boundary lipids” that are adjacent to and interacting with apoA-I cannot move freely;38 therefore, the packing defects would be generated by the fluctuation of “core lipids” not associated with apoA-I unless the lipids in the disc were closely packed. The metastability of the mixture of POPC vesicles and apoA-I molecules is in reasonable agreement with the fact that discs cannot be formed spontaneously in vivo without ABCA1 activity. The high activation barrier of disc formation suggests that the activation barrier of disc degradation is also high; this is compatible with recent reports that the denaturation of HDL involves high free energy barriers (e.g., ∆G‡ ) 17 kcal/apoA-I mol (71 kJ/apoA-I mol)) that decelerate protein unfolding and confer particle stability.50,51 Implication of ABCA1-Mediated Disc Formation. The thermodynamic stability of discs raises the possibility that ABCA1 may assemble disc particles by lowering the activation energy of disc formation, not by directly transporting individual lipids to apoA-I via energy-dependent processes. The large contribution of the formation of helices in apoA-I to the thermodynamic stability of the discs suggests that the activation barrier of disc formation occurs as a result of the deep insertion of apoA-I into the membranes, where apoA-I can increase its helicity but must expose the hydrophilic groups of the amphipathic R-helices to the apolar environment of the acyl chain regions. It is known that ABCA1-mediated lipid efflux is not specific to apoA-I and that other apolipoproteins and synthetic peptides with amphipathic helices can also efflux lipids from cells,52-54 indicating that the physicochemical properties, but not the biochemical properties, of the amphipathic helical structure are a prerequisite for disc formation. The affinity of these amphipathic helical proteins for model membranes is known to be positively correlated with their ability to remove cellular lipids.45,55 The amount of apoA-I binding to cellular membranes has been reported to be about 10-fold greater than that of apoA-I specifically binding to ABCA1, suggesting that apoA-I/lipid interactions take place during the assembly of disc particles, rather than direct apoA-I/ABCA1 interactions.56 Furthermore, many studies conducted using artificial membranes have reported that apoA-I has a higher affinity to membranes with higher curvatures due to the greater space between the phospholipid head groups into which the amphipathic R-helices of apoA-I can penetrate.34,55 It has also been shown that phaseseparating membranes can be spontaneously transformed into discs in response to the addition of apoA-I due to lattice defects that occur at the phase boundary regions.28,57,58 Taken together, these results raise the possibility that ABCA1 triggers spontaneous disc formation by changing the local environment around ABCA1 and creating packing defects, where apoA-I can deeply insert its amphipathic R-helices and extract lipids. Indeed, several studies have reported changes in the membrane environment by ABCA1. For example, Landry et al. reported that ABCA1 expression results in a significant redistribution of sphingomyelin and cholesterol from rafts to nonrafts, suggesting that ABCA1 is capable of reorganizing the nonraft microdomains on the plasma membrane and that the expansion of the nonraft microdomains plays a critical role in apoA-I-mediated lipid efflux.59 In addition, Vedhachalam et al. reported that ABCA1 lipid translocase activity creates exovesiculated lipid domains that have high curvatures, which could facilitate apoA-I binding and allow spontaneous solubilization

Thermodynamic Stability of Discoidal HDL

Figure 6. Schematic diagram of the Gibbs free energy of the POPC/ apoA-I disc and the mixture of vesicles and apoA-I molecules. The disc is thermodynamically stabilized due to the decrease in enthalpy in response to the formation of helices in apoA-I, which can compensate for the unfavorable entropy of lipids closely packed by apoA-I. The activation barrier of disc formation is high, as shown in Figure 5; therefore, the mixtures of POPC vesicles and apoA-I molecules are metastable.

to create disc particles.55 However, further studies are needed to clarify the molecular mechanism governing disc formation by ABCA1. Conclusions We compared the thermodynamic stability of POPC/apoA-I discs to that of a mixture of POPC vesicles and apoA-I molecules. The discs are found to be thermodynamically more stable than the mixtures on account of the decrease in the enthalpy of the formation of helices in apoA-I, which can compensate for the unfavorable entropy associated with lipids being closely packed by apoA-I (Figure 6). The activation barrier of disc formation is high; therefore, the mixtures of POPC vesicles and apoA-I molecules remain metastable. These results suggest that ABCA1 lowers the activation energy of disc formation by creating microdomains or protruded membrane structures. Acknowledgment. This study was supported by a Grant-inaid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (No. 22018015) and Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists (No. 202796). References and Notes (1) Fielding, C.; Fielding, P. J. Lipid Res. 1995, 36, 211. (2) Miller, G.; Miller, N. Lancet 1975, 1, 16. (3) Brooks-Wilson, A.; Marcil, M.; Clee, S.; Zhang, L.; Roomp, K.; van Dam, M.; Yu, L.; Brewer, C.; Collins, J.; Molhuizen, H.; Loubser, O.; Ouelette, B.; Fichter, K.; Ashbourne-Excoffon, K.; Sensen, C.; Scherer, S.; Mott, S.; Denis, M.; Martindale, D.; Frohlich, J.; Morgan, K.; Koop, B.; Pimstone, S.; Kastelein, J.; Genest, J. J.; Hayden, M. Nat. Genet. 1999, 22, 336. (4) Lee, J.; Parks, J. Curr. Opin. Lipidol. 2005, 16, 19. (5) Zannis, V.; Chroni, A.; Krieger, M. J. Mol. Med. 2006, 84, 276. (6) Borhani, D.; Rogers, D.; Engler, J.; Brouillette, C. Proc Natl. Acad. Sci. U.S.A. 1997, 94, 12291. (7) Wu, Z.; Wagner, M.; Zheng, L.; Parks, J.; Shy, J. R.; Smith, J.; Gogonea, V.; Hazen, S. Nat. Struct. Mol. Biol. 2007, 14, 861. (8) Gianazza, E.; Eberini, I.; Sirtori, C.; Franceschini, G.; Calabresi, L. Biochem. J. 2002, 366, 245. (9) Tricerri, M.; Sanchez, S.; Arnulphi, C.; Durbin, D.; Gratton, E.; Jonas, A. J. Lipid Res. 2002, 43, 187. (10) Maiorano, J.; Jandacek, R.; Horace, E.; Davidson, W. Biochemistry 2004, 43, 11717. (11) Li, L.; Chen, J.; Mishra, V.; Kurtz, J.; Cao, D.; Klon, A.; Harvey, S.; Anantharamaiah, G.; Segrest, J. J. Mol. Biol. 2004, 343, 1293.

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