Phospholipid Monolayers Provide Insights into ApoA-I Membrane Insertion

Phospholipid Monolayers Provide Insights into ApoA-I Membrane Insertion

J. Mol. Biol. (2011) 410, 60–76 doi:10.1016/j.jmb.2011.04.006 Contents lists available at www.sciencedirect.com Journal of Molecular Biology j o u r...

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J. Mol. Biol. (2011) 410, 60–76

doi:10.1016/j.jmb.2011.04.006 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Fluid and Condensed ApoA-I/Phospholipid Monolayers Provide Insights into ApoA-I Membrane Insertion Lionel Chièze 2 , Victor Martin Bolanos-Garcia 1 , Mathieu Pinot 2 , Bernard Desbat 3 , Anne Renault 2 , Sylvie Beaufils 2 ⁎ and Véronique Vié 2 ⁎ 1

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK Institut de Physique de Rennes, UMR-CNRS 6251 Université de Rennes 1, Campus de Beaulieu, 35042 Rennes cedex, France 3 CBMN (UMR5248 CNRS) Université Bordeaux1, ENITAB, 2 rue Robert Escarpit, 33607 Pessac, France 2

Received 26 February 2011; received in revised form 30 March 2011; accepted 2 April 2011 Available online 12 April 2011 Edited by J. Bowie Keywords: apolipoproteins; Langmuir; AFM; ellipsometry; PM-IRRAS

Apolipoprotein A-I (ApoA-I) is a protein implicated in the solubilization of lipids and cholesterol from cellular membranes. The study of ApoA-I in phospholipid (PL) monolayers brings relevant information about ApoA-I/PL interactions. We investigated the influence of PL charge and acyl chain organization on the interaction with ApoA-I using dipalmitoylphosphatidylcholine, dioleoyl-phosphatidylcholine and dipalmitoylphosphatidylglycerol monolayers coupled to ellipsometric, surface pressure, atomic force microscopy and infrared (polarization modulation infrared reflection–absorption spectroscopy) measurements. We show that monolayer compressibility is the major factor controlling protein insertion into PL monolayers and show evidence of the requirement of a minimal distance between lipid headgroups for insertion to occur, Moreover, we demonstrate that ApoA-I inserts deepest at the highest compressibility of the protein monolayer and that the presence of an anionic headgroup increases the amount of protein inserted in the PL monolayer and prevents the steric constrains imposed by the spacing of the headgroup. We also defined the geometry of protein clusters into the lipid monolayer by atomic force microscopy and show evidence of the geometry dependence upon the lipid charge and the distance between headgroups. Finally, we show that ApoA-I helices have a specific orientation when associated to form clusters and that this is influenced by the character of PL charges. Taken together, our results suggest that the interaction of ApoA-I with the cellular membrane may be driven by a mechanism that resembles that of antimicrobial peptide/lipid interaction. © 2011 Elsevier Ltd. All rights reserved.

*Corresponding authors. E-mail addresses: [email protected]; [email protected]. Abbreviations used: ApoA-I, apolipoprotein A-I; DPPC, dipalmitoyl-phosphatidylcholine; DPPG, dipalmitoylphosphatidylglycerol; DOPC, dioleoyl-phosphatidylcholine; LE, liquid expanded; LC, liquid condensed; PM-IRRAS, polarization modulation infrared reflection–absorption spectroscopy; AFM, atomic force microscopy; HDL, high-density lipoprotein; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PC, phosphatidylcholine; PG, phosphatidylglycerol; TBS, Tris-buffered saline; PL, phospholipid. 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

Insights into ApoA-I Membrane Insertion

Introduction Apolipoprotein A-I (ApoA-I) (243 amino acids, 28 kDa) belongs to the class of exchangeable apolipoproteins, a protein family characterized by their ability to move between lipoprotein particles.1–4 ApoA-I and the rest of exchangeable apolipoproteins (i.e., ApoA-II, ApoE and the apoC family: ApoC-I, ApoC-II and ApoC-III) are characterized by the presence of helical repeats of amphipathic nature. Even though all exchangeable apolipoproteins show a common rapid adsorption and form stable monolayers at interfaces, important differences in their interfacial behavior exist.5,6 Human ApoA-I is the major protein constituent of high-density lipoprotein (HDL) particles, which play a central role in the early steps of cholesterol efflux and reverse cholesterol transport. 7 Free ApoA-I takes lipids from the cell membrane to form discoidal HDL particles in a process that is facilitated by the delivery of phospholipids (PLs) from the cell membrane by the cell membrane transporter ABCA1. Discoidal HDL particles are efficient acceptors of unesterified cholesterol and are the preferred substrates of plasma glycoprotein lecithin–cholesterol acyltransferase, which catalyses the transfer of an acyl group from phosphatidylcholine (PC) from the surface particle to cholesterol, producing esterified cholesterol. Discoidal HDL reorganizes in particles called small α-migrating, spherical HDL. Lecithin–cholesterol acyltransferase and other PL transfer proteins subsequently remodel the small α-migrating, spherical HDL particles to form larger α-migrating spheres. These spherical particles contain a higher amount of ApoA-I and are the major form of HDL circulating in normal human plasma. α-migrating, spherical HDL particles are subsequently remodeled by cholesteryl ester transfer protein, which transfers esterified cholesterol toward lipoproteins of lower density. In this manner, lipid-poor ApoA-I can be released from HDL particles before they are recycled by the liver to generate new HDL particles. In contrast to ApoE, which shows an ApoA-I-like four-helix bundle organization and for which a receptor has been identified, to date, there is no evidence of the molecular interaction between ApoA-I and a membrane receptor. Nevertheless, the observation that different lipidic domains of the plasma membrane are important for global lipid efflux, that there is a preferential interaction of ApoA-I with loosely packed domains of low cholesterol content and that lipid-free ApoA-I is present in plasma and interstitial fluid8–11 prompted us to investigate the behavior of ApoA-I when exposed to membrane PLs. Studies on model vesicles (small unilamellar vesicles-large unilamellar vesicles) can bring relevant information on the dynamics of protein–lipid

61 interactions and the heterogeneity of the lipid bilayer with regard to temperature.2,12–15 Indeed, vesicle systems mimic the cell bilayer, including its curvature, although in an extremely simplified form. However, the study of vesicle systems by optical methods only brings information at the micrometer scale and makes it difficult to produce vesicle surfaces at the desired surface pressure. One popular, alternative method to characterize protein–lipid interactions consists in the study of proteins into lipidic monolayers prepared with a Langmuir trough. The isotherm of each lipid (i.e., the evolution of surface pressure π with respect to the molecular area) is performed with a movablebarrier Langmuir trough, which allows preparation of lipid monolayers at desired surface pressures by compressing initially spread molecules. It also allows the study of monolayers in several states including homogeneous fluid, condensed phases and fluid and condensed coexisting phases. In addition to surface pressure measurements, null ellipsometry can be used to determine the amount of protein adsorbed at the interface, while interfacial spectrometry [polarization modulation infrared reflection–absorption spectroscopy (PM-IRRAS)] allows the study of secondary structure changes after adsorption into the lipidic layer. Langmuir trough has the additional advantage of permitting the transference of monolayers to mica sheets for atomic force microscopy (AFM) studies. AFM can be used to obtain a topographic picture of the layer at a resolution of several tenths of nanometer16,17 on which statistical image analysis can be performed.18 We centered our study on the interaction of ApoAI with zwitterionic PL [dipalmitoyl-phosphatidylcholine (DPPC) and dioleoyl-phosphatidylcholine (DOPC)] and anionic PL [dipalmitoyl-phosphatidylglycerol (DPPG)]19,20 prepared in various physical states (from homogeneous fluid or condensed phase to states with fluid/condensed phase coexistence) with Langmuir trough. We characterized the exclusion pressure and the organization of ApoA-I in different lipid monolayers. ApoA-I organization was studied by means of spatial distribution and geometry analyses of ApoA-I clusters in the lipidic domains, within a resolution of several tenth nanometers, as well as the orientation/conformation of the protein inside the clusters.

Results ApoA-I is inserted in PL monolayers For experiments at the liquid/lipid interface, ApoA-I was injected under lipid monolayers prepared at different surface pressures as described in Materials and Methods. We refer to them as the

62 initial surface pressures, πinitial. The kinetics of protein adsorption at the interface were determined by recording the increase in ellipsometric angle δΔ and surface pressure Δπ produced by the protein at the liquid/lipid interface. Figure 1 shows the adsorption kinetics under a DPPG monolayer at πinitial = 20 mN/m. The surface pressure increase, Δπ (5 mN/m), is significant and reveals that ApoA-I inserts part of its residues in the PL monolayer.21–24 The insertion of ApoA-I in the lipidic layer is confirmed by the increase in the ellipsometric

Insights into ApoA-I Membrane Insertion

angle, which is much smaller than in the case where ApoA-I forms a continuous layer at the liquid/air interface (δΔ = 2° against Δ = 6.5°, Fig. 1). The same trend is noticed with DPPC, DOPC and DPPG for πinitial, which ranged between 8 mN/m and 30 mN/m. Figure 2 shows that, as expected,22 Δπ and δΔ decrease when πinitial increases. This behavior is attributed to the limited insertion of ApoA-I residues between polar headgroups when the lipid compactness increases (the lipid molecular area decreases since the πinitial increases). Evidence of preferential interactions with PLs

Fig. 1. Kinetics of adsorption of ApoA-I injected below a lipidic layer of DPPG. The lipidic layer is prepared by spreading the lipids at the interface and compressing the molecules to reach a surface pressure of 20 mN/m (πinitial). Top: the increase in surface pressure (Δπ = 5 mN/m) shows the presence of ApoA-I at the liquid/lipid interface and the insertion of this protein in the lipid layer (the dotted line shows initial pressure). Bottom: the increase in the ellipsometric angle after protein injection (δΔ = 2°) is much lower than that of the ellipsometric angle of the protein alone at the liquid/air interface (in the inset) (Δliquid/air = 6.5°). The dotted line shows the initial ellipsometric angle Δ. This behavior confirms the insertion of ApoA-I in the lipid layer. The arrow indicates the injection of the protein in the subphase. Inset: ellipsometric angle at the liquid/air interface after protein injection (Δliquid/ air = 6.5°).

The graph of Δπ versus initial surface pressure (πinitial) (Fig. 2) allows the extraction of the exclusion pressure πe, which is defined as the maximal πinitial of the lipid over which Δπ is null. πe values are 39 mN/m, 31 mN/m and 27 mN/m for DPPG, DOPC and DPPC, respectively. The exclusion pressure πe⁎ extracted from ellipsometric measurements confirms the relative high affinity of ApoA-I for these lipids and suggests that the interaction of ApoA-I with the negatively charged DPPG is more effective than it is with zwitterionic DPPC or DOPC. Although we observed similar δΔ and Δπ values between DOPC and DPPC for a large range of πinitial, the values of πe and πe⁎ revealed a more effective interaction of ApoA-I for DOPC than DPPC at high surface pressure. For surface pressures higher than 20 mN/m, DPPG and DPPC are totally in a liquid-condensed (LC) phase, while DOPC is in a liquid-expanded (LE) phase.25,26 Hence, the differences between DPPG and DPPC allowed us to study the effect of the lipid charge on the interaction with ApoA-I, while the differences between DOPC and DPPC enable us to study the effect of the phase on such interaction. AFM of mixed layers of ApoA-I/PLs at πinitial = 30 mN/m confirmed the behavior noticed at macroscopic scale by ellipsometry and surface pressure measurements. AFM images of these layers are shown on Fig. 3. Over these images, the background is a regular bright gray, thus indicating that all the monolayers are in a single phase. We can see on these images luminous spots, which are ApoA-I clusters (perhaps melted with lipids) of nanometer dimension and much larger than lipid clusters. The heights of the largest protein clusters are approximately 10 nm with a surface of approximately 0.015 μm 2 . We measured the surface occupied by ApoA-I in the lipid monolayer (to which we will refer to as S) in order to establish whether the lipid exhibits any difference in their affinity for ApoA-I. AFM images of ApoA-I/PL monolayers at πinitial = 30 mN/m revealed S values of 0.54% for

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Fig. 2. Increase in ellipsometric angle δΔ (right) and surface pressure Δπ (left) as a function of the initial surface pressure (πinitial) of the lipidic layer of DPPC (○), DOPC (♦) and DPPG (●) recorded after injection of ApoA-I to the subphase (final concentration, 1.5 μg/ml). Arrows show exclusion pressures πe (from surface pressure measurements) and πe⁎ (from ellipsometric measurements) for DPPC (black continuous arrow), DOPC (gray continuous arrow) and DPPG (black dotted arrow) monolayers. Values for πe and πe⁎ are greater for DPPG than for other lipids and greater for DOPC than DPPC. These results show that ApoA-I has a more effective interaction with DPPG than with other lipids and, to a lesser extent, with DOPC than DPPC. The precision is 1 mN/m for surface pressure measurements and 0.5° for ellipsometric measurements.

DPPG (anionic PLs in the LC phase) and 0.22% for DOPC (zwitterionic PLs in the LE phase) (see Table 1). The mean values of S of ApoA-I/DPPG

and ApoA-I/DOPC monolayers are considered significantly different (P b 0.05, see Table 1 for error bars). In the case of DPPC (zwitterionic PLs

Fig. 3. On the top: AFM images of mixed monolayers of ApoA-I/PLs where the initial surface pressure (πinitial) was ca 30 mN/m (field of view: 8 × 8 μm2 and z-range = 10 nm): DPPC with ApoA-I (a), DOPC with ApoA-I (b) and DPPG with ApoA-I (c). On the bottom: the same images after thresholding. Clusters are shown in red. For all lipids, phases and initial surface pressures, ApoA-I forms clusters higher than those of lipids.

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Table 1. Relative surfaces occupied by mixed ApoA-I/PL monolayers, pure PL monolayers and pure ApoA-I Lipid monolayers (πinitial) DPPC (30 mN/m) DOPC (30 mN/m) DPPG (30 mN/m) DOPC (30 mN/m) gray level 149

Relative surface occupied in mixed monolayers (%)

Residual surface in PL monolayers (%)

Relative surface occupied by protein (S) (%)

0.10 ± 0.04 0.32 ± 0.09 0.79 ± 0.34 0.19 ± 0.08

0.10 ± 0.02 0.1 ± 0.07 0.25 ± 0.24 0.08 ± 0.07

b0.05 0.22 ± 0.12 0.54 ± 0.42 0.11 ± 0.11

Each measurement (mean and standard deviation) was performed over at least eight AFM images and two samples of mixed monolayers.

in the LC phase), S is equal to the residual surface (measured in pure lipid monolayers, see Materials and Methods). Thus, we concluded that no protein clusters are present in DPPC at πinitial = 30 mN/m and that there is a grading of S at πinitial = 30 mN/m, as follows: SDPPG N SDOPC N SDPPC. This grading is in good agreement with that from exclusion pressure (πe) data, as follows: πDPPG N πDOPC N πDPPC. Hence, ellipsometric, surface pressure and AFM measurements consistently showed that ApoA-I has a higher affinity for DPPG in the LC phase than for zwitterionic lipids, regardless of the phase of the latter lipids. We also noticed that, for zwitterionic lipid monolayers in the LE phase (i.e., DOPC), protein–lipid interactions exist at higher πinitial than in the case of lipid monolayers in the LC phase (i.e., DPPC). This preference of ApoA-I for the LE phase of DOPC in comparison to DPPC in the LC phase could be due to the difference in the lateral compressibility of the lipid monolayers, the difference in the polar headgroup spacing or conformational changes in the acyl chains of both lipids. However, for each πinitial ranging between 10 mN/m and 20 mN/m, ApoA-I induces similar Δπ in DPPC and DOPC. This behavior shows that Δπ in the zwitterionic lipids DPPC and DOPC is not dependent upon the length and structure of the acyl chains. Hence, the phase preference observed in zwitterionic lipids cannot be explained by the acyl chain state difference between DPPC and DOPC. It shows that, as in the case of the lipopeptide surfactine,27 protein insertion is not due to a matching of the hydrophobic part of the protein with the lipid chain. Next, we used AFM to study mixed layers of ApoA-I/PLs at πinitial = 15 mN/m. At this surface pressure, DPPG and DPPC show LE/LC phase coexistence with LC phase domains of micrometer scale, while DOPC is in a single phase (LE). These results prompted us to investigate whether the effects of charge and phase on protein distribution are similar to those of single-phase monolayers. Figure 4 shows AFM images of ApoA-I/PL layers for πinitial = 15 mN/m. In AFM images of ApoA-I/DPPC or ApoA-I/DPPG monolayers, wide bright-gray domains represent the LC phase, whereas wide dark-gray domains represent the LE phase (since LE domains are higher than

LC domains by 1 nm). For AFM images of ApoAI/DOPC, the background appears as a regular bright gray, thus confirming that the monolayer corresponds to a single LE phase. As mentioned before, ApoA-I clusters can be seen as homogeneous luminous spots. The cluster repartition seems to be more heterogeneous for the ApoA-I/ DPPC layer than for the other ApoA-I/PL systems. This heterogeneity can be attributed to the preferential insertion of ApoA-I in LE phase domains compared to LC phase domains. We examined in detail the influence of phase composition over ApoA-I insertion in DPPC at πinitial = 15 mN/m. Since the relative surface occupied, S, is height dependant, we measured the number of protein clusters by surface unit N/s instead of S. The N/s value is 40 times higher for the LE phase than for the LC phase of DPPC (17.6 μm− 2 and 0.4 μm− 2, respectively). Hence, we observed that, in the case of adjacent micrometersized domains, a higher protein insertion in the LE phase exists in comparison to the LC phase. A plausible explanation on this trend is that the space between polar headgroups is lower in the LC phase than it is in the LE phase, which limits the insertion of ApoA-I residues in the former phase. Next, we compared ApoA-I insertion in the LE phase in a single-phase monolayer against monolayers where LE/LC coexists. For this, we analyzed the N/s value in the LE phase of DPPC and DOPC at the same πinitial of 15 mN/m. N/s values are 17.6 μm− 2 for the LE phase in DPPC and 5.4 μm− 2 for the LE phase in DOPC (see Table 2). This result shows that ApoA-I insertion in the LE phase seems to be facilitated by the higher compressibility of DPPC when DPPC is in the LE/LC phase transition. For πinitial higher than 20 mN/m, DPPC is in the single LC phase. In this case, ApoA-I insertion is more difficult to achieve than at lower πinitial. This feature might be due to the fact that the space between polar headgroups is lower in the LC phase than it is in the LE phase and/or the fact that lipid condensation dramatically reduces the space available at the interface. Therefore, we suggest that the preference of ApoA-I for the LE phase over the LC phase, which leads to a different exclusion pressure

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Fig. 4. On the top: AFM images of mixed monolayers of ApoA-I/PLs, where the initial surface pressure (πinitial) was ca 15 mN/m (field of view: 8 × 8 μm2 and z-range = 10 nm): DPPC with ApoA-I (a), DOPC with ApoA-I (b) and DPPG with ApoA-I (c). On the bottom: the same images after thresholding. For DPPC, the strong difference between the number of clusters in the LC phase (colored red) and the number of clusters in the LE phase (marked by white crosses) reveals the different ApoA-I affinities of LE and LC phases. For DOPC and DPPC, clusters are colored red for both phases.

(πe) between DPPC and DOPC, results in the spacing of the polar headgroups. In the case of DPPG monolayers we noticed that the influence of lipid phase is marginal in comparison to DPPC monolayers because higher amounts of ApoA-I are inserted in the LC phase of anionic lipids compared to the LC phase of zwitterionic lipid. The data are in good agreement with the stronger effect of charge versus phase determined by ellipsometry, surface pressure measurements and the relative surface occupied by proteins at πinitial = 30 mN/m.

Table 2. Numbers of clusters by surface unit, N/s, for DOPC (LE phase) and for LE and LC phases of DPPC Percent of Mean number image occupied of protein Lipid monolayers by the clusters (πinitial) phase (%) per image LC in DPPC (15 mN/m) LE in DPPC (15 mN/m) DOPC (15 mN/m)

Number of clusters per surface unit: N/s (μm− 2)

88

22

0.4

12

135

17.6

100

344

5.4

Details of the calculations are shown in Materials and Methods.

The lowering of polar headgroup spacing is responsible for the straightening of ApoA-I α helices PM-IRRAS measurements were performed to investigate the conformation of ApoA-I at the liquid/air interface or liquid/lipid interface in the presence of zwitterionic lipids (i.e., DPPC and DOPC). Figure 5 shows PM-IRRAS spectra recorded during the adsorption kinetics of ApoA-I at the liquid/air interface at the beginning of the kinetics (22 min after protein injection), at the beginning of the plateau of the surface pressure (71 min) and at the end of the kinetics when both π and Δ are stable. All spectra were normalized by the spectrum of the liquid/air interface without protein in the subphase, which was performed before protein injection. The normalized PM-IRRAS spectra of ApoA-I at the liquid/air interface are shown in the 1900- to 1300cm− 1 spectral range. We focused on the amide I band located between 1700 and 1600 cm− 1 and on the amide II band located between 1600 and 1500 cm− 1. Indeed, the frequencies of the maximum of signal intensity (peak) of these bands can be correlated with a particular protein conformation.28 On the three spectra shown in Fig. 5, the amide I peak is located at 1655.6 cm− 1, and the amide II peak, at 1542.8 cm− 1.

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Fig. 5. Normalized PM-IRRAS spectra of ApoA-I monolayer at the liquid/air interface: 22 min (gray line), 71 min (dark line) and 363 min. (dark dotted line) after the start of the kinetics. The positions of amid I and amid II bands peaks are, respectively, around 1655.6 cm− 1 and 1542.8 cm− 1, which reveal a predominantly α helix conformation. The AMIDI/AMIDII ratio (amide I band peak intensity over amide II band peak intensity) is around 6 when the kinetics was started since 71 min and is around 3 at the end of the kinetics (363 min). This trend shows straightening of α helices due to protein–protein interactions. Inset: measurements of surface pressure (upper curve) and ellipsometric angle (lower curve) of ApoA-I kinetics at the liquid/air interface. The vertical dotted line shows the time of acquisition of spectra.

Between the start of the kinetics and the occurrence of the plateau, the intensity of these peaks increases, thus confirming that the peaks are due to the presence of ApoA-I at the liquid/air interface. Moreover, the peak frequencies are typical of an α helical conformation.29–38 In case of a single α helix peptide, the ratio of the intensity of the amide I peak over the intensity of the amide II peak, AMIDI/AMIDII, can be related to the mean angle between the helix and the normal plane of the interface.29,39–42 In the case of protein containing multiple α helices located at the interface, the AMIDI/AMIDII ratio brings information on helix orientation, provided that the helices orient in the same direction.43 As discussed below, geometrical analysis of protein clusters suggests that this is probably true for ApoA-I helices when inserted into a monolayer. In the case of ApoA-I at the liquid/air interface, the AMIDI/AMIDII ratio value is 6 at the beginning of the plateau of the surface pressure, which corresponds to an angle close to 80° (hence, helices are nearly in the plane of the interface). This ratio drops to a value of 3 at the end of the kinetics and corresponds to an angle of approximately 60°. These results thus show that α helices lie in the plane of the liquid/air interface and that they are straightened throughout the establishment of protein– protein interactions. Next, PM-IRRAS spectra were recorded during the first 2 h of ApoA-I adsorption kinetics below DPPC at πinitial = 20 mN/m, as at this πinitial value, ApoA-I is inserted in the lipid monolayers. These spectra were normalized against the spectrum of the liquid/lipid interface, which was recorded before the injection of the protein onto the subphase. Figure

8 shows a normalized spectrum (2 h after the start of the kinetics). The maximal intensity of amide I band is around 1650.8 cm− 1 and shows that ApoA-I adopts a predominantly α helix conformation at the liquid/air interface. At the equilibrium state, the AMIDI/AMIDII ratio is the same as that calculated for the liquid/air interface (about 3), suggesting that the mean angle between helices and liquid surface normal is around 60°. We set out to investigate if the presence of oriented helices at the liquid/lipid interface can be related to the shape of ApoA-I clusters. To determine whether this is the case, we characterized the geometry of protein clusters in lipid monolayers by AFM according to a published method.18 The initial surface pressure was chosen to be 15 mN/m because the amount of protein clusters in the lipid layer is higher at πinitial = 15 mN/m than at πinitial = 30 mN/ m, allowing a proper statistical analysis. Figure 6 shows the distribution of the cluster height as a function of their surface for the three PLs DPPC, DOPC and DPPG. We refer to it as the height versus surface distribution. In all cases, it was noticed that cluster height increases with cluster surface. This effect must be produced by lateral interactions between protein segments outside the lipid layer, which result in the orientation of these segments in the same direction as the cluster surface increases. Figure 6 shows a comparison of the protein height versus surface distribution between the LC phase and the LE phase of the DPPC monolayer (Fig. 6a). As mentioned in the previous section, in DPPC monolayers, the height of clusters is lower in the LE phase than in the LC phase. We noticed that this difference increases as the surface of

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Fig. 6. Graphs of height versus surface for ApoA-I clusters in ApoA-I/PL monolayers at an initial surface pressure (πinitial) of 15 mN/m. Each point represents a cluster. On the top: (a) ApoA-I/DPPC: LC phase (●) and LE phase (○); (b) ApoA-I/DPPC: LC phase (●) and LE phase with height correction for the difference of thickness between lipid phases (+1 nm) (○); (c) ApoA-I/DPPC (LC phase) (●) and ApoA-I/DOPC (Δ) with height correction for the difference of thickness between DPPC and DOPC layers; (d) ApoA-I/DPPC (LC phase) (●) and ApoA-I/DPPG (LC and LE phases) (□).

clusters increases. We tested whether this arises from the difference in thickness between LC and LE phases; Fig. 6b shows that the protein height versus surface distribution corrected by the height difference of 1 nm between LE and LC phases of DPPC does not explain the height difference of the clusters between LE and LC phases. Furthermore, we noticed that the protein height versus surface distribution in the LC phase of DPPC and single LE phase of DOPC (the height correction detailed in Materials and Methods for DOPC is applied) is similar (Fig. 6c). It evidences that the shape of ApoA-I clusters in the LE phase depends on whether the LC phase exists in the layer (as in DPPC) or not (as in DOPC). Such behavior is attributed to the difference of the LE phase compressibility between both lipids in this surface pressure range; condensation of lipids in the

LE phase leaves enough free space for the protein to insert in DPPC, whereas in DOPC, the lack of phase condensation limits protein insertion. The different LE phase compressibility accounts for a distinct geometry of protein clusters in DPPC versus DOPC, and the larger the interfacial space available, the higher are the chances of protein molecules to spread out. Figure 7 shows the height versus surface distribution at two different πinitial values (15 mN/m and 30 mN/m). Only DOPC and DPPG were used for this analysis because in DPPC monolayers the number of protein clusters was very low at πinitial = 30 mN/m. At the resolution of our AFM images (1 pixel ∼ 250 nm2) it can be seen that whatever the lipid used, the cluster geometry was barely modified when the initial surface pressure changed.

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To summarize, we suggest that the decrease in the space between polar headgroups produces an initial straightening of helices, which allows helix–helix interactions outside the lipid layer. The charge of the polar headgroup determines the orientation of protein segments

Fig. 7. Graphs of height versus surface of ApoA-I clusters in ApoA-I/PL monolayers, at initial surface pressures (πinitial) of 15 mN/m (●) and 30 mN/m (□). Each point represents a cluster. (a) ApoA-I/DOPC with height correction for the difference of thickness between DPPG and DOPC layers; (b) ApoA-I/DPPG.

The trend we observed suggests that for surface pressure between 15 mN/m and 30 mN/m the amount of ApoA-I residues inserted in the lipidic layer is not affected by the distance between polar headgroups. Then, it seems that there is a threshold for the polar headgroup spacing where the lowering of distance between polar headgroups does not modify the geometry of clusters but results in a decrease in the amount of protein molecules in interaction with the lipid monolayer, as proved by the decrease in π and Δ. The data suggest that the presence of specific helices inserted into the lipid monolayer is necessary to stabilize ApoA-I at the liquid/lipid interface.

PM-IRRAS measurements were performed to study the conformation of ApoA-I in the presence of the charged lipid DPPG. The adsorption kinetics of ApoA-I below DPPG were recorded using PM-IRRAS under conditions identical with those described for DPPC monolayers. The spectra were normalized to the spectrum of the liquid/lipid interface recorded before protein injection onto the subphase (Fig. 8). Interestingly, ApoA-I under a DPPG monolayer at a πinitial of 20 mN/m shows a significant increase in the (negative) intensity of the dip centered at 1670.1 cm− 1, a feature not observed for ApoA-I under the DPPC monolayer, for which spectra were close to those observed at the liquid/air interface. Such feature might be produced by the increase in the thickness of the interfacial molecular film when the axis of ApoA-I helices is more perpendicular to the interface.42,44 Because this effect prevents us from determining the position of the peak of the amide I band, PM-IRRAS spectra could not provide further insight into the structure of ApoA-I in DPPG. However, geometrical analysis of protein clusters by AFM shows that when the cluster surface increases, the clusters have smaller heights in the LC phase of DPPG than in DPPC or DOPC (Fig. 6c and d). This in turn suggests that ApoA-I residues are closer to the polar headgroups of DPPG than when the protein is exposed to zwitterionic lipid layers. As pointed out in the previous section, protein cluster distribution is the same whatever the phase of DPPG is (Fig. 7). Furthermore, the distribution in the zwitterionic DOPC is remarkably different to that of the negatively charged DPPG. We thus consider that the features of protein cluster geometry between DPPG and others lipids, at least at the resolution of our AFM images, are due to the character of lipid charge rather than the lipid phase. These results are in agreement with our PMIRRAS measurements of a helix straightening in DPPG; if these helices are more deeply inserted in a monolayer of DPPG than DPPC, then the helices localize closer to the negatively charged phosphate group. These observations in turn suggest a different organization of ApoA-I in the DPPG monolayer compared to the other lipids tested.

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69

Fig. 8. Normalized PM-IRRAS spectra of mixed monolayer of ApoA-I/DPPC (dark-gray line) and mixed monolayer of ApoA-I/ DPPG (black line) with a starting surface pressure of 20 mN/m. The spectra of ApoA-I/lipids were normalized by the spectrum of the same lipid (DPPC or DPPG) monolayer at 20 mN/m. For ApoA-I/ DPPC, the position of the peak of amid I band is 1650.8 cm− 1 (amid II band peak is not located). For ApoA-I/DPPG, there is a dip at 1669.1 cm− 1 in the amid I band and a non-attributed maximum at 1440.1 cm− 1. These spectra were recorded about 140 min after the start of the kinetics. ApoA-I spectrum at the end of the kinetics of adsorption at the liquid/air interface (bright-gray line) is shown for comparison. Inset: surface pressure measurement of ApoA-I adsorption kinetics under the monolayer of DPPG (dark line) and DPPC (light dotted line). The vertical dotted line shows the time of acquisition of spectra.

Discussion Our study on Langmuir trough brings detailed information on the relative affinity of ApoA-I with PLs as a function of the physical state and charge of the layer and provides a model system for the early steps of the interaction of ApoA-I with PLs of the cell membrane. Using ellipsometry and surface pressure measurements, we have shown that ApoA-I residues are inserted between the polar headgroups of DPPC, DOPC and DPPG. Moreover, AFM of mixed ApoAI/PL monolayers revealed that ApoA-I molecules associate to form nanometer-sized clusters with heights smaller than 10 nm and surfaces smaller than 0.015 μm2. Furthermore, measurements on AFM images of the relative surface (S) occupied by ApoA-I in lipids at a πinitial value of 30 mN/m as well as measurements of exclusion pressures (πe) show that ApoA-I insertion is more effective in LE DOPC than it is in LC DPPC. Our studies also show that ApoA-I interaction is more effective with DPPG over DOPC and DPPC; ApoA-I has a stronger interaction with the LC phase of anionic lipid than the LC and LE phases of zwitterionic lipids. These results reveal a charge effect, which is in good agreement with previous reports45 and evidences that ApoA-I charge exerts a more important effect than the composition of the phase during ApoA-I insertion into lipidic monolayers. AFM image analysis also shows that ApoA-I insertion in the LE phase is facilitated during the LE/LC phase transition; insertion in LE DPPC is more effective than insertion in LE DOPC, prob-

ably due to the lower compressibility of the latter phase. In the case of zwitterionic lipids, below a minimal distance between polar headgroups (i.e., in the LC phase), protein insertion cannot occur while in the LE phase. Moreover, the amount of inserted protein is dependent upon the compressibility of the lipid layer. We deduced from these observations that ApoA-I insertion induces a lateral displacement of lipids and suggest that the compressibility of the lipid monolayer in the cell membrane might play an important role in controlling the amount of ApoA-I present at this surface, a possibility initially suggested by Ibdah and Phillips in a study performed at the macroscopic level.22 However, our results show that this model would be valid only if the space between polar headgroups allows the insertion of ApoA-I residues. Studies performed on neutral PL vesicles suggest that disorder of the lipidic layer dictates protein insertion and leads to the preferential interaction of ApoA-I with fluid phases of PLs.12 The authors suggest that ApoA-I would be localized at the frontiers between domains.12 Our data confirms the preferential interaction of ApoA-I with the fluid phase and show that ApoA-I is localized well inside the fluid phase and not at the boundaries between fluid and gel phases. We propose that in neutral PL layers the lateral compressibility of the domain is a major driving force for ApoA-I insertion. In the literature on cholesterol efflux, it has been shown that lipid depletion by ApoA-I occurs preferentially with non-raft domains, containing mainly unsaturated lipid and a low amount of

70 cholesterol46 or with raft domains, which contain mainly unsaturated lipids and a lower cholesterol/ lipid ratio.47 This supports our hypothesis that the packing of lipid chain plays a major role in the ApoA-I/lipid interaction. PM-IRRAS measurements performed at the liquid/ air interface show that the amphipathic ApoA-I helices lay at the interface plane when the available space for protein insertion is large enough. However, when lateral interactions between proteins occur, the lying of helices is prevented, and these interactions induce the straightening of α helices. Indeed, in the case of the ApoA-I/DPPC monolayer, PM-IRRAS measurements reveal that ApoA-I helices are not parallel to the liquid/lipid interface plane. AFM image analysis revealed that when the lateral compressibility of the DPPC domains decreases the cluster height for a given surface is higher. We noticed that the differences in geometry are not due to the way proteins and lipids are mixed because the cluster surfaces remain the same. The increase in cluster height is in agreement with PM-IRRAS data and shows that ApoA-I helices do not lay parallel to the interface plane. We conclude from these results that the ApoA-I helices are straightened in the DPPC monolayer and that this is likely the result of lateral interactions arising from the decrease in lateral compressibility when the surface pressure decreases. Importantly, when the lateral compressibility of the lipid monolayer becomes low enough, that is, for πinitial value ranging from 15 mN/m to 30 mN/m, no changes in the cluster geometry were noticed. This trend strongly suggests that ApoA-I could not possibly interact with PLs if some helices were not inserted in the lipid monolayer since the increase in surface pressure is due to the insertion of ApoA-I. Therefore, we postulate that ApoA-I helices can be classified in two categories: helices with high lipid affinity and helices with low lipid affinity. The insertion of high-lipid-affinity helices is necessary to make an ApoA-I/PL interaction while helices with low lipid affinity straighten at the liquid/lipid interface when lateral interactions occur (i.e., when the lateral compressibility of the lipid monolayer is low). These results are supported by previous interfacial studies performed with synthetic peptides that mimic ApoA-I fragments. Such studies revealed that only the two end helices of ApoA-I out of the eight tandem amphipathic helical domains have significant lipid affinity. 48,49 Our studies suggest that different lipid affinity between helices also occurs in full-length ApoA-I, a notion supported by a recent report where the central 3–4 repeat pair of human ApoA-I shows membrane insertion.50 AFM image analysis also revealed that protein clusters are closer to the polar headgroups of DPPG than DPPC and DOPC, thus providing evidence of

Insights into ApoA-I Membrane Insertion

the influence of the headgroup charge over the protein cluster geometry. Moreover, the charge of polar headgroups has an influence on the geometry of the protein cluster regardless of the phase of the PL monolayer. Although the different cluster geometry of DPPG is supported by PM-IRRAS and ellipsometric measurements, further studies are needed to clarify whether a common mechanism mediates the interaction of ApoA-I with anionic and zwitterionic PLs. In any case, what is clear is that the insertion of ApoA-I into the lipid monolayer is a prominent feature of the interaction of ApoA-I with anionic PLs and that the polar headgroups of DPPG establish extensive interaction with ApoA-I. Our analysis of ApoA-I cluster geometry by AFM shows that cluster heights increase with the surface of the cluster, which results in a reorientation of ApoA-I helices. Whether these interactions are the consequence or the driving force of cluster formation requires further investigation. We noticed that the interaction of ApoA-I with anionic lipids does not prevent ApoA-I clustering into the PL monolayer and that the surface of ApoA-I clusters is not smaller in DPPG than in zwitterionic lipids. Numerous papers suggest cooperative interactions between ApoA-I helices and that this may account for discriminating between intramolecular ApoA-I interactions and ApoA-I/PLs interactions.48,49,51,52 The intermolecular interactions could stabilized ApoA-I into PL monolayers and therefore contribute to the πe. In a previous work, Gillotte et al. made a link between the insertion into PL monolayers of peptides, apolipoprotein deletion mutants or fulllength apolipoproteins and the cellular PL/free cholesterol efflux that these molecules induce in human fibroblasts.52 They reported a correlation between this efflux and the exclusion surface pressure πe. Thus, intermolecular interactions may have an influence over the efflux of cellular cholesterol. Furthermore, the existence of ApoA-I clustering might be important for HDL formation since an individual HDL has at less two copies of ApoA-I. It is interesting to compare our results with studies of ApoA-I in interaction with vesicles. Concerning the conditions allowing ApoA-I insertion, a previous study by Arnulphi et al. revealed the influence of cholesterol content during the interaction of ApoA-I with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol vesicles.14 They put in evidence that the presence of cholesterol in the membrane increases the penetration of ApoA-I into the bilayer. This is in agreement with our conclusion that a lateral displacement of lipids allows ApoA-I insertion. Moreover, Prieto and Garda showed by fluorescence measurements that the Y helices of ApoA-I are oriented parallel to the lipid acyl chains when ApoA-I is inserted in the bilayer.50 However, at the present there is no proof of a link between the

Insights into ApoA-I Membrane Insertion

orientation of the Y helices and the limit on the surface occupied by proteins. Concerning the influence of the headgroup charge, results found in literature are more controversial. The influence of anionic PLs over ApoA-I and vesicle interactions was studied by Epand et al.53 These authors characterized the interactions between ApoA-I and vesicles by way of differential scanning calorimetry measurements. They wrote that the interactions between ApoA-I and PC or phosphatidylglycerol (PG) vesicles are both entropically driven and that no supplementary electrostatic interactions between PLs and ApoA-I were made by the replacement of PC by PG. However, differential scanning calorimetry studies of ApoA-I/ PC vesicle interactions do not produce the same conclusions.14 Thus the influence of the headgroup charge during PL/ApoA-I interaction seems to be more difficult to characterize on bilayer system. However, it is worth mentioning that a previous study of Spuhler et al. on lipid vesicles and model peptides that mimic the amphiphilic helices of ApoA-I supports one of the conclusions of our study.51 These authors have studied the binding of ApoA-I model peptides to lipid vesicles by binding assays and NMR experiments. These measurements allow the extraction of the value of the equilibrium constant Kp of the binding peptide/lipid. In their study, the comparison between POPC and POPC/1palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles puts in evidence that Kp is much higher in the presence of a negatively charged lipid headgroup, while the phase of the layers is the same due to the presence of identical acyl chains. This result is in accordance with our assertions concerning the influence of a negatively charged headgroup. Furthermore, Spuhler et al. indicate that after interaction with peptides, POPC vesicles do not fraction into smaller lipidic structures (as micellar structures or non-bilayer phases) and mentioned that dimyristoylphosphatidyl choline vesicles, after interaction with the same peptide, form small discoidal particles.54 Spuhler et al. attribute this fact to the higher lateral packing of the unsaturated lipid system dimyristoylphosphatidyl choline, which favors peptide–peptide interaction. They propose that the energy of peptide-lipid interaction could eventually overcome the lipid–lipid interaction energy and breaks up the extended bilayer structure into smaller domains. Such observations support our assertion concerning the role of the compressibility of the layer. The properties of ApoA-I in lipidic layers resemble the behavior of antimicrobial amphipathic peptides 5 5 and peptide-induced pores in membranes.56 Huang proposed that the antimicrobial function of the peptide results from a two-step mechanism: firstly, the peptide inserts into the vesicle membrane, its axis being parallel to the

71 plane of the interface. This results in a displacement of the polar headgroups and the thinning of the vesicle membrane. Secondly, since the thinning of vesicle membranes is energetically unfavorable, the peptide axis tends to straight when the protein-tolipid ratio of the vesicle is high and this axis becomes perpendicular to the membrane plane. According to this model, the changes lead to the formation of pores that permeabilize the membrane. We have shown that ApoA-I firstly inserts into the lipidic layer between polar head and acyl chains, inducing the displacement of lipids. We also have shown that this initial step is followed by the straightening of the inserted helices when the lateral compressibility prevents the displacement of polar headgroups. It is possible that a similar effect is caused by ApoA-I on vesicle and cellular membranes since the decrease of lateral compressibility of lipid monolayers and the thinning of membrane vesicles imply the displacement of polar headgroups. A similar mechanism might operate during the first steps of membrane solubilization. In a study on a family of amphiphilic peptides, Huang discussed the role of electrostatic interactions and proposed that these could play a regulatory role in target cell selectivity, as bacterial membrane is often negatively charged while the mammal cell membrane is mainly neutral.55 His study prompted us to investigate the influence of the replacement of PC headgroup by PG on the ApoA-I/PL interactions. We showed that favorable electrostatic interactions between ApoA-I and lipids prevent the mechanism described for PC monolayers. Indeed, the extent of electrostatic interactions between ApoA-I and lipids allows the insertion of ApoA-I in the LC phase. Now, the space between polar headgroups is not a factor that limits the insertion of helices and drives their straightening. On the other hand, the polar headgroups establish electrostatic interactions with the whole ApoA-I molecule thus preventing the straightening of helices. Our study concerned pure zwitterionic or negatively charged lipidic layers in an attempt to define how the ApoA-I/lipid interaction depends on the phase and charge of the lipid. One important conclusion is that the ApoA-I/lipid interaction is much more effective when the lipid is charged, whatever the phase of the charged lipid is. This suggests that in a heterogeneous cell membrane the presence of charged lipids, even in low amounts would strongly affect protein–lipid interaction. It also suggests that the ratio of the different lipids in the membrane is related to the establishment of productive interactions. Another important conclusion of our study is that the compressibility of the lipid layer has an influence over protein insertion into the lipid layer, thus evidencing that ApoA-I displaces lipids laterally when inserted into PL monolayers.

Insights into ApoA-I Membrane Insertion

72 The common physicochemical features shared by members of the family of exchangeable apolipoproteins determine the high affinity of these proteins for lipidic layers. At the same time, subtle differences in their amphiphilic properties and/or the local microenvironment (phase and charge of the layer) account for a distinctive behavior in the cell membrane and/or lipoprotein particles.6 Definition of the molecular details of this process is the focus of ongoing studies.

Materials and Methods Protein expression and purification Recombinant ApoA-I was expressed in Escherichia coli Rosetta 2 at 37 °C, 250 rpm, in 2× tryptone–yeast broth. When a cell density of 0.6–0.8 at OD600 nm was reached, cells were induced with IPTG. Inclusion bodies were resuspended in denaturing buffer (i.e., 10 mM Tris buffer containing 8 M urea, 25 mM imidazole and 5 mM β-mercaptoethanol, pH 8.0). The resulting solution was loaded onto one chromatographic column packed with Ni-NTA agarose previously equilibrated in the same buffer solution. After being washed with at least 20 column volumes, recombinant ApoA-I was refolded by quick dilution in Tris-buffered saline (TBS) buffer containing 1 M arginine and concentrated using Vivaspin centrifuge tubes. Refolded ApoA-I was subjected to thrombin cleavage after extensive dialysis in TBS. One unit of thrombin protease of high purity was used to digest 1 mg of recombinant ApoA-I for 6–8 h at 4 °C. Thrombin and the cleaved histidine tag were removed using a benzamidine fast flow column and a Ni-NTA column, respectively, previously equilibrated in TBS. As the final purification step, ApoA-I was loaded onto a gelfiltration column (Superdex 75, HR 26/60) and eluted in 20 mM Tris buffer and 200 mM NaCl, pH 8.0 (TBS buffer), at 1 ml/min. After analysis by SDS-PAGE and measurement of UV absorption spectra (200–300 nm), fractions containing pure ApoA-I were collected and concentrated. Pure recombinant ApoA-I was concentrated 5-fold and stored at − 20 °C. N-terminal sequence and mass spectrometry were carried out to confirm protein identity and purity of recombinant ApoA-I. Buffer and protein concentration All measurements were performed in 20 mM phosphate buffer at pH 7 (0.01 mM Na2HPO4, 0.01 mM NaH2PO4 and ultrapure water) and temperature of 20 ± 2 °C. For ApoA-I subphase concentrations superior to 3.0 μg/ml, the surface pressure value is 22 mN/m. In this case, the liquid/air interface is saturated by proteins, and it could be possible that protein–protein interactions appear in subphase. Hence, a subphase concentration of 1.5 μg/ml was chosen for all experiments, leading to a lower surface pressure of 17 mN/m. At this mass concentration, the concentration of ApoA-I monomers is 53.10− 9 M. Then, at the liquid/lipid interface and with 1.5 μg/ml of ApoA-I in subphase, we suppose that protein–lipid

interactions are favored in comparison to protein–protein interaction in subphase.43 Phospholipids Three PLs were chosen to distinguish phase effects from charge effects: two zwitterionic lipids (DPPC and DOPC) and an anionic lipid (DPPG). DPPC and DPPG have saturated acyl chains and show first-order phase transition from the LE phase to LC phase.25,26 DOPC has unsaturated acyl chains and is always in the LE phase.26 These three PLs were purchased from Avanti Polar Lipids (Alabama, US). Solutions at 0.5 mM DPPC, 0.5 mM DOPC and 0.5 mM DPPG were prepared by dissolving the PLs in chloroform solution. Before each experiment, an isotherm was performed on the PL stock solution. Monolayers and Langmuir–Blodgett techniques Experiences were performed with a computer-controlled and user-programmable Langmuir Teflon-coated trough (type 601BAM, equipped with two movable barriers of a total surface 716 cm2; Nima Technology Ltd., England). Before the experiments were started, the trough was cleaned successively with ultrapure water (Nanopure-UV), ethanol and, finally, warm ultrapure water. The trough was filled with the phosphate buffer. PLs were spread over the clean liquid/air interface between movable barriers using a high-precision Hamilton microsyringe at a pressure of 0 mN/m. After 10 min to allow evaporation of the solvent, films were compressed by moving barriers up to the required surface pressure (πi) at a rate of 20 cm2/min. Then, the barriers were maintained at a fixed position (so that the surface of the trough is constant), and the protein was injected in the subphase with a Hamilton syringe. The needle of the syringe passed under a barrier, and in this case, the PL monolayer was not perturbed. The surface pressure was measured following a Wilhelmy plate method using a filter paper connected to a microelectronic feedback system for surface pressure measurements. Values of surface pressure (π) were stable and recorded every 4 s with a precision of ±0.2 mN/m. Ellipsometry measurements were carried out with an inhouse automated ellipsometer57 in a “null ellipsometer” configuration.58 He–Ne laser beam (λ = 632.8 nm; Melles Griot) is polarized with a Glan–Thompson polarizer and reflected on the surface of the trough (incidence angle of 52.12°). After reflection on the water surface, the laser light passed through a λ/4 retardation plate, a Glan–Thompson analyzer and a photomultiplier. The analyzer angle, multiplied by two, yielded the value of the ellipsometric angle (Δ), that is, the phase difference between parallel and perpendicular polarizations of the reflected light. The laser beam probed a surface of 1 mm2 and a depth in the order of 1 μm. Initial values of the ellipsometric angle (Δ0) and surface tension of pure buffer solutions were recorded on the subphase for at least half an hour. These values have been subtracted from all data presented below. Values of Δ were stable and recorded every 4 s with a precision of ± 0.5°. For imaging pure monolayers of PLs by AFM (without injection of protein), the film was transferred to a mica

Insights into ApoA-I Membrane Insertion

support that was previously immersed in the subphase by the Langmuir–Blodgett method. The transfer speed was 0.16 mm/min. In the case of protein injection, the mixed monolayer was transferred at the equilibration state (i.e., surface pressure reached a plateau) to a mica support that was not previously immersed in the subphase unlike the case of PL monolayers. The transfer speed was always 0.16 mm/min. Pure PL monolayers and mixed monolayers of proteins/PLs were transferred to freshly cleaved mica sheets. AFM measurements Surface images of the Langmuir–Blodgett monolayers were obtained under ambient conditions using a Pico-plus atomic force microscope (Molecular Imaging, Phoenix, AZ) operating in contact mode. Two scanners of 100 × 100 μm2 and 10 × 10 μm2 were used for measurements. Topographic images were acquired in constant force mode using silicone nitride tips with a nominal spring constant of 0.06 N/m (manufacturer specified). The applied force was kept as low as possible during the scan (∼ 1 nN). AFM images of 8 × 8 μm2 size, with lateral resolution of ∼250 nm2 by pixel, are analyzed to characterize the geometry and surface occupied by protein clusters. For these images, the resolution of the height is 0.117 nm (z-range = 30 nm; 256 levels of gray). These images were exported from the AFM microscope in TIFF format. The images were analyzed with the public image processing program ImageJ†. In order to determine the surface and height of objects in the AFM images, we performed the following process. The images were similarly thresholded at gray level 140 (1.4 nm above the lipid). The pixels with a gray level above this threshold take a value of 255, while the others take a null value. Therefore, in the binarized images, the center and the surface of objects are calculated. Then, on the gray-level images, the program searches the maximal gray value around the center of the object to determine its height. Objects with surfaces greater than 0.046 μm2 were considered as typical lipid domains (i.e., nano-domains of the LC phase showing over elevations). For comparison of objects in DOPC to objects in DPPC (LC phase) and DPPG, we applied a correction to the height of objects in DOPC due to the fact that the thickness of DOPC layer is smaller than the thickness of DPPG and DPPC, with a difference of 1 nm. This correction consists of thresholding AFM images at gray level 149 (1 nm higher than the previous threshold) and then translation of the reference (height = 0 nm) of 1 nm. To select the objects in the LE phase of DPPC, we used a lower threshold than gray level 140 due to the low height of these clusters. Then, we used a threshold at gray level 125 (0.35 nm below LC phase height). Graphs of particle height versus particle surface were built from AFM images of ApoA-I/PL layers with initial surface pressures (πinitial) of 15 mN/m and 30 mN/m. To measure the total surface occupied by clusters for each lipid, which will be referred as S, we summed object surfaces, and this sum is divided by the number † http://rsbweb.nih.gov/ij/

73 Table 3. Total numbers of AFM images and protein clusters for measurements of surface occupied by protein molecules in lipid monolayers (S) Number of AFM images (number of clusters)

A-I/DPPC A-I/DOPC A-I/DPPG A-I/DOPC, gray level 149

15 mN/m

30 mN/m

6⁎ (328) 6⁎ (2065) 8 (1351) 6⁎ (1156)

9 (292) 13 (1629) 8 (1219) 13 (628)

Measures marked with asterisk (⁎) were performed with one Langmuir–Blodgett sample of monolayers; other measures were performed with two Langmuir–Blodgett samples of monolayers.

of AFM images. The result is expressed in percent of the surface of the image. However, a correction must be applied because some defects are observed on pure monolayers of PLs. These imperfections might be confused with clusters of proteins in mixed monolayers of lipids and proteins. Thus, we computed the mean surface occupied by imperfections (we refer to them as the residual surface) on at least four AFM images of pure PL monolayers, and we subtracted this surface from the surface occupied by clusters on mixed monolayers of lipids and proteins. A statistical test for bilateral comparison of means‡ was used to check as to whether the difference of the occupied surface between DPPC, DOPC and DPPG is significant (P b 0.05). Next, we measured the error produced by image resolution on surface measurements. We supposed that clusters are circular and that radius is known with a precision of 1 pixel; the convolution of tip is not significant as the error is less than standard deviation divided by 10. Measurement of S reveals better the protein occupation in lipids than the number of protein clusters by surface unit N/s. However, when the cluster height is very different between lipids or between phases, we used N/s to compare the difference of protein occupancy in the lipids. For example, to get the N/s value in the LE phase of DPPC, we measured the mean number of protein clusters in the LE phase of DPPC (total number of protein clusters in the LE phase over all AFM images divided by the number of AFM images) and divided the mean surface occupied by the LE phase over AFM images (total surface of the LE phase over all AFM images, in square micrometers, divided by the number of AFM images). For comparison of cluster density between LE and LC phases of DPPC, we distinguished protein clusters in the LE phase from the nano-domains of lipids (top of lipid domains is localized at a height ∼ 0 nm). Table 3 shows the number of AFM images and the number of particles used to measure the surface occupied by proteins and to build graphs. In situ infrared spectroscopy We studied the secondary structure of ApoA-I at the hydrophilic/hydrophobic interface with and without ‡ http://www.info.univ-angers.fr/∼gh/wstat/compmoy.php

Insights into ApoA-I Membrane Insertion

74 lipids. PM-IRRAS measurements were first performed at the liquid/air interface. In this case, the ApoA-I structure is influenced by the hydrophilic/hydrophobic interface and by protein–protein interactions at this interface. Next, PM-IRRAS measurements were performed at the liquid/lipid interface. In this case, the ApoA-I structure is influenced by the hydrophilic/ hydrophobic interface, protein–protein interactions and, now, protein–lipid interactions. Two lipids were used: DPPC and DPPG. The comparison between these two lipids permits us to know the influence of polar headgroup charge on the conformation/orientation of protein segments. PM-IRRAS combines Fourier transform infrared reflection spectroscopy of an interface with fast modulation of the polarization of the infrared radiation between parallel (p) and perpendicular (s) directions with respect to the plane of incidence.42,59 The PM-IRRAS spectrometer used has been described in detail elsewhere.59 The two-channel processing of the detected signal gives the differential reflectivity spectrum ΔR/R = (Rp − Rs)/(Rp + Rs). With an angle of incidence of 75°, transition moments in the interface plane give strong and upward-oriented bands, while transition moments perpendicular to the interface give weaker and downward-oriented bands.42 PM-IRRAS was performed using a second Langmuir trough under conditions identical to those described for ellipsometry and surface pressure measurements. The surface of the second trough was ca 95 cm2. The formation of the lipidic layers and the injection of protein were the same as described previously. In the case of protein alone at the buffer/air interface or injected below a lipidic layer, the protein concentration was 1.5 μg/ml. Spectra were recorded at 8 cm− 1 resolution on a Nicolet 850 FTIR spectrometer equipped with a photovoltaic MCT detector (SAT, Poitiers). All spectra were recorded at a scanning mirror speed of 0.47 cm s − 1 by co-adding 600 scans. The total acquisition time for each spectrum was approx. 10 min. PM-IRRAS spectra were recorded as a function of time up to several hours at the beginning of the adsorption kinetics. The spectrum of the bare liquid/air interface was systematically recorded and used as the reference to divide the spectra of the protein alone at the liquid/air interface. In the case of the mixed lipid/ ApoA-I monolayer, we recorded the spectrum of the lipidic layer before the injection of protein, and we report the spectra of the mixed lipid/ApoA-I monolayer divided by the spectrum of lipidic layer. Normalized spectra were corrected for baseline deviations.

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